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1, | SciELO

tr- '

Governo do Estado de São Paulo

Secretaria de Estado da Saúde

Coordenação dos Institutos de Pesquisa

Instituto Butantan — São Paulo-SP — Brasil

MEMÓRIAS

INSTITUTO BUTANTAN

Volume 51, número 4, 1989

São Paulo, SP - Brasil

1989

SciELO

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MEMÓRIAS do INSTITUTO BUTANTAN. (Secretaria de Estado da Saúde)

São Paulo, SP — Brasil, 1918 —

1918 - 1983/84, 1-47/48

Publicação interrompida de 1985 a 1986.

1987, 49 (1-3)

1988, 50 (1-3, supl.)

1989, 51 (1-4)

ISSN 0073-9901

MIBUAH

CDD 614.07205

Solicita-se permuta / Exchange desired

SciELO

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Mem. Inst.Butantan

51(4), 1989.

FASCÍCULO EM HOMENAGEM AO 70? ANIVERSÁRIO DO

DR. SAUL SCHENBERG, DIRETOR DO SERVIÇO

DE FISIOLOGIA DO INSTITUTO BUTANTAN.

THIS NUMBER IS A TRIBUTE TO DR. SAUL SCHENBERG,

DIRECTOR OF THE DEPARTMENT OF PHYSIOLOGY,

INSTITUTO BUTANTAN, ON OCCASION OF HIS 70th

BIRTHDAY.

Mem. Inst.Butantan

51(4), 1989.

SUMÁRIO/CONTENTS

Studies on the cross-reactivity of snake venom antisera.

Estudos sobre reatividade cruzada de antivenenos de serpentes.

D. MEBS & F. KORNALIK. 127

Hemoglobin biosynthesis in bone marrow and peripheral blood

erythroid cells of the snake Waglerophis merremii (Reptilia, Ophidia,

Colubridae)

Biossíntese de hemoglobina nas células eritróides de medula óssea'

e do sangue periférico da serpente Waglerophis merremii (Reptilia,

Ophidia, Colubridae)

Diva Denelle SPADACCI MORENA; Pasquale MORENA; Aurora

Marques CIANCIARULLO; Adolpho BRUNNER JÚNIOR ... 133

The occurrence of N-methyltaurine in the sea anemone

Bunodosoma caissarum

A ocorrência de N-metiltaurina na anênoma-do-mar Bunodosoma

caissarum

Raymond ZELNIK; Maria de Fátima Alves da SILVA; Flavia

MARTELLINI-LANDSHOFF; Akhtar HAIDER. 141

Standardization of anesthesia with pentobarbital in the snake

Bothrops jararaca.

Padronização de anestesia com pentobarbital em serpente Bothrops

jararaca.

Ana Marisa CHUDZINSKI; Marília C. L. SEELAENDER; Eva M.

A. KELEN. 147

Cross-reactivity of horse monovalent antivenoms to venoms of ten

Bothrops species.

Produção de soros hiperimunes monovalentes para venenos de

espécies de serpentes do gênero Bothrops.

Wilmar DIAS da SILVA; Rosalvo GUIDOLIN; Isaías RAW; Hisako

Gondo HIGASHI; Celso Pereira CARICATI; Josefina Farina

MORAIS; Maria Laura S. R. LIMA; Ivone Kazuko YAMAGUCHI;

Amélia Keiko NISHIKAWA; Marco Antonio STEPHANO; José

Roberto MARCELINO; José Ricardo PINTO; Maristela José

SANTOS . 153

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51(4), 1989.

Variação do comportamento biológico do tumor humano KB,

heterotransplantado seriadamente em ratos "nude" atímicos.

Variation on biological behavior of human KB tumor serially

heterotransplanted into athymic nude rats.

Leonor DENARO-MACHADO; Suely A. KFOURI; Terezinha R.

P. OGATA; Jesus Carlos MACHADO; Gilberto Alvarenga PAULA;

Lúcia Silva BARBOSA; Regina F. G. FERREIRA; Selma M. C.

N. PETRELLA. 169

Biochemical studies of the sea snake neurotoxins.

Estudos bioquímicos de neurotoxinas de serpente marinha.

Anthony T. TU & Roger A. MILLER. 177

Chromatographic purification of antivenoms and antitoxins.

Purificação cromatográfica de antivenenos e antitoxinas.

Waldely de Oliveira DIAS; Maria Izabel ESTEVES; Joana Akiro

FURUTA; Hisako Gondo HIGASHI; José Roberto MARCELINO;

Nilza Yurimi OISHI; Sally Muller Affonso PRADO; Anita Mitico

TANAKA; Celina Maria Pompeo Mome UEDA; Isaías RAW 195

Sarafotoxins, a new group of cardiovascular modulators from snake

venom

Sarafotoxinas, um novo grupo de moduladores cardiovasculares

a partir do veneno de serpente.

Elazar KOCHVA; Avner BDOLAH; Dan GRAUR; Zvi WOLLBERG 205

Local tissue damage induced by Bothrops snake venoms. A review.

Lesão local tissular induzida pelos venenos de serpente Bothrops.

Uma revisão.

José Maria GUTIÉRREZ & Bruno LOMONTE. 211

Isolation and properties of a phospholipase A2 from the venom of

the snake Bothrops moojeni (Caissaca)

Isolamento e propriedades da fosfolipase A2 do veneno de Bothrops

moojeni (Caissaca)

Antonia Paula REICHL; Solange de Toledo SERRANO; Marina

Tizuko ASSAKURA; Fajga Ruchla MANDELBAUM. 225

Coletânea de resumos de trabalhos publicados pelos pesquisadores

do Instituto Butantan (1988)

Collection of sumaries of articles published by the scientific staff

of Instituto Butantan (1988). 239

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Mem. Inst. Butantan

51(41:127-132, 1989.

STUDIES ON THE CROSS-REACTIVITY

OF SNAKE VENOM ANTISERA

D. MEBS*

F. KORNALIK* *

ABSTRACT: Cross-reactivity between antisera and snake venoms which

have not been used for immunization is a common phenomenon observed

in closely related as well as in quite distinct snake species. A polyvalent

antivenom (Behringwerke North-Africa) was found to possess considerable

neutralization potency to a North-American crotalid snake (Crotalus

adamanteus). By tractionation of Bitis arietans and of Crotalus adamanteus

venom hemorrhagic factors were isolated which were neutralized by the

antivenom indicating that there exist commom antigenic properties in

venom constituents of even unrelated snake species.

KEY WORDS: Snake venom, antivenom cross-reactivity, hemorrhagic

factors.

INTRODUCTION

Snake venoms are complex mixtures of proteins and polypeptides, which

have antigenic properties. Immunesera raised against snake venoms con-

tain, therefore, a great number of antibodies, of which some are important

to neutralize the venom's lethal effect. Cross-reactivity of antivenoms with

venoms of snakes which have not been used in immunization is a common

phenomenon 1 . This leads to the assumption that venoms of even unrelat¬

ed snakes representing different species or genera, or which are geographi-

cally separated may have common antigens.

The immunologic analysis of such complex mixtures of antigens and

antibodies can be performed by applying immunodiffusion techniques.

Precipitation lines, the reaction product between antigen and antibody, permit

conclusions about the extent of relationship between venoms. A number

of studies exist providing informations on interrelationships between spe¬

cies, on evolutionary and genetic implications 1 . However, the data obtained

Zentrum der Rechtsmedizin. University of Frankfurt, Frankfurt/Main Federal Republic of Germany.

Institute of Pathophysiology. University Karlovy. Prague, CSSR.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 25/01/1989; accepted 09/02/1989.

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MEBS, D. & KORNALIK, F. Studies on the cross-reactivity of snake venom antisera. Mem. Inst. Butan-

tan, 51(41:127-132, 1989

by these methods have limited value, if practical aspects of cross-reactivity,

i.e. cross-neutralization, are concerned. The neutralization of the lethal and

other harmful effects of snake venoms by antivenoms, which are paraspecific

to others not used in immunization can only be evaluated by in vivo test-

systems. This includes the neutralization of lethality, LD 50 , hemorrhagic,

necrotizing, defibrinating or coagulant activities in mice or rats. Whereas

data on the neutralization capacity of commercial antivenoms to the lethal

action of various snake venoms exist, neutralization studies on other ven¬

om activities are still rare. The WHO standardization program should cer-

tainly contribute to the knowledge on the potential cross-neutralization

capacity of antivenoms. 2

Beside these basic informations: which antivenom reacts with which

venom, it appears important to find an answer to the questions: what charac-

terizes common antigens and what is their role in the course of envenoma-

tion. It is quite clear that the main approach to this problem should

concentrate on those venom components (antigens) only, which are involved

in the venorrTs lethal or pathological actions.

In studies on various commercial antivenoms, their neutralizing capaci¬

ty to venom activities such as lethal, hemorrhagic, necrotizing, defibrina¬

ting and coagulant effects, striking cross-reactivities had been observed 3 ' 4 ^

For instance Behringwerke-antivenom North-Africa as well as Wyeth

Anticrotalidae-antivenom were both found to be very effective in neutraliz¬

ing the lethal activity of the venom from the African puff adder ( Bitis arie-

tans). But it has to be considered that the polyvalent Behringwerke-antivenom

only was produced by using Bitis gabonica venom as antigen among other ven¬

oms such as from Naja haje, Vipera lebetina and Echis carinatus, whereas

in Wyeth-antivenom only North-and Central-American crotalid snake ven¬

oms, such as from Crotalus atrox, C.adamanteus, C.durissus terrificus and

Bothrops atrox had been used for immunization. On the other hand, Wyeth-

antivenom was much less effective to Crotalus adamanteus venom, but

Behringwerke-antivenom shows a four times higher activity in the mouse

protection test. Therefore, one may conclude that a common antigen (or

antigens) responsible for the lethal effect must be present in both venoms.

Bitis arietans as well as Crotalus adamanteus venom produce massive

hemorrhage which eventually leads to death (at least in mice), if internai

bleeding occurs. Consequently the efficacy of both antivenoms to neutral¬

ize hemorrhagic activity was tested.

MATERIAL AND METHODS

Bitis arietans venom was purchased from D. Muller, Johannesburg, South

África; Crotalus adamanteus venom was obtained from F. Kornalik. The fol-

lowing antivenoms were used for neutralization studies: polyvalent Crotali-

dae antivenom from Wyeth Laboratories, Philadelphia, PA, USA; polyvalent

antivenom North-Africa from Behringwerke, Marburg, FRG. LD 50 was as-

sayed by intravenous injection into mice (20 g, 10 animais per dose), neu¬

tralization capacity of antivenoms by preincubating 0.1 ml venom solution

(0.9% NaCI) with 0.1 ml antiserum for 30 min at 37° C. Hemorrhagic ac-

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MEBS, D. & KORNALIK, F. Studies on the cross-reactivity of snake venom antisera. Mem. Inst. Butan-

tan, 51(41:127-132, 1989

tivity was tested in mice according to Taborska 6 . Prior intradermal venom

application 0.2 mlof51 Cr labled mice erythrocytes were injected in-

travenously. In the neutralization tests 50 Mg venom in 0.1 ml saline were

mixed with 0.1 ml antivenom or normal rabbit serum as control and, after

30 min incubation, injected intradermally in the interscapular region of mice.

The animais were sacrificed after 24 hrs and the amount of extravasai blood

of a 2.5 cm 3 area at the injection site was calculated from radioactivity. Ven¬

om fractionation was performed as described previously 7 .

RESULTS AND DISCUSSION

Under the conditions described Behringwerke-antivenom is able to com-

pletely neutralize the hemorrhagic activity of Bitis arietans and almost com-

pletely that of Crotalus adamanteus venom (Fig. 1). Wyeth-antivenom is less

active towards Bitis venom, but provides full protection in the case of Crotalus

adamanteus venom.

To identify the antigen or antigens involved in this cross-reaction Crota¬

lus adamanteus venom was fractionated by gel filtration (Fig. 2). Three main

protein-containing fractions were separated. Fraction I contained the hemor¬

rhagic principie of the venom, fraction II several enzymes (proteases, phos-

pholipase A) and fraction III represents a toxin of crotamine-like activity which

is rarely found in Crotalus adamanteus, but is present in other Crotalus

venoms 8 . It has myotoxic activity producing local myonecrosis like myotoxin

a from C. viridis viridis venom. The last two fractions are protein-free and

consist of nucleotides or similar compounds. When the LD 50 of the frac¬

tions was determined by i.v. injection, only fraction I and III exhibited marked

lethality, whereas fraction II was essentially non-toxic. Since similar toxins

like the myotoxin of fraction III are not present in Bitis venom, it was as-

sumed that the hemorrhagic factors in both venoms may be responsible

for causing cross-reactions. Preliminary experiments demonstrated that frac¬

tion III is not neutralized by both antivenoms.

Since fraction II is essentially non-toxic, it may play a minor role in en-

venomation. Therefore, the hemorrhagic principie present in fraction I was

further purified by ion-exchange chromatography on DEAE-cellulose, the

hemorrhagin obtained was a semi-pure product which was compared to

a hemorrhagin previously isolated from Bitis arietans venom 7 . Immunodiffu-

sion tests using the Ouchterlony method revealed that both crude venoms,

Bitis arietans and Crotalus adamanteus, produce several precipitation lines

when reacting with Behringwerke-antivenom (Fig. 3). But also the semi-pure

hemorrhagins from both venoms show distinct bands. Moreover, the non-

toxic fraction II produced much more precipitation lines indicating that criteria

for cross-reactivity based only on immunodiffusion tests are less useful

In preliminary tests using 50 Mg of each hemorrhagin mixed with 0.1

ml antivenom Behringwerke-antivenom was more effective (1:8 dilution) in

neutralizing Crotalus adamanteus than Bitis arietans hemorrhagin (1:4 dilu¬

tion). Wyeth-antivenom exhibited full protection (more than 1:8 dilution) to

Crotalus adamanteus, but was ineffective to Bitis arietans hemorrhagin.

What are the conclusions from these experiments? Snake venoms con-

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MEBS, D. & KORNALIK, F. Studies on the cross-reactivity of snake venom antisera. Mem. Inst. Butan-

tan, 51(41:127-132, 1989

Venom

Antivenom

Control

Bitis

arietans B W N

C.adamanteus

Residual Hemorrhagic Activity (%)

0 50 100

Fig. 1. Neutralization of hemorrhagic activity of Bitis arietans and Crotalus adamanteus ven¬

om (50 ng) by Behringwerke North-Africa (BWN) and Wyeth Anticrotalidae antivenom (W;

0.1 ml antivenom, undiluted).

Fig. 2. Gel filtration of 500 mg Crotalus adamanteus venom on a Sephadex G-75 column

(95 x 2.5cm) eluted with 0.1M ammonium acetate. Fractions of 6 ml were collected at a

flow rate of 36ml per hr. LD 50 values of crude venom (c.v.) and fraction I and III are shown

in the elution pattern, fraction II is essentially non-toxic.

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MEBS, D. & KORNALIK, F. Studies on the cross-reactivity of snake venom antisera. Mem. Inst. Butan-

tan, 51(41:127-132, 1989

Bitis arietans

Crotalus odamanteus

Fig.3. Immunodiffusion-test of Bitis arietans (BA), Crotalus adamanteus (CA) and of their

semi-pure hemorrhagins (h.) to Behringwerke North-Africa (BW) antivenom.

131

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MEBS, D. & KORNALIK, F. Studies on the cross-reactivity of snake venom antisera. Mem. Inst. Butan-

tan, 51(41:127-132, 1989

tain common antigens which are of minor importance in envenomation, but

others which may also be involved in the venom's lethal or noxious action.

From a practical point of view, demonstration of cross-neutralization seems

to be of great value, if a wider application of polyspecific antivenoms in snake-

bite cases is concerned. On the other hand, our present knowledge on cross-

reactivity and neutralization is rather poor, if one considers the number of

commercial antivenoms available and the tests which could be done with

snake venoms involved in human envenomations. Beside lethality-

neutralization this should include other test systems such as hemorrhagic,

necrotizing, coagulant and defibrinating activities. Another aspect seems

to be even more important. Since common antigens in snake venoms respon-

sible for the lethal or pathologic effects can be well identified, it should be

possible to produce an antigen mixture which includes all major venom com-

ponents leading to an antivenom which exhibits broad specificity. This could

be a mixture of purified antigens, such as neurotoxins, hemorrhagins,

coagulant factors etc., or a mixture of representative snake venoms which

contain these antigens in high concentrations. It may be a promising way

in the production of more effective antivenoms for the treatment of snakebite.

RESUMO: A reatividade cruzada de antivenenos com venenos de serpen¬

tes, os quais não foram utilizados na imunização, é um fenômeno comum

observado seja com espécies de serpentes bastante relacionadas, bem

como com espécies diferentes. Verificou-se que um antiveneno poliva¬

lente (Behringwerke North-Africa) possui considerável potência neutrali-

zante para veneno de serpente crotalidea norte-americana (Crotalus

adamanteus). Por fracionamento dos venenos de Bitis arietans e de Cro¬

talus adamanteus foram isolados fatores hemorrágicos, os quais foram

neutralizados pelo antiveneno, indicando a existência de propriedades an-

tigênicas comuns nos constituintes dos venenos, mesmo de espécies não

relacionadas.

UNITERMOS: Veneno de serpente, reatividade cruzada de antiveneno, fa¬

tores hemorrágicos.

REFERENCES

1. MINTON, S.A. Common antigens in snake venoms. In: LEE, C.Y. ed. Snake venoms. Ber-

lin, Springer Verlag, 1979 p. 847-62. (Handbook Experimental Pharmacology, 52).

2. WORLD HEALTH ORGANIZATION. Progress in the characterization of venoms and stan-

dardization of antivenoms. Offset Publication (58), 1981.

3. KORNALIK, F. & TABORSKA, E. Neutralization of lethal, local and systemic effects of snake

venoms by monovalent and polyvalent antivenoms. In: EUROPEAN SYMPOSIUM ON

ANIMAL, PLANT & MICROBIAL TOXINS, 5. Proc. Hannover, 1983. p. 9-15.

4. MEBS, D. Myotoxic activity of phospholipases A, isolated from cobra venoms; neutrali¬

zation by polyvalent antivenoms. Toxicon, 24: 1001-1008, 1986.

5. MEBS, D.; POHLMANN, S.; VON TENSPOLDE, W. Snake venom hemorrhagins; neutrali¬

zation by commercial antivenoms. Toxicon, 26: 453-458, 1988.

6. TABORSKA, E. Intraspecies variability of the venom of Echls carinatus Physiol. bohemos-

lovaca, 20: 307-318, 1971.

7. MEBS, D. & PANHOLZER, F. Isolation of hemorrhagic principie from Bitis arietans (puff

adder) snake venom. Toxicon, 20: 509-512, 1982.

8. MEBS, D; EHRENFELD, M.; SAMEJIMA, Y. Local necrotizing effect of snake venoms on

skin and muscle; relationship to serum creatine kinase. Toxicon, 2i: 393-404, 1983.

132

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Mem. Inst. Butantan

51(41:133-139, 1989.

HEMOGLOBIN BIOSYNTHESIS IN BONE MARROW AND

PERIPHERAL BLOOD ERYTHROID CELLS OF THE SNAKE

WAGLEROPHIS MERREMII (REPTILIA, OPHIDIA, COLUBRIDAE)* *

Diva Denelle SPADACCI MORENA**

Pasquale MORENA***

Aurora Marques CIANCIARULLO* *

Adolpho BRUNNER JUNIOR**

ABSTRACT Bone marrow and peripheral blood erythroid cells of the snake

Waglerophis merremii were ultrastrücturally analysed. To enhance the entry

of immature cells to the peripheral blood, erythropoiesis was stimulated

through the induction of a hemolytic anemia, by injecting saponin. Typical

hemosomes, very similar to those observed in mammalian and avian

immature erythroid cells were found, as well as some of their precursor

forms. These organelles are taken as possible sites where the integration

of heme into the four globin polypeptide chains occur, on account of their

content of hemoglobinic nature. Such entities are a specialyzed indirect

derivation from mitochondria, through successive transformations to

lamellated bodies, prehemosomal vesicles, prohemosomes and finally to

hemosomes.

KEY WORDS: Hemoglobin biosynthesis; snake hemoglobin; snake he-

mosome.

INTRODUCTION

lt has been proposed that hemoglobin (Hb) biosynthesis could occur

in hemoglobinized organelles termed hemosomes, or where heme is integrat-

ed into the four polypeptide globin chains 1 . Such organelles have a highly

dense matrix, due to their Hb content, their narrow intralamellar space has

a longitudinal disposition and in general their diameters are smaller than

the diameters of mitochondria. These studies began by the examination of

• This work was presented in the XI Coll. Braz. Soc. Electron Microscopy — Caxambu-MG-Brazil, 1987.

* * Seção de Microscopia Eletrônica — Instituto Butantan.

* * * Seção de Hematologia — Instituto Butantan.

Correspondence should be send to Dr. A. Brunner Jr. C.P. 65-01051, SP — Brazil

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 10/11/1988; accepted 02/03/1989

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SPADACCI MORENA, D.D.; MORENA, P. CIANCIARULLO, A.M, BRUNNER JÚNIOR, A. Hemoglobin

biosynthesis in bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae). Mem. Inst. Butantan, 51141:133-139, 1989.

rabbit-embryo peripheral blood erythroblasts and reticulocytes. Further in-

vestigations were carried out in order to ascertain the hypothesis on the

functional role of hemosomes. Several experimental and analytical works

showed circumstantially a constant close relationship between Hb biosynthe¬

sis and hemosome formation 2 " 5 - 7 ' 8 . Hemosomes were also found in

mouse 11 ' 12 . 14 , other rodents 13 , chicken 15 , toads ( Bufo ictericus and Bufo

paracnemis)' 10 , as well as in the fish Thymallus thymallus erythroid cells 14 .

It has been pointed out that this characteristic organelle is an indirect deri-

vation of the mitochondrion 7 ' 9 , that occurs through successive modifica-

tions to lamellated body, prehemosomal vesicle, prohemosome, and

hemosome.

This paper shows that Waglerophis merremii erythroid cells contain

hemoglobinized organelles, through cell fractionation and electrophoresis

of the organellar lysate supernatant. Results of electron microscopic studies

on some stages of hemosome formation are presented, as already partially

shown 19 , comparatively to the findings in erythroid cells of other ver¬

te b rates.

MATERIAL AND METHODS

Normal adult snakes (Waglerophis merremii) newly received by the Ins¬

tituto Butantan were submitted to hemolytic anemia. The snakes were sub-

cutaneously injected, each day with saponin in 0.75% saline solution, 3

mg/kg body weight, for three days; blood was harvested (72 h), and reticulo-

cyte countings reached approximately 30%, as examined through a modi-

fied new methylen blue technique 18 . For electron microscopy, blood was

fixed initially in 1% glutaraldehyde in 0.20 M Millonig's buffer, for 1 h at

room temperature, in order to concentrate the immature forms in the course

of normal sedimentation. After this, erythroid cells were fixed in 3%

glutaraldehyde, for approximately 2 h at room temperature, followed by os-

mic tetroxide fixation, 1% in the same buffer for 1 h at 4°C, uranyl acetate

contrastation, 40 min at room temperature, dehydration, and embedding

in Polylite 8001 9 . Thin sections were obtained in a Porter-BIum MT-1 micro-

tome, contrasted by lead citrate and examined in the Elmiskop I and Zeiss-

EM 109 electron microscopes, at 60 and 80 kV, from x 7,000 to x 50,000

magnification.

For the demonstration of Hb within the hemosomes through electropho¬

resis in polyacrylamide gel 16 , blood cells were fractionated, the hemosomes

isolated, washed and osmotically lysed, following Brunner eia/. 1 with some

modifications: Centrifugation at 4,000 xg, instead of 1,350 xg, for a com¬

plete nucleus and cell debris sedimentation without repetition of this step;

centrifugation at 12,000 xg, instead of 10,000 xg, for the organellar frac-

tion sedimentation. The supernatants of the organellar lysate and of the last

washing were concentrated ten fold, or lyophilized, and Hb was diluted to

1:50. A 2.5mA current was applied for 40 min at 5°C, and Hb bands were

identified by benzidine or tolidine reaction.

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SPADACCI MORENA, D.D.; MORENA, P. CIANCIARULLO, A.M, BRÜNNER JÚNIOR, A. Hemoglobin

biosynthesis in bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae). Mem. Inst. Butantan, 51(41:133-139, 1989.

RESULTS AND DISCUSSION

The interpretation given for the several structural aspects found in

Waglerophis merremii erythroid cells synthesizing Hb and involved in hemo-

some formation, is basically fundamented on studies carried out in mam-

malian erythroid cells 9 . A general view of a maturing bone marrow

erythroblast is shown in Fig.1, corresponding to a still highly immature stage,

as evaluated by the high number of polysomes and single ribosomes. Some

organelles modifying continuously from prohemosomes to transitional stages

followed by transformation to the hemoglobinized hemosomes, are seen.

A long organelle constituted by the three developmental stages is shown

in Fig.2, presenting a great structural similarity to the hemosomes and their

precursors found in rabbit-embryo 1 , adult rabbit 6 ' 8 , guinea-pig 3 , human 2 ,

and chicken 15 erythroid cells. A typical hemosome of a peripheral blood im¬

mature erythrocyte is seen in Fig.3; for comparison, an obliquely sectioned

mitochondrion is presented in the inset.

As to Hb biosynthesis dynamics it begin by iron uptake via transferrin

which constitute complexes through an interaction with glycoprotein recep-

tors bound at numerous sites of the cell surface, probably as happens in

mammalian erythroid cells 17 . This occurs through rhopheocytosis, consist-

ing in a Progressive invagination process of the plasma membrane (Fig.8),

giving rise to endocytic vesicles (Fig. 6). These vesicles fuse among them-

selves resulting large, highly electron-dense free ferruginous particle inclu-

sions, due to the disappearance of the limiting membrane. Afterwards, such

ferruginous inclusions, constituting an iron source for heme biosynthesis,

are involved by an initially single membrane (Fig.4); the vesicle content be-

comes amorphous and less electron-dense. The membrane which involves

the ferruginous material results from the unfolding of double lamellae origi-

nated from a lamellated body that in turn is a mitochondrial derived struc-

ture (Fig.5), as shown in tissue culture cells 7 and in rabbit erythroblasts 9 .

This lamellated body is a structural as well as probably an enzymatic com-

plex catalysing heme and the final Hb biosynthesis reactions. From this stage

onwards the captation of iron containing compounds, and of the globin chains

occur through double lamellae expansions. These expansions return con¬

stituting prehemosomal vesicles which, possibly after a rotational movement,

approaches the ferruginous material and the globin chains one another, giv¬

ing rise to condensed prehemosomal vesicles (Fig. 6) that afterwards modify

successively to prohemosomes (Fig. 7) and hemosomes 9 . At this stage the

integration of heme into the four globin chains could take place. When hemo¬

somes attain a high Hb concentration, their membranes disrupt and Hb

spread throughout the cytoplasm. This happens repeatedly until the final

cytoplasmic Hb concentration is reached for the constitution of a new ma-

ture erythrocyte. Claussen et al. 13 and Claussen 14 studying erythropoiesis

in the rodents Apodemus sylvaticus and Micromys minutus, in the mouse

Mus musculus and in the fish Thymallus thymallus found very interesting

ultrastructural aspects of Hb biosynthesis similar to those found in the course

of red blood cell maturation in other vertebrates. Flowever, there exists a

little disagreement between Claussen's 14 diagram of Hb biosynthesis and

the interpretation of Brunner et al. 9 as to the integration of the globin chains

135

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LO 11 12 13 14 15 16

SPADACCI MORENA, D.D.; MORENA, P. CIANCIARULLO, A.M, BRUNNER JÚNIOR, A. Hemoglobin

biosynthesis in bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae), Mem. Inst. Butantan, 51(41:133-139, 1989.

Fig. 1 — Bone marrow erythroblast showing a prohemosome ph continuous to a transition-

al stage f. At the upper region of the picture, the continuity of this stage (t) with the hemo-

some h can be seen; N — nucleus. Bar equals 1.0 pm.

Fig. 2 — Larger magnification of an erythroblast showing a long organelle constituted by

a prohemosome ph followed by a transitional stage í and a hemosome h; N — nucleus.

Bar equals 0.5 p m.

Fig. 3 — A typical hemosome h is seen. For comparison a mitochondrion m is shown in

the inset; N — nucleus.

Fig. 4 — Vesicle v containing a still granulated ferruginous material.

Fig. 5 — A lamellated body lb apparently rising from a mitochondrion m is shown.

Fig. 6 — Condensed prehemosomal vesicle hv constituted by three concentric double lamellae;

within the double lamellae some ribosomal particles can be observed. Arrow points to an

endocytic vesicle.

Fig. 7 — Condensed prehemosomal vesicle hv modifying to prohemosomes ph.

Bars in Figs. 3-7 correspond to 0.25 pm.

136

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SPADACCI MORENA, D.D.; MORENA, P. CIANCIARULLO, A.M, BRUNNER JÚNIOR, A. Hemoglobin

biosynthesis in bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae). Mem, Inst. Butantan, 51(41:133-139, 1989.

y tf . ri 8 -V* »

* **’ .*«- V. \\ *

^ ~ iSk-: "• .VW* “

•; • ■' v. ’ , ^

jr*v * - h

^ ■, >:■

* * . - ’ '.V^ V >?§£ • - ; . V#-, :

. . , v: r . * ' . „i ,••••: .1 *■

■1-. *-** ' ->

. -T . • ■*;. ,. - . » ■

Q h

iSf-

*K ^ ,

s^|4r

Hb|

Fig. 8 — Reticulocyte showing obliquely sectioned hemosomes h, and a Golgi complex G.

Arrow points to a rhopheocytic site. Bar equals 0.5 /um .

Fig. 9 — Electrophoretic Hb patterns levidenced by tolidine) from the hemoglobinized

cytoplasm (I), and from the supernatant of the lysed hemosomal fraction (III), showing two

bands. The concentrated supernatant of the last washing of the fraction did not present any

visible Hb band (II).

in the course of hemosomegenesis. Accordíng to these authors, the globin

chains are enclosed in the prehemosomal vesicle, together with the fer-

ruginous compounds, before prohemosome formation.

Typical hemosomes, showing similar structural characteristics as those

found in mammalian and chicken erythroid cells, are seen in Fig. 8. The

hemoglobinic nature of the hemosome content, determined through elec-

trophoresis in polyacrylamide gel is seen in Fig. 9, showing two Hb bands.

The hemosomal fraction isolated from erythroid cells homogenate and sub-

mitted to five washings, was osmotically lysed so that Hb had been released

from the organelles. The supernatant of the last washing did not present

any visible Hb band showing, therefore, that all cytoplasmic Hb had been

discarded.

137

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LO 11 12 13 14 15 16

SPADACCI MORENA, D,D., MORENA, P. CIANCIARULLO, A.M, BRUNNER JUNIOR, A. Hemoglobin

biosynthesis in bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae). Mem. Inst. Butantan, 51(41:133-139, 1989.

ACKNOWLEDGEMENTS

The Authors wish to thank Dr. M.L. Beçak for permission to use the Zeiss

EM 109 electron microscope at the Laboratory of Animal Genetics, and to

Miss Olga Brunner and Mr. Carlos Jared for their technical assistance; to

the Laboratory of Herpetology and Venoms for specimens supplying, and

to Mrs. Sibylle Heller for her editorial aid.

This work was supported by CNPq (Proc. 103829/80) and by FEDIB (Ins¬

tituto Butantan).

RESUMO: Células eritróides de medula óssea e do sangue periférico da

serpente Waglerophis merremil foram analisadas ultra-estruturalmente.

A fim de intensificar a passagem de células imaturas para o sangue peri¬

férico, a eritropoiese foi estimulada através da indução de uma anemia

hemolítica pela injeção de saponina. Hemossomos típicos, muito seme¬

lhantes àqueles observados em células eritróides de mamíferos e aves,

foram encontrados, assim como algumas de suas formas precursoras.

Nestes organelos ocorreria, provavelmente, a integração do hemo às quatro

cadeias polipeptídicas de globina, considerando o seu conteúdo de natu¬

reza hemoglobínica. Estas entidades são uma derivação especializada in¬

direta das mitocôndrias, através de sucessivas transformações para corpos

lamelados, vesículas pré-hemossômicas, pró-hemossomos e, finalmer,-

te, para hemossomos.

UNITERMOS: Biossíntese de hemoglobina; hemoglobina de serpente; he

mossomo de serpente.

REFERENCES

1. BRUNNER JR, A.; SCHWANTES, A.R.; SCHWANTES, M.L.; BEÇAK, W. Hemoglobin in

immature erythrocyte mitochondrion-like organelles. Experientia, (Basel) 23:569-571,

1972.

2. BRUNNER JR., A.; COIRO, J.R.R., SCHWANTES, M.L.; SCHWANTES, A.R. Hemosomes

and hemoglobin biosynthesis in embryo and in regressive anemias. Mem. Inst. Butan¬

tan, 37:335-344, 1973.

3. BRUNNER JR., A.; COIRO, J.R.R.; JAMRA M.; GENTER, G. Cytomorphologic findings

on the final stages of erythrocyte maturation in mammals. Rev. bras. Pesq. med. Biol,

3:441-450, 1975.

4. BRUNNER JR., A.; MITSUTANI, C.Y.; ARAÚJO PERES, C. Comparative ultrastructure

of late rabbit-embryo erythroid cells in liver and peripheral blood. Am. J. Hematol.,

2: 227-236, 1977a.

5. BRUNNER JR„ A.; RIZZO, E. de; MITSUTANI, C.Y.; RIBEIRO DE BARROS, M.A. Induc-

tion of hemosome formation and hemoglobin biosynthesis in Hela cells. Res.exp.Med.,

170: 1 7-22, 1977b

6. BRUNNER JR., A.; MITSUTANI, C.Y; BILOTTA, J.A.T., ARAÚJO PERES, C. Cytomorphol-

ogy and behavior of late bone marrow and peripheral blood erythroid cells in experimen¬

tal hemolytic anemia. Cyfo/og/a,45:411-421,1980.

7. BRUNNER JR., A.; RIZZO, E. de; MITSUTANI, C.Y.; MENDES, I.F.; RIBEIRO DE BARROS,

M.A. The mitochondrial function in hemosome formation and hemoglobin biosynthe¬

sis. Comp.Biochem.Physioi, 736:829-833, 1982.

8. BRUNNER JR., A.; SANO MARTINS, I.S.; MITSUTANI, C.Y.; BILOTTA, J.A.T.; ARAÚJO

PERES, C. A relative morphological evaluation of hemoglobin biosynthesis in peripheral

blood reticulocytes of normal and anemic rabbits. Comp. Biochem. Physiol.,

74A: 755-760, 1983a.

138

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10 11 12 13 14 15 16

SPADACCI MORENA, D.D.; MORENA, P. CIANCIARULLO, A.M, BRUNNER JÚNIOR, A. Hemoglobin

biosynthesis In bone marrow and peripheral blood erythroid cells of the snake Waglerophis merre-

mii (Reptilia, Ophidia, Colubridae). Mem. Inst. Butantan, 51(41:133-139, 1989.

9. BRUNNER JR., A.; TOLEDO BILOTTA, J.A.; SPADACCI MORENA, D.D. Mitochondria,

hemosomes and hemoglobin biosynthesis. Cell Tissue Res.,223: 215-225, 1983b.

10. CIANCIARULLO, A.M.; SPADACCI MORENA, D.D.; BRUNNER JR„ A. Aspectos mor-

fofisiológicos da maturação eritrocitária em anfíbios, correlacionados com a biossín-

tese de hemoglobina. In: COLÓQUIO DA SOCIEDADE BRASILEIRA DE MICROSCOPIA

ELETRÔNICA, 11., Caxambu, 1987. Anais Caxambu, 1987. p. 101-102.

11. CLAUSSEN, C.P. Elektronenmikroskopische Untersuchungen zur Hàmoglobinbildung bei

Trisomie 12 des Mãusefetus. Verh. Anat. Ges., 73:855-861, 1979.

12. CLAUSSEN, C.P. Elektronenmikroskopische Untersuchungen zur Dottersackerythropoese

und Hàmoglobinbildung bei fetaler Trisomie der Maus. Verh. Anat. Ges., 74: 269-276,

1980.

13. CLAUSSEN, C.P.; JÜDES, U.; CLAUSEN, H.; GROPP, A. Apodemus sylvaticus and

Micromys minutus L.; the ultrastructure of fetal red blood cells and the hemoglobin

biosynthesis. Zool. Anz., Jena, 213:322-332, 1984.

14. CLAUSSEN, C.P. Cell organelles involved in hemoglobin biosynthesis in vertebrates. In:

DUNCKER, H.R. & FLEISCHER, G., ed. Fortschritte der zoologie, vertebrate mor-

phology. Stuttgart, Gustav Fischer Verlag, 1985. v. 30, p. 397-400.

15. COIRO, J.R.R. Comparatiave study on the ultrastructure of elements of the avian and

mammalian erythrocytic series. Correlation with hemoglobin biosynthesis.

Mem.Inst.Butantan, 39: 169-206, 1975.

16. DIETZ, A.A. & LUBRANO, T. Separation and quantitation of lactic dehydrogenase isoen-

zymes by disc electrophoresis. Analyt. Biochem., 20: 246, 1967.

17. HUEBERS, H.A. & FINCH, C.A. The physiology of transferrin and transferrin receptors.

Physiol. Rev, 67:520-582, 1987.

18. MAGALHÃES OLIVEIRA, A.A. Corante "New methylen blue" para reticulócitos. São

Paulo, 1980. /Tese — Departamento de Bioquímica da USPA

19. SPADACCI MORENA, D.D.; MORENA, P; CIANCIARULLO, A.M.; BRUNNER JR„ A. Aspec¬

tos ultra-estruturais da biossíntese de hemoglobina em Waglerophis merremii. In: COLO

QUIO DA SOCIEDADE BRASILEIRA DE MICROSCOPIA ELETRÔNICA, 11., Caxambu,

1987. Anais. Caxambu, 1987. p. 105-106.

139

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10 11 12 13 14 15 16

Mem. Inst. Butantan

51 (41:141-146, 1989.

THE OCCURRENCE OF N-METHYLTAURINE IN

THE SEA ANEMONE BUNODOSOMA CAISSARUM

Raymond ZELNIK*

Maria de Fátima Alves da SILVA*

Flavia MARTELLINI-LANDSHOFF*

Akhtar HAIDER**

ABSTRACT: Significant amounts of N-Methyltaurine together with the

recently described novel purine Caissarone, were isolated from the acetonic

and methanolic extracts of the whole body wall of the sea anemone

Bunodosoma caissarum. The structure of N-Methyltaurine was deduced

by spectroscopic methods.

KEY WORDS: Sea anemones; Cnidaria; Bunodosoma caissarum Corrêa;

N-methyltaurine; caissarone.

INTRODUCTION

Sea anemones belong to the phylum Cnidaria, one of the morphologi-

cal features of which is the nematocyst or cnidocyst apparatus, also known

as stinging cells, useful for prey capture. Fishes and small invertebrates are

paralyzed 3 on the discharge of the nematocysts which may also inflict in-

jurious effects on man, ranging from mild dermatitis to severe local

necrosis 10 . Since toxicity is indicative of potent physiological activity,

these marine organisms have attracted considerable attentión because of

their potential biological value. Chemical investigations have gained growing

interest since the finding of tetramine, a curare-like toxin, in the tentacles

of Actinia equina\ The various papers appearing over the last decades in-

clude descriptions of neuro- and cardio-active polypeptides 2 , hemolytic

factors 4 ' 18 , antitumoral bioassays 8 ' 20 and organo-sulphuric 21 , organo-

-phosphoric 14 , indolic 17 and guanidinic 6 compounds.

Bunodosoma caissarum Corrêa (Anthozoa, Actiniaria) is a sea anemo-

Serviço de Química Orgânica, Instituto Butantan, C. R 65, 01051, São Paulo, Brazil.

Research and Development Centre, Sochinaz S.A., Vionnaz (VS) and Institut de Chimie Organique, Université de

Lausanne, 2 Rue de La Barre, 1005 Lausanne, Switzerland.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 28/12/1988, accepted 03/03/1989.

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10 11 12 13 14 15 16

ZELNIK, R.; SILVA, M.F.A. da; MARTELUNI-LANDSHOFF. F.; HAIDER A. The occurrence of N Methyl-

taurine in the sea anemone Bunodosoma caissarum. Mem. Inst. Butantan, 51 (4): 141-146, 1989.

ne abundant on reefs along the Brazilian coast 7 . Material released by ne-

matocyst discharge has been shown to be active on cholinoceptive

preparations' 9 and a cardiotonic polypeptide has been recovered from a me-

thanolic extract of the anemone 13 . We have recently isolated a novel puri-

ne derivative, caissarone 7, from the whole body walls of this cnidarian 24 .

The presence of further cômponents as revealed by thin-layer chromato-

graphy of a methanolic extract, led us to re-examine this marine organism

and from which we now report the isolation of N-methyltaurine 2.

CH-

I '

CH 3 -N-CH 2 -CH 2 -S0 3 H

H 2 N-CH 2 -CH 2 -S0 3 H

MATERIAL AND METHODS

Specimens were collected on the São Sebastião coast, State of São Pau¬

lo, and frozen for transport. The homogenized anemones (I.OOOg, wet weight)

were extracted with acetone (3L) and methanol (4L) at room temperature,

the extracts filtered and the combined organic phases evaporated under re-

duced pressure. The aqueous concentrate was then treated with diethyl ether

to remove pigments, lipids and sterols and lyophilized to a viscous paste

which was triturated in methanol to give a whitish precipitate (12g), contai-

ning mainly inorganic salts. The methanolic filtrate was evaporated to dryness

and the residue (40g) analyzed by t.l.c. (Silica H, Merck; methanol-water

2:1; iodine vapours), revealing the presence of at least three components,

one of which was readily identified as caissarone 7 by comparison with an

authentic sample. Table 1 summarizes the sequence of steps used in the

extraction procedure.

Melting points were determined on a Reichert-Kofler hot-stage appara-

tus and are uncorrected. Infra-red spectra were performed in KBr pellets,

using a Perkin-Elmer 737 instrument. 1 H and 13 C NMR spectra were recor-

ded on a Bruker WH-360 spectrometer, operating at 360 and 90.5MHz,

respectively, in D 2 0 solution.

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10 11 12 13 14 15 16

SciELO^o

ZELNIK, R.; SILVA, M.F.A. da; MARTELLINI-LANDSHOFF. F.; HAIDER A. The occurrence of N Methyl-

taurine in the sea anemone Bunodosoma caissarum. Mem. Inst, Butantan, 51 (41:141-146, 1989.

or by infra-red spectroscopy and the melting points of the crystallization Pro¬

ducts compareci. Most of the eluates in the course of these analyses con-

tained substantial amounts of the purine caissarone 7. The eluates from

MeOH-H 2 0 3:1 and 2:1 displayed a second component, named BC-2, and

were pooled to be rechromatographed on Sephadex LH-20. Those fractions

containing BC-2 were treated with hot methanol in which caissarone is more

soluble, thus providing a clear separation of compound BC-2, the total yield

of which amounted to 440mg (0.044%).

Recrystallization of BC-2 from H 2 0-MeOH yielded the pure compound

in the form of white needles, m.p. 250-255° (Lit. 16 :244-245°). This subs-

tance reacted positively to the nitrogen and sulphur tests and in the infra-

-red spectrum showed absorption bands at v max 3450,3100 (OH or NH 2 ),

1620 ( = NH), 1480 (CH 3 ), 1220, 1190 (S0 3 H or S0 3 -), 1050 (S = 0), 850,

780, 750cm _1 . No carbonyl bands were observed in the general range

1750-1700cm _1 . In the ''H NMR spectrum, a signal at & 2.78 (s, 3H) in-

dicated the presence of an N-methyl group and two signals at ó 3.3 (t,

J = 7 Hz, 2H) and <5 3.47 (t, J = 7 Hz, 2H) p.p.m. disclosed two adjacent

methylene groups. In the 13 C NMR spectrum, a quartet centered at 6

35.82 and two triplets centered at ô 47.32 and 49.14 p.p.m. represented

one methyl and two methylene groups, respectively, thus confirming the

above indications. From the elemental analysis, a molecular formula of

C 3 HgN0 3 S was deduced (Found: C, 25.55; H, 6.68; S, 22.89; C 3 H 9 N0 3 S

requires: C, 25.89; H, 6.52; S, 23.04%). Therefore BC-2 is a sulphur-

-containing amino-acid with an N-methyl group linked to a two-carbon atom

skeleton.

These data strongly suggest that compound BC-2 is the N-methyl deri-

vative of taurine, a fact confirmed when its IR and ] H NMR spectra were

superimposed with those of N-methyltaurine as indicated in the literature 22 .

Among the sulphur-containing amino-acids, taurine 3, a widely distri-

buted compound, associated with a broad range of physiological actions 11 ,

has been found in a number of sea anemones 21 ' 9 ' 23 ' 15 and its presence in

the tissues of marine invertebrates related to the regulation of osmotic

pressure 12 . N-methyltaurine has been isolated from sponges 5 and algae 16

but has not yet been recorded in sea anemones. This paper thus represents

the first report on the occurrence of N-methyltaurine in a sea anemone.

ACKNOWLEDGEMENTS

These studies were supported in part by grants from the Conselho Na¬

cional de Desenvolvimento Científico e Técnico-CNPq (8/82) and the Fun¬

dação de Amparo à Pesquisa do Estado de São Paulo (82/1139-2;

84/0687-1). The Centro de Biologia Marinha of the University of São Paulo

at São Sebastião (SP) is acknowledged for allowing the use of its facilities.

Two of us (MFADS and FML) are grateful to the Institute Butantan for gran-

ting post-graduate research fellowships. The authors would like to thank Ms.

Amabile K. Matida (Institute Butantan) for helpful discussions at an early

stage of this study and Dr. John McNamara (University of São Paulo) for

reading the manuscript. They also acknowledge the skillful technical assis-

tance of Mrs. Amélia S. Andreoni and Mr. Jovelino Da Silva as well as the

secretarial help of Mr. David Lopes.

144

í, | SciELO

ZELNIK, R.; SILVA, M.F.A. da; MARTELLINI-LANDSHOFF. F.; HAIDER A. The occurrence of N Methyl-

taurine in the sea anemone Bunodosoma caissarum. Mem. Inst. Butantan, 51 (4): 141-146, 1989.

RESUMO: A anêmona-do-mar Bunodosoma caissarum Corrêa foi subme¬

tida a um tratamento extrativo com acetona e metanol e os extratos cro-

matografados em colunas de Sephadex LH-20, resultando no isolamen¬

to de N-metiltaurina e de caissarone.

UNITERMOS: Anêmona-do-mar; cnidaria; Bunodosoma caissarum Cor¬

rêa; N-metiltaurina; caissarone.

REFERENCES

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lung des Tetramins, eines Giftes aus Aktinia equina. Z. Biol., 79:113, 1923.

2. BERESS, L; BERESS, R.; WUNDERER, G. Purification of three polypeptides with neuro-

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3. BERNFIEIMER, A. W.; AVIGAD, L. S.; LAI, C. Y. Purification and properties of a toxin

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1982 and references cited therein.

4. BERNHEIMER, A. W.; AVIGAD, L. S.; BRANCH, G.; DOWDLE, E.; LAI, C. Y. Purifica¬

tion and properties of a toxin from the South African sea anemone Pseudactinia va¬

ria. Toxicon, 22: 183, 1984.

5. CALDERÓN, J. S.; QUIJANO, L; GÓMEZ, F.; ACOSTA, F.; RIOS, f. Estúdios prelimi¬

nares sobre algunas esponjas dei litoral mexicano — I. Rev. Latinoamer. Quim 73-49

1982.

6. CHEVOLOT, L. Guanidine derivatives \n: Marine natural products. New York. P. J.

Scheuer, Academic Press, 1981. V.4, p.53-91.

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qüência em águas brasileiras. Mem. Inst. Butantan, 33:27, 1966.

8. DUNN, D. F.; KASHIWAGI, M.; NORTON, T. R. The origin of antitumour activity in

sea anemones. Comp. Biochem. Physiol., 50 C: 133, 1975.

9. ELLINGTON, W. R. Metabolic responses of the sea anemone Bunodosoma cavernata

(Bosc) to declining Oxygen tensions and anoxia. Physiol. Zool.,55: 240,1982.

10. FIALSTEAD, B. W. Poisonous and venomous marine animais of the world. Washing¬

ton, U. S. Government Printing Office, 1965. v. 1, p.297-371.

11. HUXTABLE, R. J.; FRANCONI, F.; GIOTTI, A. The biology of taurine. Adv. Exp Med

Biol., 217:3, 1987.

12. JACOBSEN, J. G. & SMITH JR., L. H. Biochemistry and physiology of taurine derivati¬

ves. Physiol. Rev., 48: 424, 1968 and references cited therein.

13. KELECOM, A.; SOBRIERO-KELECOM, I. C.; SOLE-CAVA, A. M. Bunodosine, a cardio-

tonic and CNS acting polypeptide from the sea anemone Bunodosoma caissarum.

IUPAC International Symposium on Marine Natural Products, 4, La Laguna Tenerife

1982.

14. KITTREDGE, J. S.; IS BELL, A. F.; HUGHES, R. R. Isolation and characterization of the

N-methyl derivatives of 2-aminoethyl-phosphonic acid from the sea anemone An-

thopleura xanthogrammica. Biochemistry, 6: 289, 1967.

15. KITTREDGE, J.S.; ROBERTS, E.; SIMONSEN, D. G. The occurrence of free

2-aminoethyl-phosphonic acid in the sea anemone Anthopleura elegantíssima. Bio¬

chemistry, 7:624, 1962.

16. LINDBERG, B. Methylated taurines and choline sulphate in red algae. Acta Chem.

Scand., 9:1323, 1955.

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5-hydroxy-tryptamine in a sea anemone. Nature, 730:658, 1957.

18. MEBS, D. & GEBAUER, E. Isolation and proteinase inhibitory, toxic and hemolytic poly¬

peptides from a sea anemone, Stoichactis sp., Toxicon, 18:97, 1980.

19. MENDES, E. G. & FREITAS, J. C. de. The responses of isolated preparations of Buno¬

dosoma caissarum (Corrêa 1964) to drugs. Comp. Biochem. Physiol., 79 C: 375

1984.

20. PETTIT, G. R.; DAY, J. F.; HARTWELL, J. L; WOOD, H. B. Antineoplastic compo-

nents of marine animais. Nature, 227: 962, 1970.

21. ROBIN, Y. & ROCHE, J. Sur la présence de taurocyamine (Guanidotaurine) chez des

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ZELNIK, R.; SILVA, M.F.A. da; MARTELLINI-LANDSHOFF. F,; HAIDER A. The occurrence of N Methyl-

taurine in the sea anemone Bunodosoma caissarum. Mem. Inst. Butantan, 51 (41:141-146, 1989.

22 .

23.

24.

Sadtler Standard Spectra, Infra-red spectrogram n? 40766 and NMR spectrum n?

10450 M, Sadtler Research Laboratories Inc., Philadelphia 1971.

SILVA, M. F. A. da; MARTELLINI-LANDSHOFF, F.; BUONONATO, M.; ZELNIK, R. A

ocorrência de taurina (ácido 2-aminoetano-sulfônico) na anêmona-do-mar Phyllac-

tis flosculifera Lesueur. Mem. Inst. Butantan. 50:77 , 1988.

ZELNIK, R.; HARAGUCHI, M.; MATIDA, A. K.; LAVIE, D.; FROLOW, F.; WEIS, A. L.

X-ray molecular structure of caissarone, a novel purine derivative from the sea ane¬

mone Bunodosoma caissarum Corrêa 1964. J. Chem. Soc. Perkin I: 2051, 1986.

146

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10 11 12 13 14 15 16

Mem. Inst. Butantan

51(41:147-152, 1989.

STANDARDIZATION OF ANESTHESIA WITH PENTOBARBITAL IN

THE SNAKE BOTHROPS JARARACA *

Ana Marisa CHUDZINSKI**

Marília C.L. SEELAENDER* *

Eva M.A. KELEN**

ABSTRACT: A standardized surgical anesthesia was established for

Bothrops jararaca snakes injected subcutaneously with 30 mg/kg of.

pentobarbital (Nembutal). Induction times ranged between 30 to 40

minutes for 63 of the 79 injected snakes (79,7%).

These uniform results are different from those obtained with

intrapleuroperitoneal injections (17 snakes), currently used.

KEY WORDS: Subcutaneous anesthesia, anesthesia in snakes with

pentobarbital, Bothrops jararaca anesthesia.

INTRODUCTION

A standardization of anesthesia during experiments with the Crotalidae

snake Bothrops jararaca was required for surgical procedures. A long-lasting,

stable State of anesthesia and a fast induction time were needed.

Inconstant results are reported for anesthesia after intrapleuroperitoneal

(IPP) or intramuscular (IM) injections of pentobarbital in different snakes 1 - 2 .

Karlstrom and Cook 5 suggested that results suffer the influence of factors

as size, individual physiology, condition of the snake at the time of injec-

tion, room temperature, site of injection and degree of handling of the animal.

The subcutaneous (SC) route for pentobarbital administration has not

been reported routinely, although it has been used for anesthesia with an

ultrashort-acting barbiturate, Brevital Sodium 6 and with Ketamine

Hydrochloride 4 . The SC injection of Brevital Sodium lead to a short induc¬

tion time and a short-lasting anesthesia, which allowed surgical procedures

Presented at the 2nd Annual Meeting of the Federation of Societies of Experimental Biology (FESBE), 1987.

' "Seção de Fisiopatologia Experimental. Instituto Butantan.

Correspondente to: Dr. Eva M.A. Kelen.

Instituto Butantan — C.P. 65 — 01051 — São Paulo-SP, Brasil.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 04/5/1989; accepted 18/7/1989.

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CHUDZINSKI, A.M. ;SEELAENDER, M.C.L; KELEN, E.M.A. Standardization of anesthesia with pento-

barbital in the snake Bothrops jararaca. Mem. Inst. Butantan, 51 14): 147-152, 1989.

when 15 mg/kg body weight were administered in Thamnophis sirtalis and

Natrix sipedon sipedon 6 .

The efficiency of the subcutaneous administration of pentobarbital for

the anesthesia of Bothrops jararaca snakes is here reported.

MATERIAL AND METHODS

The anesthetic employed was Sodium Pentobarbital (Nembutal, Abbott

Laboratories) as a 5% solution prepared from capsules containing lOOmg.

The salt was dissolved in distilled water with the help of a magnetic stirrer

and stored at 4°C after centrifugation. Bothrops jararaca snakes, 42 males

(70 to 400g, mean of 154g) and 54 females (90 to 655g, mean of 260g),

received each, an optimal dose which was standardized for these snakes

as 30mg/kg body weight, either subcutaneously or intrapleuroperitoneally.

The administration of the anesthetic was performed while an operator im-

mobilized the snake by holding head and tail, and a second operator made

the injection. IPP injections (17 snakes) were made ventrally, into the distai

third of the body, avoiding the pericardial region. The SC injections (79

snakes) were made at the junction of ventral and dorsal scales, approxi-

mately at 10cm below the heart. The presence of a gap, felt with the nee-

dle, indicates the space between skin and muscle to be injected.

Three main stages of anesthesia were arbitrarily established:

I- Sedation: slow movements as response to pressure of the tail with a hook.

Presence of righting reflex. Normal size pupils, easily distinguished in the

light brown colored eyes of this snake.

II- Partial anesthesia: incapacity of promoting serpentine movements. Loss

of righting reflex. Tail-withdrawal reflex on pressure still present. Semi-con-

tracted pupils.

III- Deep anesthesia: loss of tail-withdrawal reflex, lack of any sort of response

to pressure of the hook. Contracted pupils.

The stage of anesthesia required was stage III which lasted for a mini-

mum of three hours. Experiments were carried out at different ambient tem-

perature ranges. No artificial respiration was needed. The snakes were not

fed over a period of, at least, 96 hours before anesthesia.

RESULTS

Table 1 indicates the number of anesthetized snakes related to time

(minutes) of anesthesia induction at the levei of stage III. Results are ex-

pressed in ten minutes intervals. Among the 79 subcutaneously injected

snakes, 63, weighing from 70 to 655 g, were anesthetized within the range

of 30 to 40 minutes (79,7%). The stage of anestesia was demonstrated

to be stable and no supplementation of dose was ever necessary. The ef-

fect of the anesthetic lasted for at least three hours but the exact time of

recovery was not observed with the exception of two animais, which reco-

vered within 48 hours. None of the 79 animais died under this treatment.

The stage of recovery from anesthesia was considered when the snake was

back to all the reflexes affected during anesthesia (stages I through lll).

Among the 17 intrapleuroperitoneally injected snakes, increasing times

of induction were directly proportional to increasing body weight. For ex-

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barbital in the snake Bothrops jararaca. Mem. Inst. Butantan, 51(41:147-152, 1989.

ample, 3 snakes, weighing 106 to 127 g were anesthetized after 30 minutes,

while 4 others, weighing 161 to 400g reached stage III within 50 minutes.

Stage III suffered oscilations in three animais. Recovery took 23 to 48 hours

and 5 of the snakes died before’recovery. Figures 1 and 2 illustrate the differ-

ent behaviour of subcutaneously and intrapleuroperitoneally injected

Bothrops jararaca snakes, in relation to the time of induction of stage III.

TABLE 1

Anesthesia of Bothrops jararaca snakes with pentobarbital. Time of induction to the levei

of stage III in 10 minutes intervals after intrapleuroperitoneal (IPP) or subcutaneous (SC)

injections of a 5% anesthetic solution. Dose: 30 mg/kg body weight.

Induction

time of

Number of

snakes

Body weight g

stage III

minutes

mean

range

—

360

206-564

179

75-353

288

70-655

SC 50

196

120-335

—

415

345-485

—

369

no induction

—

118

106-127

195

140-250

IPP 50

288

161-400

02*

253

200-305

405

—

01*

431

no induction

272

204-445

* Oscilation of stage III

149

CHUDZINSKI, A.M. ;SEELAENDER, M.C.L; KELEN, E.M.A. Standardízation of anesthesia with pento-

barbital in the snake Bothrops jararaca. Mem. Inst. Butantan, 51 (41:147-152, 1989.

FIG. 1: Distribution of Bothrops jararaca snakes according to time of induction of deep

anesthesia (stage III) in 10 minutes interval, after subcutaneous injections of pentobarbital

(30 mg/kg body weight).

4 r

30 40 50 60 70 80 90

TIME OF INDUCTION (MINUTES)

FIG. 2: Distribution of Bothrops jararaca snakes according to time of induction of deep

anesthesia (stage III) in 10 minutes interval, after intrapleuroperitoneal injections of pen¬

tobarbital (30mg/kg body weight).

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barbital in the snake Bothrops jararaca. Mem. Inst. Butantan, 51(41:147-152, 1989.

DISCUSSION

Results demonstrate that a standard dose of the anesthetic (Nembutal)

per unit of body weight, can be used with no need for supplementation when

SC administration is performed in Bothrops jararaca snakes. According to

these results, about 80% of the snakes reached stage III of anesthesia wi-

thin 30 to 40 minutes, their body weight ranging from 70 to 655 g. Experi-

ments were performed at different seasons, when the temperature variation

was from 3.5 to 34.6°C. From this, it is evident that subcutaneously induced

anesthesia with pentobarbital is not influenced by size variation or by am-

bient temperature modification during the procedure.

The criteria of stages arbitrarily established to follow intensity degrees

was a helpful step for the standardization procedure. It was also used by

Genevois et at 3 . with slightly different definitions.

The results obtained with IPP injections of pentobarbital in 17 Bothrops

jararaca corroborate the data from literature, concerning the influence of

weight variation in snakes under a same anesthetic dose, since larger ones

had an elongated induction time as demonstrated in table 1.

The possibility that the intense integumentary vascularization in

snakes 7 , in which circulation is modified during thermoregulation 8 , might

be involved in the regular absorption of the anesthetic by the SC route should

be further investigated. Inconsistent results in the IPP induced anesthesia

may be due to difficulties in defining the site and the depth of injection, in

view of the extense and not well delimited pleuroperitoneal cavity, in which

the diaphragm is lacking. The risk of organ injury must be also mentioned.

The standardization obtained for Bothrops jararaca with the SC route

of injection of Nembutal indicates this administration as extremely convenient

when deep anesthesia is required.

ACKNOWLEDGMENTS

Thanks are due to Augusto S. Abe, State University of São Paulo, Cam¬

pus of Rio Claro-SP, and Andreas Moser, Visiting Biologist from Basel, Swit-

zerland, for fruitful discussion. A.M.C. was recipient of grant no.TB/1439-80

from FEDIB at the time of the standardization experiments. M.C.L.S.

was

under a scholarship from FAPESP (n° 86/3057-4). The skillful technical as-

sistance of Jorge Abel Ferreira in the contention of animais is gratefully ac-

knowledged as well.

RESUMO: Foi obtido um método padronizado de anestesia cirúrgica para

serpentes Bothrops jararaca, injetadas com 30mg/kg de pentobarbital

(Nembutal) por via subcutânea. O tempo de indução variou entre 30 e

40 minutos para 63 de 79 serpentes estudadas (79,7%). Estes resulta¬

dos uniformes contrastam com os obtidos com a via intrapleuroperitoneal

(17 serpentes), normalmente adotada.

UNITERMOS: Anestesia subcutânea, anestesia em serpentes com pen¬

tobarbital, Bothrops jararaca anestesia.

REFERENCES

1. BETZ, W.T. Surgical anesthesia in reptiles, with special reference to the water snake.

Natriz rhombifera. Copeia,2: 284-287, 1962.

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CHUDZINSKI, A.M. ;SEELAENDER, M.C.L; KELEN, E.M.A. Standardization of anesthesia with pento-

barbital in the snake Bothrops jararaca. Mem. Inst. Butantan, 5^41:147-152, 1989.

2. CLARK, H. Scientific apparatus and laboratory methods; embryonic series in snakes. Scien¬

ce, 85:569-570, 1937.

3. GENEVOIS, J.P.; AUTEFAGE, A.; FAYOLLE, P.; CAZIEU, A.; BONNEMAISON, P. L'anés-

thesie des espèces insolites en pratique vétérinaire courante. Revue Méd. Vét.,

134:471-479, 1983.

4. JACKSON, O.F. & COOPER, J.E., Anesthesia and surgery. In: COOPER J.E.; JACKSON

O. F., eds. Diseasesofthereptilia. London, Academic Press, 1981. v.2, p. 535-549.

5. KARLSTROM, E.L. & COOK JR., S.F. Notes on snake anesthesia. Copeia, 1:57-58, 1955.

6. WANG, R.; KUBIE, J.L.; HALPERN, M. Brevital sodium; an effective anesthetic agent

for performing surgery on small reptiles. Copeia, 4:738-743, 1977.

7. WHITE, F.N. A functional interpretation of the integamentary vessels of snakes. Herpeto-

lógica, 13:127-130, 1957.

8. WHITE, F.N. Circulation. VI. The role of the cardiovascular system in thermoregulation.

In 1 GANS, G., ed. Biology of the reptilia. London, New York, Academic Press, 1976.

v. 5, p.312-317.

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Mem. Inst. Butantan

51 (41:153 - 168, 1989.

CROSS-REACTIVITY OF HORSE MONOVALENT ANTIVENOMS TO

VENOMS OF TEN BOTHROPS SPECIES

Wilmar DIAS da SILVA**

Rosalvo GUIDOLIN*

Isaias RAW* * *

Hisako Gondo HIGASHI*

Celso Pereira CARICATI*

Josefina Farina MORAIS*

Maria Laura S.R. LIMA*

Ivone Kazuko YAMAGUCHI *

Amélia Keiko NISHIKAWA*

Marco Antonio STEPHANO*

José Roberto MARCELINO*

José Ricardo PINTO*

Maristela José SANTOS*

ABSTRACT: Horses were immunized with B. alternatus, B. atrox, B. cotiara,

B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. moojeni, B.

neuwiedi and B. pradoi venoms. Antibodies recognizing the venom

antigenic components were either immunochemically detected by the

enzyme-linked immunosorbent, double immunodiffusion and quantitative

precipitation methods or biologically by the assays measuring the venoms

indirect hemolytic and lethal toxic activities. Specific and cross-reacting

antibodies against the Bothrops venoms were found in all ten monovalent

antivenoms. Modifications in the serum electrophoretic patterns

characterized by a reduction of the albumin peak and by a correspondent

increase of the y-globulins with a patent or no modification of the a or [)

globulins were found in these sera.

KEY WORDS: Antivenoms, Bothrops venoms, monovalent Bothrops

antivenoms, cross-reactivity reactions.

Seção de Concentração e Fracionamento de Soros.

Laboratório Especial de Imunoqulmica.

Centro de Biotecnologia.

Instituto Butantan —C.P. 65 — 01051 — São Paulo-SP-Brasil.

The "Comissão de Soros, Ministério da Saúde, Brasil", has recommended studies to determine the satisfactory

Bothrops venoms mixture to be used as antigen to produce the corresponding antivenom. Therefore, a study

group at the "Instituto Butantan, São Paulo, Brasil" was convened and charged with these studies and this paper

contains the result obtained.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 8/5/1989; accepted 18/7/1989.

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species. Mem. Inst. Butantan, 51(41:153-168, 1989.

INTRODUCTION

Horse antivenoms against Bothrops venoms have been produced using

as antigen a mixture composed of venoms from seven of this snake spe¬

cies. At least ten well characterized snake species, belonging to the genus

Bothrops, have been identified in Brazil and it is well known that the ven¬

oms from these snakes as complex mixtures, contain various enzymes in

addition to toxic elements. There are evidences that some of these compo-

nents are largely distributed among the majority whereas others are res-

tricted to some species. This raises questions concerning the ability of

antisera routinely produced for therapeutic purposes to neutralize the rele¬

vam components responsible for the pathophysiologic action of the venom

as a whole. In order to clarify these practical posed questions two experimen¬

tal protocols were delineated: a) production of monovalent antisera against

each of the ten Bothrops venoms and analysis for the presence of antibod-

ies against the components found both in the venom used for immuniza-

tion and in the venoms from the other Bothrops species; and, b) to test in

parallel assays, the polyvalent Bothrops antivenoms routinely prepared in

the Instituto Butantan for therapeutic purposes for their capacity to com¬

bine with the components present in each individual Bothrops venom.

ABBREVIATIONS

LD 50 .lethal dose 50%

BVM. Bothrops venoms mixture

FCA.Freund's complete adjuvant

FIA.Freund incomplete adjuvant

PBS.phosphate saline buffer

BSA.bovine serum albumin

NPGB.p-nitrophenyl-p' guanidino benzoate

PMSF.phenyl-methyl sulphonyl-phluoride

EDTA.ethylene-diamine tetraacetic acid

MATERIAL AND METHODS

Animais. Adult horses with 400-450kg of body weight maintained at

the São Joaquim farm, Instituto Butantan were to produce both, the monova¬

lent and the Bothrops polyvalent antivenoms. Outbred albino mice weigh-

ing 18-22 g from Biotério Central, Instituto Butantan were used for the

neutralization test.

Venoms. Bothrops venoms from the B. alternatus, B. atrox, B. cotiara,

B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. moojeni, B. neu-

wiedi and B. pradoi were pooled venoms from several adult snakes main¬

tained in the "Seção de Venenos" of the Instituto Butantan. Venoms were

extracted, desiccated and stored at 4°C before use. Stock Solutions of ven¬

oms from each Bothrops species were, as otherwise indicated, prepared

by diluting 100mg of dried venom in 10 ml of 0.15M phosphate buffer at

pH 7.2. The Bothrops venom mixture used to produce the polyvalent an¬

tivenoms was prepared by adding one volume 10mg/ml solution of B. jararaca

venom to an equal volume of a 10 mg/ml solution containing equal parts

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DIAS da SILVA, W, et al. Cross-reactivíty of horse monovalent antivenoms to venoms of Bothrops

species. Mem. Inst. Butantan, 51 (4): 153-168, 1989.

of venoms from B. alternatus, B. neuwiedi, B. jararacussu, B. cotiara, B.

moojeni and B. pradoi. The immunizing mixtures were prepared just before

use, by diluting stock Solutions to a final concentration of 5 mg/ml in: a)

Freund's complete adjuvant (FCA); b) Freund's incomplete adjuvant (FIA);

c) alginate; d) 0.15 M NaCI. The LD 50 values for each Bothrops species were

previously determined by the "Seção de Controle" of the Instituto Butan¬

tan by the probit analysis Finney 4 . The values for each Bothrops species

are indicated in parenthesis: B. alternatus (66.0 Mg); B. atrox (145.0 ^g);

B. cotiara (46.79 Mg); B. erythromelas (70.0 Mg); B. insularis (50.0 Mg);

B. jararaca (33,0 Mg); B. jararacussu (76.0 Mg); B. moojeni (115.2 Mg);

B. neuwiedi (35.5 Mg) and B. pradoi (61.56 m9>-

Antivenom production. Monovalent antivenoms were obtained from

horses subcutaneously injected with one Bothrops venom each: B. alterna¬

tus (horse n° 120), B. atrox (horse n° 144), B. cotiara (horse n° 134), B.

erythromelas (horse n° 25 and 37), B. insularis (horse n° 54 and 154), B.

jararaca (horse n° 141), B. jararacussu (horse n? 162), B. moojeni (horse

n° 290), B. neuwiedi (horse n° 980) and B. pradoi (horse n° 131). The

animais were previously sensitized by a primary stimulus with 5.0 mg of

the venoms in FCA. Six months later horses were reinjected subcutaneous¬

ly with 3 mg of the corresponding venom in 10 ml of alginate. After three

months a third reinjection was performed with 5 mg venom in FIA followed

by three injections of 2,5mg venom in 0.15 M NaCI, at 7 days intervals. Seven

liters of blood were collected one week later and the plasma separated and

stored at 4°C. Polyvalent plasma with antibodies, against the mixture of ven¬

oms from B. alternatus, B. cotiara, B. jararaca, B. jararacussu, B. moojeni,

B. neuwiedi and B. pradoi were obtained by immunizing horses with BVM

essentially by the same immunization schedule.

Purification of horse IgG and its F(ab') 2 fragments. All purifications were

carried out using the same pool horse plasma.

IgG. IgG was prepared as described by Stempuch and Audran 14 . Horse

hyperimmune plasma at a concentration of 70 mg/ml, was heated at 56°C

for 15 min and subsequently centrifuged at 900 x g for 10 min. The pH

of the supernatants was adjusted to 5.0 with 0.1 N acetic acid and caprylic

acid (Merck, Darmstadt) was then added, to a final concentration of 8.7%,

under vigorous stirring for 30 min at room temperature. After centrifuga-

tion (15 min, 1000 x g) the supernatant was filtered through a 0,45 pm

Millipore membrane (Millipore Corporation, Bedford, MA 01730), and dia-

lysed overnight against 0.85% NaCI at 4°C. The IgG preparation was stored

at -20 °C. F(ab') 2 . The method. of Pape 12 Slightly modified was used to ob-

tain F(ab') 2 fragments from both monovalent or polyvalent horse hyperim¬

mune plasma according to the procedures established by the Instituto

Butantan (unpublished data): Briefly Bothrops antivenom plasma diluted to

40 mg/ml with distilled water was heated at 30°C. The pH was adjusted

to 3.1 with 2.5 M HCI, and pepsin (INLAB, São Paulo, Brazil) was then ad¬

ded under slow stirring (5.0 gr per liter of plasma). The mixture was incubated

for 40 min at 30°C, followed by the addition of tetrasodium pyrophosphate

(Labsynth, São Paulo, Brazil) (0.1% final concentration) and toluene (INLAB,

São Paulo, Brazil) (0.1% final concentration). Temperature was then elevat-

ed to 55°C and the pH was adjusted to 5.2 with 20% NaOH. After adding

ammonium sulphate (Quimis, São Paulo, Brazil) to a final concentration of

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17.5%, the solution was stirred during 90 min at 55°C and then centrifuged

for 10 min at 900 x g. The supernatant was removed and the pH adjusted

to 6.9-7.1 with NaOH. Ammonium sulphate was again added to the mix-

ture to a final concentration of 28% which was stirred at room temperature

for 60 min. After centrifugation, the precipitate was resuspended in 0.85%

NaCI and the resulting solution dialysed for 48h against 0.85% NaCI at 4°C.

The F(ab') 2 rich preparation was stored at -20°C. The protein content was

measured by the method of Lowry et al. 9 using bovine serum albumin as

a standard. The preparations obtained were termed monovalent or polyva-

lent Bothrops antivenoms.

The ELISA method Theakston et al. 15 One hundred u! of Bothrops ven¬

oms (1 u g/ml) were absorbed to the wells of Nuclon plates (Delta, Den-

mark), at 4°C, overnight, blocked with 3% BSA in phosphate buffered saline

containing 0.05% Tween 20 for 3h at room temperature. The plates were

washed with 0.05% of BSA in phosphate buffered saline containing 0.05%

of Tween 20. One hundred p I of several dilutions of each serum sample

(1/100-1/12,000) were added to the wells and allowed to incubate for 45

min at room temperature. The plates were washed again as mentioned and

100 pl of peroxidase conjugated anti-mouse immunoglobulin diluted 1/1,000

(Cappel, Cochanville, Pa) were added to each well. Plates were incubated

for 45 min at room temperature. After washing the wells as previously

described, 100 pl of ortho-phenylenediamine (Sigma Co., U.S.A) (1 mg/ml)

and 4 pl of H 2 0 2 were added to wells and plates were allowed to stand

at room temperature for 15 — 20 min before spectrophotometric determi-

nation of colour change.

Double Immunodiffusion Ouchterlony 11 . Agarose was dissolved in 0.056

M sodium veronal buffer, pH 8.6, at 0.8% concentration and overlaid on

glass plates. The plates contained one center well and eight outer wells (3mm

in diameter) at 4mm distance. Twenty microlliters of Bothrops crude ven¬

oms were added to central wells and tested against eight dilutions of the

corresponding horse antisera pipetted in the outer wells. The plates were

allowed to develop for 24h at room temperature and stained with Coomas-

sie blue. Antivenoms were arbitrarily classified as weak reactors (WR) and

strong reactors (SR) according to the presence of precipitin bands at dilu¬

tions up to 1:64 and 1:128, respectively.

Cellulose acetate electrophoresis. Electrophoresis of horse sera obtained

immediately before and after the immunization with each Bothrops venom

was performed in 0.04M sodium veronal buffer pH 8.6 during 25 minutes

at 200 Volts. Strips were stained for 8 minutes in 0.5% amidoblack 10B

dissolved in a mixture of 47.5% methanol and 5% acetic acid, and des-

tained in the same mixture. They were dried in absolute methanol, and treated

with a solution containing 85% methanol, 14% acetic acid, 1% glycerol.

Absorbance was determined in a densitometer. In control samples the typi-

cal plasma protein bands were clearly seen: albumin and the globulins al,

a 2, p 1, p 2, y 1 and r 2.

Ability of Bothrops antivenoms to neutralize the indirect hemolytic ac-

tivity of Bothrops venoms. 0.3 ml of packed sheep erythrocytes were washed

four times with 0.15 M NaCI and pelleted. To these pellets were added 0.3

ml of egg yolk diluted 1:4 in 0.1M NaCI containing 0.25 ml of 0.01 M CaCI 2 ,

resuspended and mixed with 25 ml of 0.8% agarose dissolved in phosphate-

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buffered saline solution, pH 8.1. 18 ml of this solution were overleyed on

40 x 180 mm glass plates and allowed to solidify. 4mm diameter wells were

punched in the middle of the plates 40mm apart and filled either with 15 m I

of a standard solution of venoms previously standardized to give hemolytic

haloes of 20 mm or a mixture containing these amounts of venom plus the

antivenoms at different dilutions. The plates were incubated at 37°C for

24h, the hemolytic haloes measured and the percentage of inhibition was

calculated.

Quantitative precipitin reaction Heidelberger 6 . The IgG immunoglobu-

lins used were obtained from the different Bothrops monovalent antivenoms

purified with aid of caprylic acid and the corresponding Bothrops venoms

were used as the source of antibody and antigen, respectively. The IgG prepa-

rations containing 1 mg protein/ml were dissolved in phosphate-saline bufíer

(PBS) pH 7,4 and diluted 1:4 before use. In order to minimize the possible

IgG cleavage by the proteolytic enzymes present in the Bothrops venoms

this buffer contained 5 p-nitrophenyl — p'-guanidino benzoate (NPGB),

5 uM phenyl-methyl sulphonyl-phluoride (PMSF) and lOmM ethylene-

diamine tetracetic acid (EDTA). To a series of tubes (13 x 70 mm) were ad-

ded 0.5 ml of IgG solution and 0.5 ml of the corresponding Bothrops ven¬

om solution containing, 20, 40, 80, 160, 320, 640, 1,000 and 1,280 ng,

mixed and allowed to stand overnight at 4°C. Controls containing IgG or

the highest amount of venom alone were rumning in parallel. The precipi-

tates were washed three times with cold PBS, ressuspended in 0.1 N NaOH

and the total protein contents determined by the Lowry's method

(1951) 9 .Knowing the amounts of antigen (Ag) added in each tube, the cor¬

responding values for antibody (Ab) and the ratios Ab:Ag were calculated.

Graphics were constructed by plotting the Ag values on ordinates (Y) and

the ratios Ab:Ag (X) on abscissae. A straight line was obtained following

the equation Ab = a —bx, or Ab = a —b(x) 2 in whic "x" is the amount

of Ag added and the "a" and "b” constants representing the intercept of

the line on the "Y" axis and "slope", respectively. Using these values

graphicaly obtained and the values for the known maximal Ag precipitated

as substitutes for "a", "b” and "X", respectively, in the above derived equa¬

tion, the maximal values for specific Ab in each monovalent IgG prepara-

tion were obtained. As the venoms are complex mixtures of several

immunogenic components and the correspondent antisera shall have anti-

bodies specific for distinct epitopes and antigens, the precipitin curves ob¬

tained in each venom-antivenom assay was logistically transformed in order

to determine their parallelism and coincidence. The values for Ab on the

precipitin curves were applied to the equation y = In. Ab/2,Q 00_ , Bus-

sab 2 . t-Abl2,ooo

lhe linear function y=A ( ^g of venom) + B was obtained where A

is the angle coeficient and B the linear coeficient, these values being calcu¬

lated by the "SAS square minimal program Ray 13 . The parallelism and the

coincidence among the retified curves were determined by the F test Choi 3 ,

according to the criteria used by Murata 10 in their analyses of horse anti-

Crotalus durissus terrificus venom when assayed against gamma radiated

venom.

Mouse protection test. This assay was performed strictly obeying the

following conditions and methodology:

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DIAS da SILVA, W. et al. Cross-reactivity of horse monovalent antivenoms to venoms of Bothrops

species. Mem. Inst. Butantan, 51(41:153-168, 1989.

a) Outbred albino mice with 18-22g body weight were used.

b) The venom Solutions containing 5 LD 50 were incubated with an equal

volume of different antivenom dilutions (dilution factor of 1.2) at 37°C for

30 min. Control mixtures containing 0.15M NaCI instead of antivenom were

running in parallel. Eight mice per dilution point or for the control were in-

traperitoneally injected with 0.5 ml of each solution, the animais being ob-

served for 48h and the number of alive animais recorded. The probits were

calculated with the aid of the percentage of survival values obtained for each

serum dilution, plotted on a semi-log paper (in abscissae) against the volume

of antivenom (in ordenates) and the 50% mortality determined. With the

aid of the formule TV-1 in which TV = 5 LD 50 in ^g, the amount of ven¬

om in mg was calculated.

RESULTS

Antibodies detected by the ELISA assay.

According to their reactivity the Bothrops monovalent antivenoms were

classified for descriptive purposes as weak reactors (WR), intermediate reac-

tors (IR) and strong reactors (SR), with antibody titers below 64 x 10 3 , up

to 128 x 10 3 and over this latter values, respectively. From the data pre-

sented in Table 1 three observations can be drawn: a) B. cotiara venom pro-

duces.WR antisera against all venoms; b) B. alternatus and B. insularis

venoms produce SR antisera either when tested against the specific venom

or in cross-testing one against each other; and c) the remaining venoms

produce IR or WR antisera both in specific and in cross-testing assays.

Antibodies detected by the double diffusion method.

The Bothrops monovalent antivenoms were also classified as WR (ti¬

ters up to 64) and SR (titers over 128) according to their maximal dilution

giving visible precipitin bands. Table 2 shows: a) B. cotiara produced SR

antiserum against all venoms; b) B. alternatus, B. insularis, B. jararaca, B.

moojeni, B. neuwiedi and B. pradoi produced SR antisera at least when tested

against one of the Bothrops venoms; and, c) B. alternatus, B. atrox, B. eryth-

romelas and B. jararacussu venoms produced WR antisera.

Electrophoretic profiles of horse Bothrops antivenoms.

In comparison with normal horse serum the relative quantities of the

serum proteins in Bothrops antivenoms as determined by electrophoresis

on cellulose acetate strips were the following:

a) Albumin was unequivocally reduced in B. atrox, B. moojeni and in

BVM antivenoms but only slightly reduced in B. cotiara, B. insularis, B. jara¬

raca, B. jararacussu and B pradoi or unchanged in B. alternatus and B. neu¬

wiedi antivenoms; b) excepting for B. cotiara, B. insularis and B. moojeni,

° -globulins were decreased in the (ileg.) Bothrops antivenoms; c) the /? -

-globulins, excepting for B. atrox, B. cotiara, B. jararaca, B. pradoi and BVM

antivenoms, /^-globulins were reduced; and, d) the y -globulins, excepting

for the B. erythromelas antivenom, were increased in the other Bothrops

antivenoms. Table 3 and Fig. 1 show these results.

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TABLE 1

Antibody production in horses immunized with different Bothrops venoms, measured by the enzyme

linked immuno-absorbent assay (ELISA) and classified as strong (SR), intermediate (IR) and weak reac-

tors (WR)*

Bothrops

antivenoms

Bothrops Venoms

alt

atr

cot

ery

ins

jar

jsu

moo

neu

pra

B. al terna tus

(alt)

QÕU

B. atrox

(atr)

f WR

B. cotiara

(cot)

[ win

B. erythromelas

(ery)

□ã ]

B. insularis

(ins)

ÜSKJ

B. jararaca

(jar)

□O

B. jararacussu

(jsu)

□a 3

B. moojeni

(moo)

r in i

B. neuwiedi

(neu)

L E J

B. pradoi

(pra)

'WR, trters of 64 x 10 3 ; IR. titers of 64 x 10 3 to 128 x 10 3 ; SR, titers over 128 x 10 3

TABLE 2

Cross-reactivity of Bothrops antivenoms with different Bothrops venoms as determined by

double immuno-diffusion and classified accordingly as strong reactor (SR) and weak reac-

tors (WR)‘

Bothrops

antivenoms

Bothrops Venoms

alt

atr

cot

ery

ins

jar

jsu

moo

neu

pra

B. alterna tus

(alt)

1 WR 1

B. atrox

(atr)

TwrT

B. cotiara

(cot)

[ SR

| SR

B. erythromelas

(ery)

| WR ]

B. insularis

(ins)

1 WR 1

B. jararaca

(iar)

( WR3

B. jararacussu

(jsu)

□B

B. moojeni

(moo)

[ wiT]

B. neuwiedi

(neu)

[ WR ]

B. pradoi

(pra)

Qwr ]

‘Strong reactors (SR): titers of 128 and over; weak reactors (WR): titers up to 64.

I, | SciELO

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species. Mem. Inst. Butantan, 51(41:153-168, 1989.

Fig. 1: Cellulose acetate electrophoresis of sera obtained from normal (panei a) or from

hyperimmunized horses with venoms of B. alternatus (panei b), B. atrox (panei c), B. co¬

fiara (panei d), B. erythromelas (panei d), B. insularis (panei e), B. jararaca (panei f), B.

jararacussu (panei g/, B. moojeni (panei i), B. neuwiedi (panei j), B. pradoi (panei I) and

the mixture of Bothrops venoms composed of 50% of a solution containing B. jararaca

venom and 50% of a solution containing equal parts of venoms from B. alternatus, B.

cotiara, B. jararaca, B. moojeni, B. neuwiedi and B. pradoi.

Samples of horse sera were applied on cellulose acetate strips and electrophoresed in

0.04M sodium veronal buffer, pH 8.6, for 25 min at 200V. The numbers at right at each

panei indicate, from the top to the bottom, the concentration (%) values for albumin

and for the globulins « 1 and « 2, /? 1 and p 2, y 1 and y 2.

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TABLE 3

Electrophoretic distribution of proteins from horses hyperimmunized with Bothrops venoms.

Comparison with sera from non immunized horses.

Bothrops

antivenoms

Horse plasma proteins (%)

albumin

Globulines

(1)

B. alternatus

33.1

16.1

16.7

34.1

(2)

B. atrox

23.9

13.9

28.6

33.6

(3)

B. cotiara

27.6

17.9

24.4

30.1

(4)

B. erythromelas

45.5

15.1

15.6

23.8

(5)

B. insularis

27.9

17.9

22.1

32.1

(6)

B. jararaca

25.5

15.4

25.7

33.4

(7)

B. jararacussu

27.4

14.5

15.0

43.1

(8)

B. moojeni

21.8

16.6

16.4

45.2

(9)

B. neuwiedi

32.1

15.3

12.3

40.3

(10)

B. pradoi

25.1

12.9

24.9

37.1

Mixture of (2) + (5) +

(7) +

(8) + (9) (BUM)

20.5

14.9

30.2

34.4

Normal horse serum

34.3

18.1

27.4

20.2

ANTI-INDIRECT HEMOLYTIC ACTIVITY

The in vitro ability of different Bothrops antivenoms to neutralize the indirect

hemolytic activity of Bothrops venoms was assayed. The following results

were obtained: a) B. cotiara venom, at least under the conditions used in

these experiments, was free of indirect hemolytic activity even when high

concentrations up to 60 pg were used; b) the anti-fl. alternatus serum at

1:100 dilutions was able to block 50% of the hemolytic activity present in

the venom from all nine Bothrops species tested; c) the antisera against

the venoms from the other Bothrops species have very weak or no anti-

hemolytic activity; d) the anti-hemolytic activity present in the Bothrops poly-

valent antivenom was manifested against the B. alternatus venom but not

against the venom from the other Bothrops species.

Antibodies detected by the quantitative precipitin assay

Fig. 2 shows that in all Bothrops venom-antivenom Systems analyzed

with the addition of increasing quantities of venoms the amount of the cor-

responding antigen-antibody precipitate increases until it reaches a maxi-

mum and then declines. Inspection of the shapes of these curves shows

that both the maximum quantity of precipitated antibodies and the antigen-

antibody ratio vary for those representing the antivenom analyses against

the individual Bothrops venoms or against the BVM mixture of Bothrops ven¬

oms used to produce antivenoms for therapeutic purposes, shows that both

the maximum quantity of precipitated antibodies and the ratio antigen-

antibody vary. In order to ascertain if each antivenom has comparable precipi-

tation activity against the specific venom and against the Bothrops venom

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DIAS da SILVA, W. et al. Cross-reactivity of horse monovalent antivenoms to venoms of Bothrops

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mixture the corresponding curves were transformed in straight lines and the

"F test" applied on their respective intercepts and slopes. The values were

calculated for the intercepts and slopes and for the parameters used to de¬

termine "Fa" and "Fb” used to verify the parallelism and coincidence among

the straight iines for each individual antivenom assayed against the specif-

ic venom and against the Bothrops venoms Table 4. The values for "Fa"

and "Fb" being below the calculated criticais F t4 = 7.71 and F r5 = 6.61

respectively, indicate parallelism and coincidence of the corresponding

precipitin curves experimentally obtained for each venom-antivenom assayed

in parallel against the specific-venom and the BVM mixture. Accordingly,

all curves are parallel with these parameters while all but the B. cotiara venom-

antivenom system are coincidents. Table 5 shows the Ag mx , the Ab mx

precipitated, and the ratio Ab mx : Ag mx for all monovalent and polyvalent IgG

antibodies against Bothrops venoms. The data suggest that: a) the precipi-

tation curves obtained with IgG are similar to those obtained with whole

horse antisera and described many years ago Fleidelberger 6 , against Clos-

tridium tetani or Corynebacterium diphtheriae toxins, since insoluble precipi-

tates were not detected in large Ab excess; b) as expected those values

vary accordingly to the venom used for immunization; c) the Ab:Ag molecular

ratios can not be calculated since the venoms are complex mixtures and

the molecular weight for most of their components has not yet been deter-

mined; d) the IgG antibodies produced by a mixture of Bothrops venoms

form good amounts of Ag-Ab complexes with a high Ab:Ag ratio.

Venom neutralization. Table 6 shows these results. Three observations

can be drawn from these assays: a) There was a good correlation among

the results obtained when the specific and the B. jararaca venom where

used for the monovalent antivenoms produced with B. insularis, B. alterna-

tus and B. atrox venoms; b) B. pradoi, B. atrox and B. jararacussu seen not

to be good immunogens in terms of their capacity to produce neutralizing

antibodies, as compared with the other venoms; c) The horse sera anti-fl.

pradoi and anti-6. neuwiedi venoms were more active against B. jararaca

venom than against the corresponding venoms used for their production.

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00 IbO 320 640 1200

tiqen (pg)

20 40 80

IbO 320 640 1200

(pg>

B. i ororoco

Pool of Bothrops venoms

Fig. 2: Quantitative precipitin reaction between crude

Bothrops venoms and horse antibodies to the venom

componentes. 0.5ml of horse IgG dissolved in phospha-

te saline buffer (PBS) (1.0mg/ml) pH 7.4, was added to

0.5ml of PBS containing different amounts of the speci-

fic venom and incubated overnight at 4°C. The precipi-

tates were washed in cold PBS, ressuspended in 0,5ml

of 0.1 N NaOH and the total protein determined. Control

samples containing only venom or IgG were included.

The ratio of antibody (Ab) to venom (Ag) found in each

precipitate was plotted (axis y) against the amount of ve¬

nom (axis x).

Each horse antivenom serum was assayed in parallel

against the specific_yenom (-), against the B.

jararaca venom (• • •), or against the mixture of

Bothrops venoms ( •••••• ) .

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DIAS da SILVA, W. et al. Cross-reactívity of horse monovalent antivenoms to venoms of Bothrops

species. Mem. Inst. Butantan, 51(41:153-168, 1989,

TABLE 4

Values for F criticai of "Fa" and "Fb" used to verify the parallelism and the coincidence

of the precipitin curves obtained for each Bothrops venom-antivenom system assayed both

against the specific and the Bothrops venom mixture.

Fa*

Fi4 = 7.71

Fi .5 = 6.61

(1) B. alternatus

0.07

0.91

(2) B. atrox

0.79

0.02

(3) B. cotiara

0.45

16.10

(4) B. erythromelas

0.03

0.44

(5) B. insula ris

2.19

1.38

(6) B. jararaca

5.19**

0.52**

(7) B. jararacussu

0.12

0.26

(8) B. moojeni

0.73

1.03

(9) B. neuwiedi

0.23

3.34

(10) B. pradoi

0.03

0.06

The F criticai for "Fa" and "Fb" values with = 0.05 were calculated accordingly

to the "SAS programme square minimum" (Ray, 1982)

The F criticai for "Fa" and "Fb" of B. jararaca were F-. .,

( = 0,05)

10.13 and F 1 4 = 7.71

TABLE 5

Quantitative precipitin titration of IgG isolated from Bothrops antivenoms listed in decreas-

ing order of their Ag: Ab complex (protein in precipitates) at the equivalence 30ml.

IgG Ab

to:

A 3max (venom)

added ( g) a

Total

precipitated

protein ( g)

Ab max dgG)

precipitated

protein ( g)

Ratio

Ab:Ag

(1) B. cotiara

640

1.490

850

1.33

(2) B. alternatus

320

1.340

1.020

3.19

(3) B. moojeni

640

1.255

615

0.96

(4) B. neuwiedi

640

1.243

603

0.94

(5) B. erythromelas

160

1.195

1.035

6.47

(6) B. pradoi

160

1.080

920

5.75

(7) B. insula ris

320

1.030

710

2.22

(8) B. jararacussu

160

885

725

4.53

(9) B. jararaca

320

790

470

1.47

(10) B. atrox

505

425

5.31

(11) Mixture of (21 +

(5) + (7) + (8) + (9) b

160

1.075

915

5.72

, and Ab precipitated in g of total protein as determined

max

a Values correspond to Ag n

by the Lowry's method.

b Solution of 50% B. jararaca and 50% of equal parts of the other Bothrops venoms desig-

nated by the numbers in parenthesis.

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DIAS da SILVA, W. et al. Cross-reactivity of horse monovalent antivenoms to venoms of Bothrops

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TABLE 6

Determination of the amount of specific and Bothrops jararaca venoms neutralized by 1 .Oml different

Bothrops antivenoms as estimated by the mouse protection test. The monovalent sera are listed in

decreasing order of their neutralizing potency.

Bothrops

antivenoms

Neutralized venoms

antivenoms mg/ml

alt

atr

cot

ery

ins

jar

|SU

moo

neu

pra

B. alternatus

(alt)

9.5

1,44

4,48

4.8

4.72

9.1

0,91

0,82

5,14

0.77

B. atrox

(atr)

1,15

1,09

1.87

1,42

0,94

0,97

1,54

0,74

B. cotiara

(cot)

2,20

0,83

8,1

3,37

0,53

0,97

0,92

2,96

0,65

B. erythromelas

(ery)

2,50

1.73

2,65

5,80

2,86

2,70

0,86

0,82

0,88

B. insularis

(ins)

2,65

8,0

0,86

0,82

0,88

B. jararaca

(jar)

4,0

1,20

2,55

6,30

3,40

0,91

0,82

2,06

1,03

B. jararacussu

(jsu)

0,84

1.0

0,53

0,97

3,28

B. moojeni

(moo)

0,77

1,41

2,19

1,62

1,45

2.17

0,91

4,8

3,70

2,96

B. neuwiedi

(neu)

1.78

1,20

2,65

5,6

4,86

5,05

0,91

0,82

2,81

1,02

B. pradoi

(pra)

0,85

0,83

2,66

1,38

3,43

0,97

1,18

4,30

1,31

The mixture containing monospecific anti -Bothrops venom or 0.15M NaCI plus equal volumes of different dilutions of the specific or

the B. alternatus (1), B atrox (9), B. cotiara (2), B. pradoi (8), B. insularis (5), B. /araracussu (7) or B. moojeni (8) was incubated at

37°C for 30 min. Half milliliter of each mixture was injected i.p. in groups of 8 mice, the animais maintained under observation for •

48h, the mortality ratio determined and the amount of neutralized venom in terms of mg calculated as a function of their correspondem

probits (Finney, 1971) .

DISCUSSION

When injected in horses according to the routine schedule used in Ins¬

tituto Butantan to produce antivenoms for therapeutic purposes, all ten

Bothrops venoms, B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. in¬

sularis, B. jararaca, B. jararacussu, B. moojeni and B. pradoi were able to

induce strong immune response as detected by the presence of high quan-

tities of specific antibodies in their respective antivenom.

Antibodies, specific for the Bothrops venom components, were assayed

by five different methods: the enzyme-linked immunobsorbent assay (ELI¬

SA), the double immunodiffusion, the quantitative precipitation, the neu-

tralization of indirect hemolytic action of the venoms and the neutralization

of the venom toxic effects (NVTE).

The data obtained from ELISA and double immunodiffusion assays

demonstrated that all Bothrops antivenoms contain high quantities of anti¬

bodies either reacting against antigenic components found in the venom

used to immunize the animal or cross-reacting with components present

in the other venoms. The discrepancies observed among the titers obtained

by the ELISA and by the double immunodiffusion assays can be explained

by the sensitivity limit inhibited by these two methods: for example, the ELI¬

SA assay is capable of detecting antibodies in the range of ^ g to ng N

Ab/ml whereas in the double immunodiffusion assay this limit goes up to

5 to 10 pg N Ab/ml Bier et al. 1 .

The high quantities of antibody in the Bothrops antivenoms was con-

firmed by the quantitative measurement of precipitins. Each antivenom was

assayed by the precise analytical method introduced by Heidelberger 6 ,

either against the specific venom, or against the B. jararaca venom or against

the BVM. Fig. 2 shows that the precipitation curve comprises three distinct-

segments: an initial ascending portion, a plateau corresponding to the precipi-

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DIAS da SILVA, W. et al. Cross-reactivity of horse monovalent antivenoms to venoms of Bothrops

species. Mem. Inst. Butantan, 51(41:153-168, 1989.

tation maximum, and a descending terminal segment. The shape of these

curves (floculation type) is very similar to that described for horse antisera

to diphteria and tetanus toxins since they do not appear to go through the

origin (Heidelberger, 1949). The presence of a soluble antigen and antibody

complex at the antibody excess region as well as in the antigen excess region

explaing this kind of curve. The mechanism acounting for the floculation

type of curve may be related with the floculation antibody heterogeneity

and with the relative binding affinities of these antibodies as compared to

the precipitin antibodies. Inspection of the three floculation curves obtained

for each Bothrops antivenom indicates that they follow similar patterns. There

are, therefore, indications suggesting that the antibody population found in

each antivenom recognizes similar antigens found either in the specific venom

or in the B. jararaca one as well as in the BVM mixture. To assert this suspi-

cion the floculation curves obtained for each horse monovalent Bothrops

antivenom assayed both against the corresponding venom and the BVM

were transformed in straight lines and their intercepts and slopes were sub-

mitted to F test. With the aid of the parameters used to calculate the values

for "Fa" and for "Fb" and their corresponding "criticai F" (Table 4), it was

possible to conclude that all paired curves were parallel and coincident. The

B. cotiara antivenom pair was an exception for although being parallel it was

not coincident.

With the exception of B. atrox antivenom the amount of antigen anti¬

body complexes found at the zone of equivalence with the other nine

monovalent antivenoms was over 1,0 mg/ml of the total protein content.

These values were comparable to those obtained with the polyvalent an¬

tivenom (Table 5).

The immunochemical assays indicate therefore that each Bothrops ven¬

om per se was able to induce the production of antibodies capable of mutu-

ally cross-react against one each other. The presence of such complex

antibody populations was reflected on their ability to neutralize the biologi-

cal properties of the venoms. For instance, the monovalent antivenoms with

higher number of precipitating properties detected by quantitative meas-

urement of antigen-antibody interactions (anti-fí. alternatus, anti -B. cotiara,

anti-fí insularis, anti-fi erythromelas and anti-fí moojeni) possess also strong

neutralizing activity against the venom lethal effects. On the other hand,

the B. jararacussu antivenom contains both small amounts of precipitating

antibodies and low neutralizing activity (Table 5 and 6). Flowever, such corre-

lation was not found when the anti-indirect hemolytic activity was assayed

Lachmann et al., Gutierrez et al., 8 ' 5 ; such activity was almost exclusive of

the B. alternatus antivenom.

The increase of the v -globulins at the end of the immunization proce-

dure is compatible with the high quantities of antibodies detected in the

Bothrops antivenoms.

In conclusion: a) all ten Bothrops venoms were able to induce antibody

formation wich mutually cross-react as assayed by three different im¬

munochemical methods; b) the venoms from B. alternatus, B. jararaca, B.

moojeni and B. neuwiedi, four of the most spread species of Bothrops snakes

in Brazil Hoge et al, 7 , behaved as good immunogens for horses and the an¬

tivenoms elicited, although in different degree, are capable to neutralize the

lethal activity present in the other venoms; c) in contrast, B. jararacussu

venom behaved as poor immunogen and its lethal activity was not efficiently

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blocked neither by the specific antivenom nor the antivenoms produced by

the other Bothrops venoms. These results agree well with previous work

showing that venoms from six Bothrops species contain common antigenic

components (Villarroel et. al 1tj ; Villarroel et al 17 ). Taken together the obser-

vations described in this work indicate that the Bothrops venom mixture to

be used to immunize horses to produce antivenom for therapeutic purposes

should contain venoms from B. alternatus, B. jararaca, B. jararacussu, B.

moojeni and B.neuwiedi. Table 6 shows that the antivenoms elicited by these

five Bothrops venoms contain antibodies capable to neutralize the lethal

activities for mice present in all ten Bothrops venoms used in this work.

ACKNOWLEDGEMENTS

The authors thank Dr. a Yoko Murata (Instituto de Pesquisas Energéticas

e Nucleares, São Paulo, SP) for her advice in the statistical analysis of the

precipitin curves; Dr. a Eva M.A. Kelen for criticai advice during the prepara-

tion of this manuscript; Mrs. Inete Pereira e Mss. Elaine Rodrigues for typing

the manuscript.

RESUMO: Cavalos foram imunizados com veneno de B. alternatus, B. atrox,

B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B.

moojeni, B. newiedi, e B. pradoi. Anticorpos específicos para componentes

antigênicos dos venenos foram detectados pelos métodos imuno-

enzimático, dupla-difusão e precipitação quantitativa enquanto que os an¬

ticorpos neutralizantes foram analisados pelos métodos da hemólise in¬

direta em placas e pela neutralização de seus efeitos letais. Anticorpos,

tanto específicos como dando reações cruzadas com venenos botrópi-

cos foram encontrados em todos os dez soros monovalentes. Modifica¬

ções nos padrões eletroforéticos, caracterizadas por uma redução no pico

da albumina e por um correspondente aumento das y -globulinas com

modificações ora acentuadas ora pouco perceptíveis nas frações das a

e p globulinas, foram detectadas em todos esses soros.

UNITERMOS: Antivenenos; antissoros botrópicos monovalentes; venenos

botrópicos.

REFERENCES

1. BIER, O.G.; DIAS DA SILVA, W.; GOTZE, D.; MOTA, I. Antigen antibody interaction.

In: Fundamentais of immunology. 2. ed. Berlin, Springer-Verlag, 1986, p. 179

2. BUSSAB, W.0. Análise de variância e de regressão. São Paulo, Atual, 1986. p. 92-100.

3. CHOI, S.C. Introductory applied statistics in Science. Englewood Cliffs, N.J., Prentice-

-Hall, 1978. p. 123-159.

4. FINNEY, D.J. Probít analysis. 3. ed. Cambridges, Cambridge University, 1971.

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for testing the potency of the polyvalent anti-venom produced in Costa Rica. Toxi-

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7. HOGE, A.R.; ROMÁNO-HOGE; S.A.R.W.L. Sinopse das serpentes peçonhentas do Bra¬

sil. 2. ed. Mem. Inst. Butantan, 42/43: 373-496, 1978/79.

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bra venom factor from phospholipase A 2 contaminant. Immunology, 31: 961-968,

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ment with the Folin phenol reagent. J. Biol. Chem., 193: 265-275, 1951.

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species. Mem. Inst. Butantan, 51(41:153-168, 1989.

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cleares)

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26: 507, 1949.

POPE, C.G. The action of proteolytic enzymes on the antitoxins and proteins in immu-

ne sera. I. True digestion of the protein. Sr/f., J, exp. Pathol., 20: 132, 1939.

RAY, A.A., ed. SAS user's guide; statistics. 1982 edition. cary, North Caroline, SAS

Institutte, 1982.

STEINBUCH, M. & AUDRAN, R. The isolation of IgG from mammalian sera with the

aid of Caprylic acid. Arch. Bioch. Biophy., 134: 279 - 284, 1969.

THEAKSTON, R.D.G.; LLOYD-JONES, M.J.; REID, H.A. Micro-ELISA for detecting and

assaying venom and venom-antibody. Lancet, 2: 639 — 641, 1977.

VILLARROEL, M.S.; ZELANTE, F.; FURLANETTO, R.S.; ROLIM-ROSA, R. Contribui¬

ção ao estudo imunoquímico de venenos botrópicos: I. Análise comparativa dos com¬

ponentes antigênicos de seis espécies de venenos frente a seus respectivos

antivenenos, através das técnicas de dupla difusão e imunoeletroforese em gel de

ágar. Mem. Inst. Butantan, 38: 13-30, 1974.

VILLARROEL, M.S.; FURLANETTO, R.S.; ROLIM-ROSA, R.; ZELANTE, F.; NAVAS, J.

Contribuição ao estudo imunoquímico de venenos botrópicos. II. Análise comparati¬

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Mem. Inst. Butantan, 38: 31-40, 1974.

168

SciELO

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IVtem. Inst. Butantan

51(41:169 - 176, 1989.

VARIAÇÃO DO COMPORTAMENTO BIOLÓGICO DO

TUMOR HUMANO KB, HETEROTRANSPLANTADO

SERIADAMENTE EM RATOS "NUDE" ATÍMICOS*

Leonor DENARO-MACHADO**

Suely A. KFOURI**

Terezinha R. P. O GATA* *

Jesus Carlos MACHADO**

Gilberto Alvarenga PAULA***

Lúcia Silva BARBOSA***

Regina F. G. FERREIRA***

Selma M. C. N. PETRELLA* *

RESUMO: Foram analisados, estatisticamente, os resultados obtidos de

estudo de 18 passagens seriadas do tumor humano KB transplantado em

ratos nude atímicos. Foram abordados os aspectos de crescimento ou

regressão da massa tumoral, bem como a caquexia por vezes detectada

no receptor.

UNITERMOS: Rato nude atímico. Tumor humano KB. Hetero-transplante.

INTRODUÇÃO

Em animais "nude” atímicos, a pega inicial de heterotransplantes de

tumores humanos é mais facilmente obtida a partir de linhagens de cultu¬

ras celulares do que quando os transplantes são realizados com tecidos fres¬

cos oriundos de biópsias 4 .5,6,8,9 p 0 r outro lado, a baixa transplantabilidade

continuada desses heterotransplantes tem dificultado estudos sobre a va¬

riação do comportamento biológico dessas neoplasias transplantadas, bem

como a análise dos mecanismos imunológicos de defesa desses animais

frente ao tumor.

Trabalho realizado com auxílio do CNPq, FAPESP e FEDIB.

Instituto Butantan — Divisão çte Patologia — Seção de Anatomia Patológica.

Instituto de Matemática e Estatística — USP. CEA do Departamento de Estatística.

Instituto Butantan — C P. 65 — 01051 — São Paulo-SP — Brasil.

Dedicado ao Dr. Saul Schenberg em homenagem ao seu 70° aniversário.

Recebido para publicação em 21-6-1989; aceito em 16-10-1989.

169

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10 11 12 13 14 15 16

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atfmicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

No presente trabalho, apresentamos os resultados obtidos em uma sé¬

rie de 18 passagens de heterotransplantes de tumor humano KB (carcino¬

ma epidermoide de boca) em ratos "nude" atímicos, realizados no perío¬

do de 1985 a 1987, analisando-se principalmente o aspecto do

desenvolvimento da massa tumoral, seja no crescimento ou regressão, com

algumas considerações sobre a caquexia por vezes detectada no receptor.

Nosso objetivo principal foi o de avaliar, através da análise estatística

dos resultados, o grau de associação existente entre as variáveis estuda¬

das e a malignidade do tumor no decorrer das passagens seriadas.

MATERIAIS E MÉTODOS

Animais: Foram utilizados, para os heterotransplantes, 134 ratos nude

atímicos adultos (» 1 mês de idade), de ambos os sexos. Esses animais

foram mantidos antes e durante a fase de experimentação em condições

especiais de biotério, em sala equipada com unidade de ventilação estéril

e temperatura e umidade do ar aproximadamente constantes. Alimento e

água eram previamente esterilizados e oferecidos "ad libitum".

Tumor KB: Os animais foram heterotransplantados com tumor huma¬

no KB, uma linhagem de carcinoma epidermoide de boca humano, manti¬

do em cultura desde 1955 7 .

Inoculações: A primeira inoculação, designada como passagem zero

(PO), foi realizada a partir de tripsinização da cultura e ajuste da suspensão

celular para 1 x 10 6 células/ml, tendo sido inoculado um volume final de

1 ml/animal. As demais inoculações foram realizadas por passagem seria¬

da de fragmentos de cerca de 1 mm 3 do tumor de um animal a outro, com

auxílio de trocater, após excisão da massa em crescimento, em condições

de assepsia e fragmentação da mesma em meio Eagle. Tais passagens se¬

riadas foram codificadas de PI a P18 e agrupadas para fins de análise esta¬

tística, de modo a conter aproximadamente o mesmo número de animais.

Todas as inoculações foram realizadas subcutaneamente na região axi¬

lar esquerda dos animais.

As seguintes variáveis foram analisadas no desenvolvimento do tumor

transplantado.

1. Crescimento de massa tumoral

A primeira visualização objetiva de um abaulamento na região inocula¬

da é tida como o início de crescimento do tumor transplantado. A incidên¬

cia assim considerada dessa pega foi de 78%. O tempo decorrido (em dias)

desde a inoculação até a visualização do abaulamento variou de 6 a 46 dias.

Quando a massa tumoral atinge um tamanho que dificulta a própria lo¬

comoção do animal (cerca de 4,5cm no seu maior eixo) (Foto 1),' este é

sacrificado e sua massa é extirpada e retransplantada em outros animais.

170

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DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

Foto 1: Exemplar de rato nude portador de massa tumoral (tumor KB), desenvolvida na

região de inoculação do transplante.

2. Regressão:

Em cerca de 12% dos casos, após o crescimento constatado pelo abau-

lamento, atingindo em média 4,0cm no seu diâmetro maior, o tumor so¬

freu processo de regressão, culminando com o desaparecimento total da

massa anteriormente detectada. O tempo (em dias) para a ocorrência de

tal fenômeno, considerado desde o aparecimento da massa até o início de

seu desaparecimento, variou de 21 a 78 dias.

3. Caquexia:

Trinta e quatro dos animais inoculados (25%) entraram em estado de

caquexia e foram então sacrificados para análises complementares. Des¬

ses animais, 19 haviam apresentado massa e 15 não. O tempo decorrido

para atingir esse estado variou de 22 a 108 dias, a contar do dia em que

foram inoculados.

As variáveis acima descritas foram analisadas estatisticamente por tes¬

tes de homogeneidade (qui-quadrado), em tabelas de contingência e tes¬

tes para o risco relativo.

SciELO

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

RESULTADOS

Associando-se em tabela de contingência as variáveis "aparecimento

de massa tumoral" contra "sucessão de passagens", a análise estatística

desses resultados revela que há uma dependência entre esses dois fatores,

ou seja, parece haver indícios de que passagens mais iniciais (P0-P6) tive¬

ram menos sucesso de pega que passagens intermediárias (P7-P9) ou fi¬

nais (P10-P18), quando transplantadas (Tabela I).

TABELA I

GRUPOS DE PASSAGENS

Aparecimento

da massa

P0-P4

P5-P6

P7-P9

P10-P18

Total

Não

(24%)

(38%)

(14%)

(22%)

Sim

(76%)

(62%)

(86%)

(89%)

105

(78%)

Total

134

p = 0.04

Tabela I: Distribuição dos ratos conforme o grupo de passagem e o aparecimento ou não

da massa tumoral.

Associando-se em tabela de contingência as variáveis "tempo para apa¬

recer a massa" contra "sucessão de passagens", as estimativas obtidas

permitem concluir que o tempo para aparecer a massa tumoral aumenta

nos grupos de passagens P5-P9, em relação ao grupo de passagem P0-P4,

e diminui no grupo P10-P18 em relação aos grupos P5-P9 (Tabela II).

TABELA II

Tempo p/aparecer

a massa (em dias)

GRUPOS DE PASSAGENS

P0-P4

P5-P6

P7-P9

P10-P18

Total

6-8

(34%)

(15%)

(13%)

(34%)

9-12

(28%)

(20%)

(29%)

(50%)

13-17

(28%)

(40%)

(38%)

(16%)

Total

105

p = 0.024

Tabela II: Distribuição dos ratos conforme o grupo de passagem e o tempo para aparecer

a massa.

172

j j SciELO

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

Associando-se em tabela de contingência as variáveis "aparecimento

da regressão" contra "sucessão de passagens", conclui-se não haver in¬

dícios de que a regressão da massa tumoral dependa da passagem utiliza¬

da no transplante (Tabela III). Note-se que a proporção de "não

aparecimento de regressão" varia muito pouco de um grupo de passagem

para outro.

TABELA III

GRUPOS DE PASSAGENS

Aparecimento

da regressão

P0-P4

P5-P6

P7-P9

P10-P18

Total

Não

(90%)

(85%)

(96%)

(81%)

(88%)

Sim

(10%)

(15%)

( 4%)

(19%)

(12%)

Total

105

p = 0.45

Tabela III: Distribuição dos ratos que desenvolveram crescimento tumoral, conforme o grupo

de passagem e a ocorrência ou não de regressão.

Associando-se em tabela de contingência as variáveis "tempo para apa¬

recer a regressão" contra "sucessão das passagens", conclui-se que há

indícios de que há dependência direta entre esses fatores, embora não te¬

nha sido possível detectar como se comporta essa dependência, devido

ao número insuficiente de ratos que apresentaram regressão (Tabela IV).

TABELA IV

GRUPOS DE PASSAGENS

Tempo p/aparecer

a regressão (em dias)

P0-P4

P5-P6

P7-P9

P10-P18

Total

21-55

56-78

Total

p = 0.07

Tabela IV: Distribuição dos ratos conforme o grupo de passagem e o tempo para aparecer

a regressão.

O teste de homogeneidade em tabela de contingência entre as variá¬

veis "aparecimento da regressão" e "tempo para aparecer a massa" não

mostrou indícios de dependência entre essas duas variáveis (Tabela V).

173

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10 11 12 13 14 15 16

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem, Inst. Butantan,

51(41:169-176, 1989.

TABELA V

TEMPO PI APARECER A MASSA (EM DIAS)

Aparecimento

da regressão

0-8

9-12

13-17

18-47

Total

Não

Sim

Total

105

p = 0.40

Tabela V: Distribuição dos ratos conforme o tempo para aparecer a massa e o apareci¬

mento ou não da regressão.

Da mesma forma, quando as variáveis "tempo para aparecer a mas¬

sa" e "tempo para aparecer a regressão” são confrontadas num diagra¬

ma de dispersão (Fig. 1), conclui-se não haver indícios de que o tempo

para aparecer a massa dependa linearmente do tempo para aparecer a re¬

gressão (correlação = — 0,063).

C 24

16.0

14.0

12.0

10.0

8.0 -

6.0 -

o o

C 25

20 .

35.

50.

65.

80.

Figura 1: Diagrama de dispersão entre as variáveis "tempo para aparecer a massa'

(C 24) e "tempo para aparecer a regressão" (C 25).

174

2 3 4 5 6 SCÍELO 10 n i2 13 14 15 16

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

Com relação ao fator "caquexia", aplicando-se o teste não paramétri¬

co para o risco relativo 1 , conclui-se que não há indícios de que a passa-

gem-em que o tumor foi inoculado, ou que o tempo para aparecer a massa

tumoral, influenciem no aparecimento da caquexia.

DISCUSSÃO E CONCLUSÕES

0 valor de um modelo biológico experimental depende de sua estabili¬

dade, seja concernente às semelhanças existentes com o modelo original,

seja concernente à sua manutenção e repetitividade a longo termo.

No modelo rato nude atímico transplantado com tumor humano a es¬

tabilidade consiste na permanência da identidade original do tumor huma¬

no e a repetitividade consiste em passar esse tumor no animal por período

de tempo suficiente que permita investigações biológicas do transplante

e do transplantado, ou mesmo estudos básicos terapêuticos.

Wallace et al, 71 11 estudaram o crescimento de células KB em hams¬

ters recém-nascidos tratados com soro anti-timocítico anti-hamster (ATS)

ou soro normal de coelho (NRS). Uma única injeção de ATS pode promo¬

ver a formação de tumor KB metastático numa maior proporção de ani¬

mais, enquanto que apenas poucos animais dos tratados com NRS

desenvolviam pequenos tumores que ocasionalmente regrediam.

Sudo, 87 10 também relatou o transplante dessa mesma linhagem KB

em camundongos nude, porém esplenectomizados e irradiados.

Nosso modelo experimental rato nude atímico/tumor humano KB sem

nenhum outro artefato, vem sendo mantido em passagens seriadas em nos¬

so laboratório desde 1985 3 , encontrando-se atualmente na passagem 29.

Este nosso estudo, concernente à estabilidade do modelo a longo ter¬

mo, compara bioestatisticamente as diferenças observadas entre as pas¬

sagens iniciais do tumor e as passagens mais avançadas.

Assim, essa análise indica que, à medida que o número da passagem

aumenta, há uma maior probabilidade de aparecer a massa tumoral no ani¬

mal transplantado, mostrando haver uma interação entre a estrutura anti-

gênica da célula tumoral e a competência do microambiente do hospedeiro.

Corroborando ainda essa interação transplante/hospedeiro, pudemos

verificar que em passagens intermediárias e finais o tempo requerido para

o aparecimento do crescimento tumoral foi menor que nas passagens

iniciais.

As regressões tumorais observadas (12%) podem ser explicadas pe¬

los mecanismos de defesa individuais do hospedeiro, atuando contra o cres¬

cimento tumoral. Essa regressão não se mostrou dependente da passagem

mais ou menos avançada do tumor, nem do tempo requerido para o apa¬

recimento do crescimento tumoral, nem ainda há indícios de que o tempo

para aparecer a massa tumoral dependa do tempo para aparecer a re¬

gressão.

No que tange o fator caquexia, presente na maioria dos ratos inocula¬

dos, após um período variável de 22 a 108 dias, não há indícios de que

a passagem em que o tumor foi inoculado influencie sobre esse fator e nem

há indícios de que o tempo levado para aparecer a massa tumoral influen¬

cie no aparecimento do estado de caquexia.

Os resultados no caso do crescimento da massa tumoral, vieram cor-

175

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10 11 12 13 14 15 16

DENARO-MACHADO, L; KFOURI, S.A.; OGATA, T.R.P.; MACHADO, J.C.; PAULA, G.A; BARBOSA,

L.S.; FERREIRA, R.F.G.; PETRELLA, S.M.C.N. Variação do comportamento biológico do tumor hu¬

mano KB, heterotransplantado seriadamente em ratos "nude" atímicos. Mem. Inst. Butantan,

51(41:169-176, 1989.

roborar achados histológicos que sugerem o aumento da malignidade do

tumor seriadamente transplantado 2 .

Assim, à medida que as passagens seriadas se sucederam, os valores

médios do volume cariométrico das células componentes do tumor, au¬

mentaram significantemente e, da mesma forma, o número de mitoses au¬

mentou consideravelmente.

ABSTRACT: The results obtained from the study of 18 serial passages

of human tumor KB transplanted in athymic nude rats have been statisti-

cally analysed. Aspects of growth or regression of the tumoral mass, as

well as the cachexy sometimes detected in the receptor, have been ap-

proached.

KEY WORDS: Athymic nude rat. Human tumor KB. Heterotransplant.

REFERÊNCIAS BIBLIOGRÁFICAS

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VID, M. L. V. M. G.; KFOURI, S. A.; CARNEIRO, S. M. Heterotransplante de células

KB em ratos nude atímicos II. Estudos morfológicos. Acta Oncol. Bras., 8: 21-25, 1988.

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KFOURI, S. A.; MACHADO, J. C. Heterotransplante seriado de linhagem celular tu¬

moral humana (KB) em ratos nude atímicos. I. Estudos biológicos. Acta Oncol. Bras.,

7:115-1 17, 1987.

4. GIOVANELLA, B. C.; STEHLIN, I. S.; WILLIANS, L. I. Heterotransplantation of human

malignant tumors in the nude thymusless mice. II. Malignant tumors induced by in-

jection of cell cultures derived from human solid tumors. J. Nat. Câncer Inst.,

52: 921-930, 1974.

5. GIOVANELLA, B. C.; STEHLIN, I. S.; WILLIANS, L. I. et al. Heterotransplantation of

human câncer into nude mice: a model system for human câncer chemotherapy. Cân¬

cer, 42: 2.269-2.281, 1978.

6. GIOVANELLA, B. C.; YIN, S. O.; STEHLIN, I. S. et al. Development of invasive tumors

in the ''nude'' mouse after injection of cultured human melanoma cells. J. Nat. Cân¬

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7. HARRY, E. Propagation in a fluid medim of a human epidermoid carcinoma, strain KB

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11. WALLACE, R.; VASINGTON, P. I.; PETRICCIANI, I. C. Heterotransplantation of cultu¬

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176

1, | SciELO

Mem. Inst. Butantan

51(41:177-193, 1989.

BIOCHEMICAL STUDIES OF THE SEA SNAKE NEUROTOXINS

Anthony T. TU

Roger A. MILLER

ABSTRACT: Sea snakes belonging to the family Hydrophiidae are marine-

adapted, serpents found widespread in tropical and subtropical costal

waters of the Indian and Pacific Oceans. Sea snake venom is a mixture

of different proteins and contains potent neurotoxins. The LD B0 for sea

snake venom can be as low as 0.01 mg/Kg. The purified type I postsynaptic

neurotoxins consist of 60-62 amino acid residues with four disulfide bonds.

They are basic proteins with isoelectric points of 9 to 10 and range in

molecular weight from 6,000 to 8,000. Sea snake neurotoxin work in this

laboratory has centered on the postsynaptic neurotoxin of Laticauda

semifasciata, Lapemis hardwickii, Pelamis platurus and Acalyptophis

peronii. Sea snake neurotoxins show considerable homogeneity in their

amino acid sequences with many invariant residues. Raman studies

indicate the neurotoxins are a mixture of beta sheet and beta turns with

no alpha helical secondary structure. The origin of lethality comes from

the fact the sea snake neurotoxin strongly binds to the acetylcholine

receptor at the neuromuscular junction which leads to muscle paralysis

and respiratory arrest. Chemical modification of the conserved tryptophan

residue has lead to the loss of the specific binding of the acetylcholine

receptor and the loss of toxicity, but the modified toxin retained the ability

to bind to neurotoxin antibodies. This suggested that neurotoxins can be

converted into toxoids. A single tyrosine residue, some arginine and lysine

residues are also essential to neurotoxicity. In addition to specific residues,

some regions of polypeptide backbone are also important for toxicity.

KEY WORDS: Neurotoxins, Venoms, Acetylcholine Receptor.

INTRODUCTION

The scope of this review and discussion is restricted mainly to the work

in this laboratory related to sea snake neurotoxins and interaction with acetyl¬

choline receptors (AChR) in order to restrict the length of the review. There

Department of Biochemistry Colorado State University Fort Collins, CO 80523.

Dedicated to Dr. Saul Schenberg's 70th birthday

Received 04/7/1989; accepted 02/8/1989.

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are many review articles on this subject, and readers are encouraged to read

these for an overall view of neurotoxins. 1 " 4

The sea snake is a marine-adapted serpent belonging to the family of

Hydrophiidae and they are found widespread in tropical and subtropical

Coastal waters of he Indian and Pacific Oceans, however they are not found

in the Atlantic Ocean. (Fig. 1) There are many varieties of sea snakes with

different colors, shapes, and sizes. They are well adapted for the marine

environment and have a flat tail and a salt gland. There are two subfamilies

within the family ffydrophiidae. They are Hydrophiinae and Laticaudinae.

The two types of sea snakes have distinct differences in their ventral scales.

The former usually does not have ventral scales distinguishable from the

surrounding scales, whereas the latter has wide ventral scales. These differ¬

ences in ventral scale patterns eventually dictate their habitats. The reason

a snake can crawl is due to the movement of its ventral scales. Since

Hydrophiinae do not have ventral scales, they spend their entire lives in the

sea. On the other hand, the Laticaudinae can swin in the sea and crawl on

the beach and rocks.

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Sea snake venom is a mixture of different proteins, of which some are

toxic and some components are relatively nontoxic. Because a venom con-

tains highly toxic postsynaptic neurotoxins venom as a whole exhibits high

toxicity. The potent neurotoxins in their venoms can cause muscle paraly-

sis and respiratory failure of a victim which may lead to morbidity or death.

Most of the sea snake work in this laboratory has centered on the ve¬

nom from four species of sea snakes: Laticauda semifasciata captured in

the waters near the Philippines (Fig. 2A), Lapemis hardwickii captured in

the Gulf of Thailand (Fig.2B), Pelamis platurus captured in the Pacific Coastal

waters of Costa Rica (Fig. 20 and Acalyptophisperonii captured in the Gulf

of Thailand (Fig. 2D).

Fig. 2. (A-D) Photographs of selected sea snakes

A Laticauda semifasciata

179

B Lapemis hardwickii

TU, A.T. & MILLER, R.A. Biochemícal studies of the sea snake neurotoxins. Mem. Inst. Butan-

tan,51(41:177-193, 1989

C Petamis platurus

T * ' . ■r

* / . ;

D Acalyptophis peronii

The yellow-bellied sea snake, Pelamis platurus, which is considered to

be the most widespread sea snake in the world, is found in most of the Coastal

waters shown in Figure I. 5 Acalyptophis peronii is the third most common

sea snake in the Gulf of Thailand. The appearance, color pattern, and other

morphological characteristics of this snake are quite different from other

sea snakes. This sea snake is readily recognizable by its horn-like lifted-up

scale near the eye. 1 - 6 ' 7

The remainder of this review will summarize the work in this laboratory

in understanding the structure-function of sea snake neurotoxins and inter-

action studies with the AChR.

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Sea snake venom seems to lack cytolytic activity. When venoms of var-

ious sea snakes (E. schistosa, L. semifasciata, L. hardwickii, and R platu-

rus) were added to the cell cultures of KB cells, Yoshida sarcoma cells, and

normal peritoneal cells, no lysis was observed. 12

Most sea snake neurotoxins consist of 60-62 amino acid residues with

four disulfide bonds. These are the type I or short-chain neurotoxins. However,

several type II or long-chain toxins were also isolated. Both types I and II

are postsynaptic toxins, but type II toxins have five disulfide bonds. Some

neurotoxins have structures between type I and II; they contain four disul¬

fide bonds but have many other features similar to type II neurotoxins. 1

Most sea snake venoms seem to contain only the postsynaptic neurotox-

in. Only in Enhydrina schistosa venom, which also possesses a postsynap¬

tic toxin, was a presynaptic type found and identified as phospholipase A.

Therefore, to classify neurotoxins solely on the basis of their disulfide bonds

or amino acid sequences is an oversimplification. 1

There are four disulfide bonds in short-chain (Type I) neurotoxins which

means that there are eight half-cystines. However, all Hydrophiinae toxins

have nine half-cystines. An extra cysteine residue can be readily detected

from the Raman spectrum as the sulfhydryl group shows a distinct S-H

stretching vibration at 2578 cm -1 . 13 ' 14 Some Laticaudinae toxins do not have

a free cysteine residue as in the case of L. semifasciata toxins. These two

types of postsynaptic neurotoxins are the most commonly found neurotox¬

ins in Hydrophiidae venoms and in Elapidae (cobras and kraits) venoms.

Table 1 summarizes some of the basic characteristics of selected toxins

purified and studied in this laboratory. 6 'Ti4-i6

TABLE 1

BIOCHEMICAL CHARACTERISTICS OF SELECTED POSTSYNAPTIC NEUROTOXINS DONE

IN THIS LABORATORY

Neurotoxin

LD 50

#=res/molec

Laticauda semifasciata

Toxin b

6,800

> 9.5

0.05 mg/Kg

Lapemis hardwickii

Lapemis Toxin

6,800

9.6

0.01 mg/Kg

Peiamis platurus

Toxin b

6,800

8.7

0.185 mg/Kg

Acalyptophis peronii

Major Toxin

6,600

> 9.5

0.125 mg/Kg

Acalyptophis peronii

Minor Toxin

6,600

> 9.5

0.10 mg /Kg

(i.v. mice)

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The amino acid sequences of many sea snake neurotoxins have been

determined. The complete amino acid sequence of the neurotoxins se-

quenced in this laboratory are listed in Table 2. 6 - 7 ' 16 " 18 There are two toxic

fractions in the Acalyptophis peronii venom, the most toxic and abundant

fraction was isolated and termed major toxin. The A. peronii minor toxin

was identified and compared to that of the major toxin. The only difference

between the major and the minor toxins is in the 43rd residue. The major

toxin at this position contains glutamine, while the minor toxin contains glu¬

tã mic acid. 6 ' 7

TABLE 2

AMINO ACID SEQUENCES OF SEA SNAKE POSTSYNAPTIC NEUROTOXINS COMPLETED

IN THIS LABORATORY

Laticauda semifasciata

Toxin b

RICFNQHSSQPQTTKTCPSGQSSCYHKQWSDFRGTIIERGCGCPTVKPGIKLSCCESERCNN

Lapemis hardwickii

Lapemis Toxin

MTCCNQQSSQPKTTNCAESSCYKKTWSDHRGTRIERGCGCPQVKPGIKLECCHTNECNN

Pelamis platurus

Toxin b

MTCCNQQSSEPKTTTNCAESSCYKKTWSDHRGTRIERGCGCPQVKSGIKLECCHTNECNN

Acalyptophis peronii

Major Toxin

MTCCNQQSSQPKTTTNCAGNSCYKKTWSDHRGTIIERGCGCPQVKSGIKLECCHTNECNN

Acalyptophis peronii

Minor Toxin

MTCCNQQSSQPKTTTNCAGNSCYKKTWSDHRGTIIERGCGCPEVKSGIKLECCHTNECNN

In order to understand the exact mechanism of the neurotoxic action,

it is important to know the secondary structure of the neurotoxins as weíl.

It is now known that postsynaptic neurotoxins attach to the a-subunits of

acetylcholine receptor (AChR). 1 The conformation of sea snake neurotox¬

ins has been extensively studied. The results of these studies are sumrna-

rized in Table 3.5- 16 ' 18 ' 19

Raman spectroscopic examination of pelamis toxin b indicates that the

toxin contains a considerable amount of antiparallel /3-structure, p -turn, and

random coil without «-helix as the amide I band appears at 1673cm _1 and

the amide III band at 1246 cm -1 . Circular dichroic studies also indicate a

typical p sheetstructure. The pelamis toxins bis a typical postsynaptic neu-

rotoxin as it binds to the AChR competitively with a well known toxin, a-

bungarotoxin. 16

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TABLE 3

CONFORMATION OF SEA SNAKE NEUROTOXINS AS DETERMINED BY RAMAN

SPECTROSCOPY IN THIS LABORATORY

Venom

Toxin

Conformation

Enhydrina schistosa

Major toxin

Mixture of P turn and p sheet and no a

helix

Lapemis hardwickii

Lapemis toxin

Mixture of p turn and P sheet and no a

helix

Pelamis platurus

Pelamis toxin a

Mixture of /3 turn and p sheet and no a

helix

Pelamis platurus

Pelamis toxin b

Mixture of P turn and p sheet and no a

helix

TABLE 4

CHEMICAL MODIFICATION OF SEA SNAKE NEUROTOXINS IN THIS LABORATORY

Residue Toxin: Conclusion

Arginine

L. semifasciata

toxin a: No loss of toxicity when 1 of 3 residues modified

toxin b: No loss of toxicity when 1 of 2 residues modified

Lysine

L. semifasciata

toxin a: No loss of toxicity when 3 of 4 residues modified

toxin b: No loss of toxicity when 4 of 5 residues modified

Tryptophan

E. schistosa

Major toxin: Loss of toxicity

L. hardwickii

Lapemis toxin: Loss of toxicitv

L. semifasciata

Toxins a & b: Loss of toxicity

Tyrosine

L. hardwickii

Lapemis toxin: Loss of toxicity

Sulfhydryl group

P. platurus

Pelamis toxin: Less toxic but retains toxicity,

still bind to AChR

Disulfide bond

P. platurus

Pelamis toxin: Loss of toxicity

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The amide I band and III band for Enhydrina schistosa toxin were at 1672

cnr 1 and 1242 cm' 1 , respectively. These wave numbers are characteristic

for anti-parallel P sheet structure. The presence of p-sheet structure found

by Raman spectroscopic study was later confirmed by X-ray diffraction study

on Laticauda semifasciata toxin b. 19

Sea snake short-chain toxins have a molecular weight of only 6,800.

The small size with four disulfide bonds makes these toxins very compact

and stable molecules. Therefore, when the Pelamis platurus toxin is sub-

jected to heat treatment at 100 °C and subsequent cooling, it does not

change its conformation substantially. Amide I and III bands and S-S stretch-

ing vibration did not change by heat treatment. 5

The four disulfide bonds of the neurotoxin are clustered in one area and

there is a protruding loop. It is suspected that this loop is the one that plays

an important role in binding to the AChR. The four disulfide bonds are be-

lieved to be important for maintaining the specific conformation and have

been studied extensively. The conformation of the disulfide bond in C-C-S-

S-C-C network is gauche-gauche-gauche conformation at the S-S stretch-

ing vibration appearing at 510-512 cm -1 of Raman spectrum. 1 ' 21

Figure 4 shows the two-dimensional structure and tertiary structure of

Lapemis hardwickii lapemis toxin adapted from the more well studied toxin

b of Laticauda semifasciata venom. 22 Note the three major structural loops

of the tertiary structure termed loop A, B, and C from the amino terminal

to the carboxyl terminal respectively.

Fig. 4. Tertiary view of Lapemis hardwickii Lapemis toxin

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In order to elucidate structure and function relationships, some amino

acid residues were chemically modified and the effects of this modification

on toxicity or acetylcholine receptor binding ability of the postsynaptic neu¬

rotoxins were investigated, Chemical modification of sea snake neurotox¬

ins is summarized in Table 4 .IT 19 - 23

The amino acid residues in neurotoxins which are important for neuro-

toxic action are still not entirely clarified. Some neurotoxins contain one free

SH group, while others do not. From this fact, it would be logical to assume

the sulfhydryl group is not essential. This was actually proven to be the

case.

When N,N'-1,4-phenylenedimaleimide was used for modifying the sulf¬

hydryl group in pelamis toxin, 2 mole of toxins combined with 1 mole of

the reagent. With the sulfhydryl group modified, the S-H stretching vibra-

tional band at 2578 cm -1 disappeared. The modification of the single sulf¬

hydryl group did not alter the binding ability to the AChR or Toxicity (Fig.

5). 20

jsl

| 2 [

sni

Pelamis toxin a

N,N-1,4-phenylenedimaleimide

Modified Pelamis toxin a

(Dimerl

Fig. 5. Chemical modification study example on neurotoxin

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Disulfide bonds, however, are important in maintaining the particular toxin

structure and have been shown to be essential for toxicity. When all four

disulfide bonds are reduced and alkylated, the neurotoxin loses its toxicity. 24

The one residue most extensively studied is tryptophan. Since it was

easily modified, it indicated that the tryptophan residue is exposed. Raman

spectroscopic analysis of a sea snake neurotoxin indicated that a single tryp¬

tophan residue is indeed exposed. The tryptophan residue lies in the im-

portant loop consisting of segment 4. Modification of the tryptophan

residue induces the loss of AChR binding ability as well as the loss of

toxicity. 14,19,20,22

There is only one tyrosine residue in some sea snake neurotoxins. This

residue is usually quite difficult to modify, but once it is modified, the toxici¬

ty is lost. 23

Arginine and lysine are believed to be important, but results are not clear

because sea snake neurotoxins contain several residues of these amino

acids. 14

The acetylcholine receptor connects the nerve impulse from the axon

to the muscle by receiving a nerve transmitter, acetylcholine. The receptor

consists of five subunits of which two are identical; it is expressed as

a 2 í3 y d • It is known that the subunit is the site for the acetylcholine bind¬

ing and also for its antagonist, snake postsynaptic neurotoxin. It is general-

ly recognized that the subunits p, y , and ó are also essential to maintain

the integrity of the acetylcholine receptor. In order to further understand

the role of the subunits in the acetylcholine receptor function, the subunits

were cross-linked with dimethylsuberimidate which cross-links NH 2 . The

cross-linked acetylcholine receptor does not dissociate into its components

and retains the binding activity to Lapemis toxin, a postsynaptic toxin from

sea snake Lapemis hardwickii ve nom. This indicates that convalently linked

acetylcholine receptor subunits retain their biological function as long as

the neurotoxin binding site is not blocked. 25

Formation of cross-linked AChR is evident from the high molecular weight

band shown in the sodium dodecylsulfate polyacrylamide gel electrophore-

sis (SDS-PAGE). Without crosslinking, AChR subunits are separated into four

subunits, a ,p , y, and ó . In order to examine the biological activity of the

cross-linked AChR, lapemis toxin is iodinated with 125 l and the mutual bind¬

ing was examined. In each experiment, lapemis toxin without crosslinking

was used as the control. The effect of AChR concentration on lapemis tox¬

in binding was studied and the cross linked AChR showed very weak

binding. 26

DISCUSSION

The origin of lethality comes from the fact the sea snake neurotoxin

strongly binds to the acetylcholine receptor at the neuromuscular junction.

Thus, there is a parallel relationship between the affinity and toxicity as ex¬

pressed by LD 50 When the structure of sea snake neurotoxins is compared

with that of land snakes (Elapidae), they are very similar. Fortunately, sea

snake injects less venom in a bite; thus fatalities due to sea snake are fewer

than those of land snakes on a per bite basis. 1

The binding mechanism of neurotoxins to acetylcholine receptors is still

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unclear. Neurotoxins are fairly small polypeptides (6800-8000 Daltons), and

many of their primary structures are already known. However, owing to the

large size of the multi-subunit AChR, detailed binding sites of the receptor

are still less well understood. 1

Sea snake neurotoxins show considerable homogeneity in their amino

acid sequences. There are many invariant residues. That there is greater

similarity in sequences within a subfamily and more differences between

the two subfamilies of sea snakes is very interesting since these Chemical

data agree well with morphological differences between the two subfa¬

milies. 1

Similarity in the structures of sea snake neurotoxins are also reflected

in immunologic similarities. Usually the antibody (antiserum or antivenin)

produced against sea snake venom neutralizes the toxicities of other sea

snake venoms quite well. Sea snake neurotoxins are not only similar among

themselves but also closely resemble neurotoxins from some land snake

venoms, such as those of the Elapidae (cobras and kraits). Therefore, it is

advantageous to discuss the neurotoxins of Elapidae and Hydrophiidae to-

gether for a better understanding of their structure and function. The hig-

her toxicity of Hydrophiidae venoms compared with that of Elapidae venoms

is due to the higher concentration of neurotoxins in Hydrophiidae venoms. 1

Chemical modification of each residue in the sea snake neurotoxin indi-

cated the tryptophan-modified neurotoxin abolished the lethality and neu-

romuscular blocking activity, thereby indicating the essential role of

tryptophan residue. It is of interest to note that both tryptophan and tyrosi-

ne residues are conserved and located at exactly the same position in the

amino acid sequence regardless of the species of sea snake. When the trypto¬

phan residue was modified with N-bromosuccinamide, the toxicity of the

sea snake neurotoxins disappeared; however, they still retained the ability

to bind to neurotoxin antibody. This suggested that neurotoxins can be con-

verted into toxoids. 19

Sea snake venoms contain potent neurotoxins that bind almost irrever-

sibly to the postsynaptic acetylcholine receptors. Neurotoxins are the most

extensively studied proteins of all the components present in sea snake ve¬

noms. Sea snake toxins are basic proteins with isoelectric points of 8.7-10.

They range in molecular weight from 6,000 to 8,000. The neurotoxin is the

main protein fraction although other proteins with molecular weights up to

29,500 can be found in Pelamis platurus. The AChR is composed of five

subunitis, a 2 p Y ó . A neurotoxin attaches to the a subunit. Since there

are 2 moles of the <* subunits, 2 moles of neurotoxins attach to 1 mole

of AChR. A neurotransmitter, acetylcholine (ACh), also attaches to the a

subunit. When the ACh attaches to the AChR, the AChR changes confor-

mation, opening up the transmembrane pore so that cations (Na + ,K + ) can

pass through. By this mechanism the depolarization wave from a nerve is

now conveyed to the muscle. The difference between neurotoxin and ACh

is that the former's attachment does not open the transmembrane pore.

As a consequence, the nerve impulse from a nerve cannot be transmitted

through the postsynaptic site (Fig. 6).

Since neurotoxins are basic proteins and the AChR is acidic, it would

be logical to assume that the binding of these two proteins is due to acidic

and basic protein interactions. Yet, simple acidic and basic protein binding

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cannot explain the extremely high affinity of these two proteins based so-

lely on the ionic State. Therefore, there must be some other factor involved

in the binding. One such factor is iikely to be complementary topography

of the two molecules, which allows the two proteins to lock firmly into each

other.

2 /

AC hl

Ãch

Postsynaptlc

Neurotoxln

Fig. 6 Diagrammatical structure of Acetylcholine Receptor ACh (acetycholine)

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The binding is very tight as stated and suggests the following working hypo-

theses by these authors. As mentioned the ionic State and topography of

the two molecules (neurotoxin, a -AChR) play a major role In the recogni-

tion and binding. The importance of the invariant tryptophan residue of the

neurotoxin may play a role in receptor recognition. The neurotoxin molecu-

le would then lock the receptor in the closed conformation effectively bloc-

king the receptor function to transmit the electrochemical neuronal impulse

to the musculature leading to paralysis. Figure 7 diagrammatically depicts

the interaction of neurotoxin with the o subunit of the AChR which is im-

bedded in the lipid bilayer of the muscle cell. The branched structure repre-

sents the carbohydrate moiety on the exterior surface of the muscle cell.

These authors believe that there may occura disulfide bond exchange when

acetylcholine binds and the opening of the ionic channel occurs due to the

conformational change in the two a subunits of the AChR.

Fig. 7 Diagrammatical view of the a subunit of the Acetylcholine Receptor

Neurotoxin may serve the same role but locks the receptor in the clo¬

sed conformation. The acetylcholinesterase would be ineffective to remo¬

ve the neurotoxin as it does acetylcholine. This would therefore be an

irreversible blockage of the receptors. The specificity and irreversibility of

this action by neurotoxin probably explains the high toxicity of these mo¬

lecules.

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An alternative hypothesis, which is very similar to the above but may

account better for the very tight binding known to occur between the AChR

and neurotoxin, is that the disulfide bonds under local reducing environ-

ment actually exchange between receptor and neurotoxin molecule giving

a covalent bonded receptor toxin interaction (Fig. 8). The invariant trypto-

phan may play a role in providing the localized reducing environment of the

AChR criticai disulfide bonds and the neurotoxin. Experiments are currently

being designed to probe these hypothesis

NEUROTOXIN

RECEPTOR

Fig. 8 Expanded view of the Disulfide (S-S) Exchange of Neurotoxin and AChR

ACKNOWLEDGMENT

This work was supported by U.S. Army Medicai Research and Develop-

ment Contract DAMD17-89-Z-9019. The views, opinions, and/or findings

contained in this review are those of the author(s) and should not be cons-

trued as an official Department of the Army position, policy, or decision un-

less so designated by other documentation.

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- 193, 1989.

RESUMO: Serpentes marinhas pertencentes à família Hidrofidae são ser¬

pentes adaptadas à vida aquática e são encontradas dispersas nas águas

costeiras das zonas tropical e subtropical dos oceanos Indico e Pacífico.

O veneno da serpente marinha é uma mistura de várias proteínas e con¬

tém neurotoxinas potentes. A DL 50 para o veneno da serpente marinha

pode chegar até o baixo valor de 0,01 mg/kg.

As neurotoxinas pós-sinápticas do tipo I purificadas consistem de 60-62

resíduos de amino-ácidos com quatro pontes de disulfeto. São proteínas

básicas com pontos isoelétricos de 9 a 10 e de pesos moleculares va¬

riando de 6.000 a 8.000 daltons. Pesquisas deste laboratório foram foca¬

lizadas sobre neurotoxinas pós-sinápticas dos venenos de Laticauda

semifasciata, Lapemis hardwickii, Pelamis platurus e Acalyptophis peronii.

As neurotoxinas marinhas mostram grande homogeneidade das seqüên-

cias dos seus amino-ácidos com muitos resíduos invariáveis. Estudos Ra-

man indicam que as estruturas secundárias das neurotoxinas se compõem

de P -pregas e p -curvas sem a -hélices.

A letalidade provém do fato das neurotoxinas marinhas se ligarem forte¬

mente ao receptor de acetilcolina da junção neuromuscular o que con¬

duz à paralisia muscular e perda respiratória. Modificação química do

resíduo de triptofano acarreta a perda da ligação específica ao receptor

da acetilcolina e a perda da toxicidade, porém a toxina modificada retém

a propriedade de se ligar aos anticorpos da proteína nativa. Isso sugere

que as neurotoxinas podem ser convertidas em toxóides.

Outros resíduos de amino ácidos como uma tirosina, algumas argininas

e lisinas também são essenciais à toxicidade. Além desses resíduos es¬

pecíficos de amino-ácidos, algumas regiões do esqueleto polipeptídico

são também importantes para a toxicidade.

UNITERMOS: Neurotoxinas, Veneno, Receptor de acetilcolina.

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venoms of sea snake Lapemis hardwickii and Enhydrina schistosa. J. Biol. Chem.,

250: 1782-1785, 1975.

14. TU, A.T.; HONG, B.S.; SOLIE, T.N. Characterization and Chemical modifications of toxins

192

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TU, A.T.; MILLER, R.A.; Biochemical studíes of the sea snake neurotoxins. Mem. Inst. Butantan, 51(4)177

- 193, 1989.

isolated from the venoms of the sea snake, Laticauda semifasciata , from Philippi-

nes. Biochemistry, 10: 1295-1304, 1971.

15. TU, A.T. & HONG, B.S. Purification and Chemical studies of a toxin from the venom

of Lapemis hardwickii (Hardwick's sea snake). J. Biol. Chem., 246: 2772-2779, 1971.

16. MORI, N.; ISHIZAKI, H.; TU, A.T. Isolation and characterization of Pelamis platurus

(Yellow-bellied Sea Snake) Postsynaptic Isoneurotoxin. J. Pharnn. Pharmacol., 41:

331-334, 1989.

17. TSERNOGLOU, D.; PETSKO, G.A.; TU, A.T. Protein sequencing by Computer graphics.

Biochim. Biophys. Acta, 491: 605-608, 1977.

18. FOX, J.W.; ELZINGA, M.; TU, A.T. Amino acid sequence of a snake neurotoxin from

the venom of Lapemis hardwickii and the detection of a sulphydryl group by laser

Raman spectroscopy. FEBS Lett., 80: 217-220, 1977.

19. TU, A.T. & HONG, B.S. Purification and Chemical studies of the toxic component of

Enhydrina schistosa (common sea snake) venom. J. Biol. Chem., 246: 1012-1016,

1971.

20. ISHIZAKI, H.; ALLEN, M.; TU, A.T. Effect of sulfhydryl group modification on the neu-

rotoxic action of a sea snake toxin. J. Pharm. Pharmacol., 36: 36-41, 1984.

21. TU, A.T. Raman Spectroscopy in biology; principies and applications. New York, John

Wiley. 448p.

22. ALLEN, M. & TU, A.T. The effect of tryptophan modification on the structure and func-

tion of sea snake neurotoxin. Mol. Pharmacol., 27: 79-85, 1985.

23. RAYMOND, M.L. & TU, A.T. Role of tyrosine in sea-snake neurotoxin. Biochim. Biophys.

Acta, 285: 498-502, 1972.

24. TU, A.T.; LIN, T.S.; BIEBER, A.L. Purification and Chemical characterization of the major

neurotoxin from the venom of Pelamis platurus. Biochemistry, 14: 3408-3413, 1975.

25. MORI, N. & TU, A.T. Cross-linking of Acetylcholine Receptor subunits and its effects

on sea snake neurotoxin binding. Biochem. Archiv., 4: 85-89, 1988.

26. LIN, N. Q. & TU, A.T. (in press)

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Mem. Inst. Butantan

51(41:195 - 203, 1989.

CHROMATOGRAPHIC PURIFICATION OF ANTIVENOMS AND

ANTITOXINS +

Waldely de Oliveira DIAS*

Maria Izabel ESTEVES*

Joana Akiro FURUTA**

Hisako Gondo HIGASHI**

José Roberto MARCELINO**

Nilza Yurimi OISHI*

Sally Muller Alfonso PRADO**

Anita Mitico TANAKA*

Celina Maria Pompeo Mome UEDA*

Isaias RAW*

ABSTRACT: Commercial preparations of antivenoms and antitoxins

produced in horses, that are essentially pure F (ab)2 immunoglobulin

preparations, were submitted to ion-exchange chromatography. For anti-

crotalic, anti-bothropic and anti-tetanic sera it is possible to remove

40-60% of the inactive globulins.

KEY WORDS: Antivenoms; tetanus; antisera; antitoxins.

INTRODUCTION

Antivenoms and antitoxins used for the treatment of snakebite, arthro-

pods poisoning, diphteria or tetanus, are usually produced by immuniza-

tion of horses, followed by treatment of the hyperimmune plasma with crude

pepsin, and purification by heat denaturation and ammonium sulfate

precipitation 3 . Previous investigations from this laboratory have shown that

the product of crude pepsin digestion at pH 3.2 is a F (ab) 2 - like product

from immunoglobulins hydrolyzed by para-pepsins present in the crude pep¬

sin preparation, which is stable under the conditions used for heat

denaturation 8 .

# Centro de Biotecnologia

* * Seção de Concentração e Fracionamento de Soros

Instituto Butantan — C.P. 65 — 01051 — São Paulo-Brasil.

+ Supported by grant 4.84.236 from FINEP-PADCT.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 8/10/1988; accepted 3/8/1989.

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DIAS, W; de 0. et al. Chromatografic purification of antivenoms and antitoxins. Mem. Inst. Butantan,

51(41:195-203, 1989.

In the present paper we report further purification of the antisera by chro-

matography, that removes part of the inactive immunoglobulins.

MATERIALS AND METHODS

Hyperimmune plasma and serum: Hyperimmune plasma wõò prepared

by the immunization of horses with snake venoms, purified diphteria or teta-

nus toxins. They constitute the sera supplied by the Institute for human use

processed by the method of Harms. 3

Chromatographic resins: Cibracon blue agarose and Blue Sepharose were

obtained from Amicon, Pharmacia and Sigma Chemical Co, and S-Sepharose

from Pharmacia.

Electrophoresis: SDS-acrylamide gel electrophoresis was carried out in

7% acrylamide 2 . Electrophoresis on cellulose acetate was carried out by

the standard method used for human serum electrophoresis 4 .

Immunodifusion was carried out according to Ouchterlony 9 . Identifica¬

tion of the heavy and light y -immunoglobulin chains using immunodifu¬

sion was assayed by Prof. S. Kochwa at Mount Sinai School of Medicine.

Neutralizing activity : Antivenom activity was assayed by testing the ef-

fects of mixtures of a standard volume of the antiserum or one of its frac-

tions with different (or several) dilutions of the venom in pigeons 1 . Tetanus

and diphteria antitoxin activity was assayed in guinea-pigs by the methods

established by the US National Institutes of Health 6 ' 7 .

Protein determination: was estimated by the absorbance at 280 nm 5 .

RESULTS AND DISCUSSION

Chromatographic purification of hyperimmune anti-Crotalus venom plas¬

ma and serum

In attempting to purify ant\-Crotalus venom plasma and serum, affinity

chromatography was performed by using Cibracon blue-agarose, which is

known to bind albumin but not globulins 10 . When using "Matrex-blue", the

blue agarose produced by Amicon, two fractions were unexpectedly sepa-

rated from the globulin fraction of plasma (P) and of serum (S) (Figure IP

and IS) and only the second globulin fraction (B) had a venom neutralizing

activity. When Matrex-blue was replaced by blue-agarose or blue-Sepharose

from other sources, the separation of the globulins did not occur. Appar-

ently, the difference among those resins is due to the Cibracon-blue used

for their preparation, as this dye is not a chemically defined product.

On SDS-acrylamide gel electrophoresis the two serum globulin fractions

seem to be identical, presenting two broad bands with an average molecu¬

lar weight of 28.000-30.000 and 32.000-35.000 respectively.

Immunodifusion shows that the bands present in both fractions are heavy

and light y -globulin chains.

Electrophoresis on cellulose acetate showed that the two fractions have

different mobility (Figure 2) thus differing in their electrical charges. This

suggested that it would be possible to separate them by ion-exchange chro-

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51(41:195-203, 1989.

Fig. 1. Chromatography of plasma (P) or serum (S) on a Matrex-blue agarose column (2.5

x lOcm). 10 ml of horse hyperimmune plasma or serum containing 0.7 g and 0.32 g of pro-

tein respectively, were diluted to 30 ml with ImM potassium phosphate buffer pH 6.8. Plas¬

ma and serum globulins were eluted with the same phosphate buffer, and plasma albumin

with 0.15 M sodium chloride. A: globulin sub-fraction 1; B: globulin sub-fraction 2; C: albumin.

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DIAS, W; de 0. et al. Chromatografic purification of antivenoms and antitoxins. Mem. Inst. Butantan,

51(41:195-203, 1989.

matography. S-Sepharose, a cation exchanger, at pH 5.5, separated two

fractions (Figure 311), the first one (A) containing all the venom neutralizing

activity and the precipitating antibodies present in the whole serum, detec-

table by immunodifusion analysis (Figure 4).

By varying the pH for the S-Sepharose chromatography it was possible

to obtain more fractions (Figure 31, II, III). The yields and purifications of

these preparations are shown in table I.

TOTAL

Fig. 2. Cellulose acetate electrophoresis of the two serum globulin fractions obtained after

fractionation of anti -Crotalus venom serum by Matrex-blue agarose.

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51(41:195-203, 1989.

Fig. 3. Chromatogram on S-Sepharose column 2.2 x 10 cm, of 10 ml of anti-Crotalus se-

rum, containing 0.32 g of protein diluted to 30 ml with 0.01 M potassium phosphate buffer

at indicated pH, eluted with a gradient 0-0.15 M sodium chloride in the same buffer, for

4 hours, at a rate of 80 ml/hour.

' v<

Fig. 4. Immunodifusion analysis of fractions A and B obtained after chromatography of anti-

crotalic sera by S-sepharose chromatography and elution at pH 5.5 The wells contain ven-

om (V), anti-crotalic sera (S), and fractions A or B.

Purification of other antisera

Table I shows similar ion exchange chromatographies for anti -Bothrops

venom serum, and for tetanus and diphteria antitoxins (figures 5, 6, 7). It

is interesting to observe that a better purification, with a good yield, was

obtained for ar\l\-Crotalus venom serum at pH 6.0, for ani\-Bothrops serum

at pH 5.5 and for tetanus antitoxin at pH 4.0. The fractions obtained from

diphteria antitoxin had a lower specific activity than the whole serum, and

the activity was scattered into different fractions.

The treatment of snakebites requires the administration of 50 to lOOml

of antivenom. As shown in this paper, almost half of the protein administered

is inactive and it can be removed by a single column chromatography, al-

lowing not just to remove inactive foreign proteins, but to reduce the volume

of the antivenom administration.

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51(41:195-203, 1989.

It should be pointed out that it is possible to separate inactive from ac¬

tive v -globulin fractions by ion exchange chromatography. This process is

effective with anW-Crotalus and anti -Bothrops venom sera, that contain an-

tibodies against a large number of toxins. It is also effective for teta nus an-

titoxin, that is prepared with a highly purified tetanus toxin. However, it seems

to be ineffective for diphteria serum, also prepared with a purified toxin. While

for the first three it seems that the active antibodies are eluted together in

the first fractions, thus being more negative than the inactive fractions, this

does not occur with anti-diphteria sera.

Fig. 5. Chromatography of 10 ml of anti-Soíhrops venom serum containing 0.2 g of protein

under the same conditions as described in Figure 3.

Fig. 6. Chromatography of 10 ml of anti-tetanus toxin serum containing 0.60 g of protein

under the same conditions as described in Figure 3.

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51 (4): 195-203, 1989.

TABLE 1

PURIFICATION OF HYPERIMMUNE SERA BY ION-EXCHANGE CHROMATOGRAPHY

10 ml of hyperimmune sera were chromatographed on a 2.5 x 10 cm column of S-Sepharose

equilibrated with 0.01 M potassium phosphate-acetate buffer pH 6.0, 5.5 or 0.01 M potas-

sium phosphate-acetate buffer pH 4.0.. and eluted for 4 hours at a rate of 80 ml/hour, with

a gradient of 0-0.15M sodium chloride in the same buffer. The amount of protein added to

the column was 0.32 g for aná-Crotalus venom serum, 0.2 g for anti -Bothrops venom sera,

0.6 g for tetanus antitoxin and 0.68 g for diphteria antitoxin.

% recovery

ANTISERUN pH

fraction

of the neutralizing

activity

Crotalus venom —

initial

100

6.0

5.5

4.0

Bothrops venom

initial

100

6.0

5.5

100

4.0

Tetanus toxin

initial

8.1

100

6.0

11.7

12.9

10.8

5.5

15.0

4.0

9.0

28.0

Diphteria toxin

initial

17.5

100

6.0

9.6

16.7

10.3

5.5

9.5

11.9

3.8

4.0

6.8

8.0

4.4

* Fractions not tabulated had less that 30%

SA — specific activity is the venom neutralizing activity/A 280nm

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51(41:195-203, 1989.

RESUMO: Preparação comercial de antivenenos e antitoxinas produzidas

em cavalos, que são preparações puras de F (ab)2 de imunoglobulinas,

foram submetidas à cromatografia de troca iônica. Para os soros anticrotáli-

co, antibotrópico e antitetânico é possível remover 40-60% de globuli-

nas inativas.

UNITERMOS: antivenenos, tétano, anti-soro, antitoxinas.

REFERENCES

1. BRAZIL, V. Dosagem do valor antitóxico dos soros antipeconhentos. Rev. méd. São

Paulo, 70:457-462, 1907.

2. BREWER, J.M.; PESCE, A. J.; AMSWORTH, R. N. Experimental techniques in bio-

chemistry. New Jersey, Prentice Hall, 1974.

3. HARMS, A. J. Purification of antitoxic plasma by enzyme treatment and heat denatu-

ration. Biochem. J., 742:390-397, 1948.

4. HENRY, J.R.; CANNON, D. C.; WINKELMAN, J. W. Clinicai chemistry principies and

tecniques. New York, Harper & Row, 1974.

5. LAYNE, E. Spectrophotometric and turbidometric methods for protein determination.

In: COLLOWICK, S.P. KAPLAN, N.O. Methods in Enzymology. New York, Academic

Press, 1957 v.3 p. 447-454.

6. NATIONAL INSTITUTE OF HEALTH. Minimum requirements: diphteria antitoxin. 2nd

revision. Washington, US Department of Health, Education and Welfare, 1946.

7. NATIONAL IINSTITUTE OF HEALTH. Minimum requirements: tetanus toxin. 4th revi¬

sion. Washington, US Department of Health, Education and Welfare, 1952.

8. OISHI, N.; UEDA, C. M. P. M.; DIAS; W. O.; LATRILLA; C. M. L. ; RAW, I. The possi-

ble role of para-pepsin in the preparation of anti-venom sera. Mem. Inst. Butantan,

49: 41-45, 1987.

9. OUCHTERLONY, 0. Difusion- in gel methods for immunological analysis. Progress in

Immunology, 5: 1-78, 1958.

10. TRAVIS, J.; PANNEL, R. Selective removal of albumin from plasma by affinity chro-

matography. Clin. Chim. Acta., 49: 49-52, 1973.

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Mem. Inst. Butantan

51 141:205 - 210, 1989.

SARAFOTOXINS, A NEW GROUP OF CARDIOVASCULAR

MODULATORS FROM SNAKE VENOM

Elazar KOCHVA*

Avner BDOLAH

Dan GRAUR

Zvi WOLLBERG

ABSTRACT: A new group of toxins from the venom of the snake Atrac-

taspis, the sarafotoxins, are highly homologous with the endothelins that

originate from the endothelium of mammalian blood vessels. Both groups

of compounds are 21 amino acid peptides that affect the cardiovascular

system and also bind to various regions of the brain. The sarafotoxins may

have originated from endogenous modulators of the cardiovascular system

that evolved into toxins in the venom glands of Atractaspis.

KEY-WORDS: Sarafotoxins, Endothelins, Atractaspis, Snake Venom.

Several components of snake venoms served as tools for the elucida-

tion of natural processes in various tissues. The best known components

are the postsynaptic neurotoxins from Elapidae venoms — the alpha-

bungarotoxins and alpha-cobrotoxins. These toxins bind strongly to the

acetylcholine receptors of the neuromuscular junction of striated muscles

and were used in order to identify, isolate and characterize these receptors

(Albuquerque et a/. 1 ).

During the last year (1988), several isotoxins from the venom of the

Burrowing Asps genus Atractaspis, were shown to mimic natural products

from mammalian blood vessels, the endothelins (Bdolah ef a/., 4 - 5 Wollberg

et a/., 151

The Burrowing Asps are now considered to belong to a separate fami-

ly, the Atractaspididae (Fig. 1), that differ considerably from the other veno-

Incumbent: The Rose and Norman Lederer Chair in Experimental Biology.

Department of Zoology. George S. Wise Faculty of Life Sciences.

Tel-Aviv University. Tel-Aviv, Israel.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 7/6/1989: accepted 14/8/1989.

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KOCHVA, E.; BDOLAH, AGRAUR, D; WOLLBERG, Z. Sarafotoxins, a new group of Cardiovascular

modulators from snake venom. Mem. Inst. Butantan, 51(41:205-210, 1989.

mous and non-venomous snakes in many respects (Underwood and

Kochva, 14 ). The venom of Atractaspis has a characteristic composition and

contains both high and low molecular weight components. Some of these,

hemorrhagin, protease and phospholipase A 2 are found also in the venoms

of other snakes (Ovadia, 12 ). However, about 1 13 of the venom consists of

a new type of highly lethal low molecular weight toxins (Fig. 2; Kochva et

a/., 11 ). These toxins were first labeled S 5 and S 6 , in order of their elution

from a G-50 Sephadex column; they showed one and two bands, respec-

tively, in acrylamide gel electrophoresis. A preliminary sequence of S 5 was

later identified as sarafotoxin c, while Sg was found to contain mainly

sarafotoxins a and b. These three toxins were named after the common

Flebrew name of the Israel Burrowing Asp, SARAF Ein-Gedi and were shown

to be highly homologous isotoxins that contain 21 amino acid residues with

two disulphide bridges (Fig. 3; Takasaki et a/., 13 ). A search of the several

protein and nucleic acid sequence data banks yielded no meaningful similar-

ities with any of the published sequences. Flowever, the mammalian en-

dothelins, that are also composed of 21 amino acids, are highly homologous

with the venom sarafotoxins (Yanagisawa et at., v , Graur et al. 6 ).

Fig. 1. Atractaspis engaddensis, Flaas 1950.

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KOCHVA, E.; BDOLAH, A.; GRAUR, D; WOLLBERG, Z. Sarafotoxins, a new group of Cardiovascular

moduíators from snake venom. Mem. Inst. Butantan, 51 (4):205-210, 1989.

T u b e no.

Fig. 2. Sephadex G-50 profile of Atractaspis engaddensis venom. Ten to 30 mg lyophilised

venom were dissolved in 1 ml of 0.05 M NH 4 C0 3 , applied onto a 0.9 x 152 cm

column, and eluted with the same solution at a rate of 2ml/hr, in 1 ml fractions. S,

contains L-amino acid oxidase and hemorrhagin; S 2 contains protease(s); S 3 con-

tains phospholipase A 2 ; S 5 contains mainly SRTX-c; S 6 contains SRTX-b and

SRTX-a.

SARAFOTOXIN-ENDOTHELIN SEOUENCES

Cys-Ser-Cy3-Lys-Asp-Met-Thr-A3p-Lys-Glu-Cys-Leu-A3n-Phe-Cvs-Hi3-Gln-Asp-Val-Ile-Trp SRTX-a

Cys-Ser-Cys-Lys-Asp-Met-Thr-Asp-Lys-Glu-Cys-Leu-Tyr-Phe-Cvs-HiB-Gln-Asp-Val-Ile-Trp SRTX-t

Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Prie-Cys-Hia-Gln-Asp-Val-Ile-Trp SRTX-c

Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-Tyr-Cvs- His-Leu-Asp-Ile-Ile-Trp ET-3

Cys-Ser-Cya-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp ET-1

Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cvs-His-Leu-Asp-Ile-Ile-Trp ET-2

15 10 15 20

Fig. 3.Amino acid sequences of sarafotoxins and endothelins (Takasaki et. al. 13 1988; Yanagisawa

et al. 1 7 , 1988).

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KOCHVA, E.; BDOLAH, A.; GRAUR, D; WOLLBERG, Z. Sarafotoxins, a new group of Cardiovascular

modulators from snake venom. Mem. Inst. Butantan, 51141:205-210, 1989.

To date, three endothelins were identified ín the human gene, ET-1,

ET-2 and ET-3 (Hiley. 7 ). ET-1 was the first to be isolated from porcine en-

dothelium and was subsequently identified in human material; ET-3 was

found in the rat. The most remote members of each group, SRTX-a and

c, on the one hand, and ET-3 on the other, still share between them 11

amino acids (52%).

The sarafotoxins and endothelins are also similar in their pharmacolo-

gy and only a few differences were found between them (Wollberg et

al., 15 ). Their major function, known so far, is linked with the cardiovascu¬

lar system through their powerful vasoconstrictor effects on many blood

vessels. In addition, both the sarafotoxins and endothelins induce a posi¬

tive inotropic effect in human and animal heart muscles. When injected in

vivo, the SRTXs and ETs show a series of disturbances in the ECG that con-

sist of a remarkable but transient slope elevation of the S-T segment and

an increase in the amplitude of the R-wave (Fig. 4). At the same time, a

severe A-V block develops, starting with the prolongation of the P-R inter-

val (first degree), followed by "dropped beats'' (second degree) and by

complete atrioventricular dissociation (third degree) that leads to cardiac

arrest. These symptoms are characteristic of severe coronary insufficien-

cy, which most certainly plays a crucial role in the toxicity of the SRTXs.

However, experiments with isolated heart preparations show that these tox-

ins may also directly affect the conduction system of the heart and thus

contribute for their toxicity (Wollberg et al . 16 ).

When injected i.v. into mice, both SRTX-b and ET-1 are highly toxic

with an approximate LD 50 of 0.01 5^ g/g (Bdolah et al. 4 ).

In addition, both SRTXs and ETs bind to and compete for the same recep-

tors and induce phosphoinositide hydrolysis in the heart and brain (Ambar

et al., 2 ; Ambar et al., 3 ; Kloog et aí. 8 ; Kloog et al. 9 ).

The structural and functional similarities between SRTXs and ETs have

elicited extensive experimental work of a comparative nature, with the SRTXs

being used as probes for the elucidation of the role of ETs in the regulation

of blood pressure and other physiological and pharmacological phenome-

na of the cardiovascular system. Only time will tell whether the SRTXs will

achieve the levei of scientific importance of their elapid counterparts, the

postsynaptic neurotoxins.

The high levei of homology between the SRTXs and ETs suggests a

common phylogenetic origin for the endothelín/sarafotoxin family of pep-

tides (Fig. 5). Although endothelins are yet to be identified in non-mammalian

vertebrates, they are most probably present in snakes. From the evolution-

ary point of view, it appears that a product already found in other tissues

has evolved and adapted to a new function in the venom glands to help

in the hunting for food. A similar phenomenon was recognised in other ven¬

om components, where enzymes, such as proteases and phospholipase

A 2 , evolved into toxic hemorrhagins and presynaptic neurotoxins, respec-

tively (Kochva, 10 ).

208

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KOCHVA, E.; BDOLAH, A.; GRAUR, D, WOLLBERG, Z. Sarafotoxins, a new group of Cardiovascular

modulators from snake venom. Mem. Inst. Butantan, 51(41:205-210, 1989.

Note added in proof: Two additional members of the endothelin/sara-

fotoxin family were recently described (see Bdolah, E.; Wollberg, Z;

Fleminger, GKochva, E. SRTX-d, a new native peptide of the endo-

thelin/sarafotoxin family. FEBS Lett, 256:1-3, 1989.

RESUMO: Um novo grupo de toxinas identificadas no veneno da serpen¬

te Atractaspis, as sarafotoxinas, tem alta homologia com as endotelinas,

originárias do endotélio dos vasos sanguíneos de mamíferos. Os dois gru¬

pos de compostos são peptídeos, contendo 21 aminoácidos, que afe¬

tam o sistema cardiovascular e se ligam a várias regiões do cérebro. As

sarafotoxinas podem ter-se originado de moduladores endógenos do sis¬

tema cardiovascular, que se transformaram em toxinas nas glândulas ve-

neníferas da Atractaspis.

UNITERMOS: Sarafotoxinas, Endotelinas, Serpente Atractaspis-veneno.

REFERENCES

1. ALBUQUERQUE, E.X.; ELDEFRAWI, A.T.; ELDEFRAWI, M.E. The use of snake toxins

for the study of the acetylcholine receptor and its ion-conducting modulator. In: LEE,

C.Y. ed. Snake Venoms, Berlin, Springer Verlag, 1979. p. 377-402.

2. AMBAR, I.; KLOOG, Y; KOCHVA, E.; WOLLBERG, Z.; BDOLAH, A.; ORON, U.; SOKO¬

LOVSKY, M. Characterization and localization of a novel neuroreceptor for the pepti¬

de sarafotoxin. Biochem. Biophys. Res. Comm., 157: 1104-1110, 1988.

3. AMBAR, I.; KLOOG, Y; SCHVARTZ, L; HAZUM, E.; SOKOLOVSKY, M. Competitive inhi-

bition between endothelin and sarafotoxin; binding and phosphoinositide hydrolysis

in rat atria and brain. Biochem. Biophys. Res. Comm., 158: 195-201, 1989.

4. BDOLAH, A.; WOLLBERG, Z.; AMBAR, I.; KLOOG, Y.; SOKOLOVSKY, M.; KOCHVA, E.

Disturbances in the cardiovascular system caused by endothelin and sarafotoxin. Bio¬

chem. Pharmacol., 38: 3145-3146, 1989.

5. BDOLAH, A.; WOLLBERG, Z.; KOCHVA, E. Sarafotoxins: a new group of cardiotoxic

peptides from the venom of Atractaspis, in snake venoms. A.L. Harvey, Ed. Encyclo-

pedia of Pharmacology and Therapeutics, Pergamon Press, 1989, (in press).

6. GRAUR, D.; BDOLAH, A.; WOLLBERG, Z.; KOCHVA, E. Homology between snake ve¬

nom sarafotoxins and mammalian endothelins. Israel J. Zool., 35: 171-175, 1988/9.

7. HILEY, C.R. Functional studies on endothelin catch up with molecular biology. Trends

Pharmacol. Sei., 10: 47-59, 1989.

8. KLOOG, Y.; AMBAR, I.; SOKOLOVSKY, M.; KOCHVA, E.; WOLLBERG, Z.; BDOLAH, A.

Sarafotoxin, a novel vasoconstrictor peptide: Phosphoinositide hydrolysis in rat heart

and brain. Science, 242: 268-270, 1988

9. KLOOG, Y; AMBAR, I.; KOCHVA, E.; WOLLBERG, Z.; BDOLAH, A.; SOKOLOVSKY, M.

Sarafotoxin receptors mediate phosphoinositide hydrolysis in various rat brain regions.

FEBS Lett., 242: 387-390, 1989.

10. KOCHVA, E. The origín of snakes and evolution of the venom apparatus. Toxicon, 25:

65-106, 1987.

11. KOCHVA, E.; VILJOEN, C.C.; BOTES, D.P. A new type of toxin in the venom of snakes

of the genus Atractaspis (Atractaspidinae). Toxicon, 20: 581-592, 1982.

12. OVADIA, M. Isolation and characterization of a hemorrhagic factor from the venom of

the snake Atractaspis engaddensis (Atractaspididae). Toxicon, 25: 621-630, 1987.

13. TAKASAKI, C.; TAMIYA, N.; BDOLAH, A.; WOLLBERG, Z.; KOCHVA, E. Sarafotoxins S 6 :

several isotoxins from Atractaspis venom that affect the heart. Toxicon, 26: 543-548,

1988.

14. UNDERWOOD, G. & KOCHVA, E. On the affinities of the Burrowing Asps (Serpentes,

Atractaspis), 1989 (submitted).

15. WOLLBERG, Z.; BDOLAH, A.; KOCHVA, E. Vasoconstrictor effects of sarafotoxins in

rabbit aorta: Structure-function relatioships. Biochem. Biophys. Res. Comm., 162:

371-376, 1989

16. WOLLBERG, Z.; SHABO-SHINA, R.; INTRATOR, N.; BDOLAH, A.; KOCHVA, E.; SHA-

VIT, G.; ORON, Y; VIDNE, B. A.; GITTER, S. A novel cardiotoxic polypeptide from

Atractaspis engaddensis venom: Cardiac effects in mice and isolated rat and human

heart preparations. Toxicon, 26: 525-534, 1988.

17. YANAGISAWA, M.; KURIHARA, H.; KIMURA, S.;TOMOBE, Y;KOBAYASHI, M.; MITSUI,

Y; YAZAKI, Y; GOTO, K.; MASAKI, T. A novel potent vasoconstrictor peptide produ-

ced by vascular endothelial celis. Nature, 332: 411-415, 1988.

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Mem. Inst. Butantan

51(41:211-223, 1989.

LOCAL TISSUE DAMAGE INDUCED BY BOTHROPS

SNAKE VENOMS. A REVIEW

José Maria GUTIERREZ

Bruno LOMONTE

ABSTRACT: This review focuses on the local effects induced by Bothrops

venoms. These envenomations are characterized by myonecrosis,

hemorrhage and edema which develop rapidly after venom inoculation.

Myonecrosis is caused (a) directly, due to the direct action of myotoxins

on the plasma membrane of muscle cells, and (b) indirectly, through the

development of ischemia resultam from drastic alterations induced by these

venoms on both microvasculature and intramuscular arteries. Regarding

myotoxins, there is a group of closely related muscle damaging

components that can be classified as "myotoxins with phospholipase A 2

structure", although some of them lack this enzymatic effect. These

myotoxins have been purified from the venoms of Bothrops asper, B.

jararacussu, and. B. nummifer. Local hemorrhage in bothropic

envenomations results from the action of acidic metalloproteins of relatively

high molecular weight which act directly on the capillary vessels inducing

extravasation. Five hemorrhagic toxins have been isolated an characterized

from the venoms of B. jararaca and B. neuwiedi. Local edema is also a

typical feature after injection of Bothrops venoms. This effect is probably

due to: (a) Direct action of venom components on the microvasculature,

increasing the permeability of capillaries and venules, and (b) the effect

of endogenous mediators released by venom components. Among these

mediators are histamine, prostaglandins, kinins, C3a and C5a. Besides

edema, inoculation of Bothrops venoms elicits a prominent cellular

inflammatory infiltrate. The need of a comprehensive approach in the study

of snake venom-induced local tissue damage is stressed.

KEY WORDS: Bothrops venoms, myonecrosis, myotoxins, hemorrhage,

edema.

Instituto Clodomiro Picado. Facultad de Microbiologia. Universidad de Costa Rica, San José, Costa Rica.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 4/4/1989. accepted 04/9/1989.

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INTRODUCTION

The large majority of snakebites in Latin America are inflicted by spe-

cies classified in the genus Bothrops 5 ' 8 ' 65 . Despite the existence of evident

intraspecific and interspecific variations in the composition and pharmaco-

logical activities of their venoms 3.15,30 they jnduce a qualitatively similar

pathophysiological picture, characterized by: (a) Immediate and prominent

local tissue damage, i.e. myonecrosis, hemorrhage and edema 17 ' 65 ; (b)

cardiovascular alterations, especially hemorrhage, and hypovolemic

shock 2 ' 65 ; (c) coagulation disorders, most frequently defibrination 2,5,65- anc j

(d) renal alterations which might evolve into acute renal failure 1 ' 2 - 69 .

If antivenom administration is initiated rapidly after envenomation, neu-

tralization of systemic effects is usually achieved successfully, but neutrali-

zation of local tissue damage is a more difficult task. In a number of snakebite

cases, lack of neutralization of local effects results in permanent sequelae,

i.e. tissue loss. Due to the relevance of local effects in envenomations in¬

duced by snakes of the genus Bothrops, several research groups have studied

this problem from different perspectives. In this work we will review their

findings, and some conclusions regarding the pathogenesis of local tissue

damage by these venoms will be presented.

LOCAL MYONECROSIS

Myonecrosis is a common consequence of envenomations by species

of Bothrops 2,5,56,65 Experimental studies have observed this activity in the

venoms of B. asper, B. nasuta, B. nummifer, B. godmani, B. lateralis, B.

ophryomegas B. picadoi and B. schlegelii from Costa Rica 14,16,28,44,68, anc j

of B. jararaca, B. neuwiedi 44 , B. jararacussu 58 and B. alternatus 57 from

Brazil.

The development of muscle damage has been studied with the venoms

of B. asper 16 - 19 , B. jararacussu 58 and B. alternatus 57 .

In the first two cases myonecrosis was evident from the beginning, and

necrotic fibers were characterized by the presence of amorphous clumped

masses of myofibrils alternating with empty spaces. In the case of B. alter¬

natus venom, necrotic fibers presented a "waxy hyaline appearance".

Homma and Tu 28 proposed a classification of venom-induced myo¬

necrosis based on the morphology of the necrotic fibers 24 hr after injec-

tion of the venoms. "Myolytic" type of necrosis was characterized by fibers

where myofibrillar material appeared clumped and alternating with empty

spaces in the cytoplasm. ''Coagulative'' type of necrosis, on the other hand,

was typified by cells whose myofibrillar material had a more hyaline appear¬

ance and a homogeneous distribution. According to these authors, the ven¬

oms of B. atrox and B. schlegelii induced a "myolytic" necrosis, whereas

those of B. nasuta and B. picadoi induced a "coagulative" myonecrosis;

the venom of B. nummifer caused a "mixed" myonecrosis, i.e. presenting

both myolytic and coagulative necrotic fibers. In 1980, Gutiérrez and

Chaves 14 used these criteria to characterize myonecrosis induced by several

Costa Rican crotaline venoms.

As further studies clearly demonstrated, this classification is an over-

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simplification of a complex and dynamic pathologic phenomenon. For in-

stance, when muscle tissue was taken at various time intervals after i.m.

injection of B. asper venom, it was observed that the morphology of necrotic

fibers changed 19 . In the first 3 hr there was a predominance of necrotic

fibers with "clumped" myofibrillar material alternating with cytoplasmic

spaces devoid of myofilaments ("myolytic fibers"). However, after the sixth

hour, the large majority of necrotic fibers contained a more hyaline cytoplasm,

with myofibrillar material distributed in a more uniform fashion in the cellu-

lar space 19 . Thus, the morphological type of necrosis is different depend-

ing upon the time of tissue sampling. Similar findings have been made

recently with the venoms of Naja naja, Crotalus virídis and Crotalus atrox 51 .

Despite the lack of validity of this "myolytic" vs "coagulative" classifi-

cation of myonecrosis, experimental work performed with the venoms of

B. jararacussu 58 , B. asper 19 and B. alternatus 57 strongly suggested that

there are, at least, two different ways by which Bothrops venoms affect mus¬

cle cells: (a) directly, by the action of "myotoxins" which probably affect

the integrity of skeletal muscle plasma membrane 19 ' 57 ; (b) indirectly,

through an ischemic condition that develops in skeletal muscle secondarily

to the disruptive action of venoms on the vasculature 19 - 57 . Morphologically,

cells affected by myotoxins initially presented clumping of myofibrils and

subsequently became hyaline in appearance 19 ' 27 ' 58 . In contrast, other

necrotic cells, probably affected by ischemia, presented a hyaline morphol¬

ogy from the beginning 19 ' 57 . Similar observations were made by Ownby and

Colberg 51 , with the venoms of Crotalus atrox and Crotalus viridis, describ-

ing these cells as "moth-eaten" on the basis of their morphology in sec-

tions made from plastic-embedded tissue.

In the past, the study of myonecrosis induced by snake venoms was

limited by the lack of reliable quantitative assays to estimate the extent of

muscle damage. Moreover, the fact that only histology allowed these studies

precluded many laboratories to isolate and characterize myotoxins. In re-

cent years, several techniques have been used in the quantitation of venom-

induced myonecrosis, such as: (a) quantitation of serum leveis of the en-

zyme creatine kinase and, more specifically, of the isozyme CK-

MM 10,16,18,44,47,54- (j-,) quantitation of the release of creatine kinase in vitro

from preparations of skeletal muscle incubated with venoms 23 ' 45 ; (c) quan¬

titation of the residual content of creatine kinase in muscle injected with

venoms or toxins 26 ; and (d) quantitative histological estimation of muscle

damage, by counting the number of necrotic and surviving cells 33 - 65 .

MUSCLE DAMAGE DUE TO THE DIRECT ACTION OF MYOTOXINS

Five myotoxins have been purified to homogeneity from Bothrops ven¬

oms: B. asper myotoxin I 18 , B. asper myotoxin II 35 , B. nummifer myotox-

in 22 , and two myotoxins from the venom of B. jararacussu, one of which

was named "bothropstoxin" 29 . They had molecular weights of

13,000-16,000 and basic isoelectric points, showing similarities in their amino

acid composition. When analyzed by polyacrylamide gel electrophoresis un-

der reducing and non-reducing conditions, it was observed that some of

them were dimers whereas others were predominantly monomers 22 - 35 .

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A puzzling finding regarding these myotoxins has to do with their en-

zymatic activity. Two of them (B. asper myotoxin I and one of B. jararacussu

myotoxins) showed phospholipase A 2 activity, whereas the rest of them

lacked this effect. 18 , 22 , 29,35

However, all of these myotoxins had biochemical characteristics very

similar to those of phospholipases A 2 . For instance, B. nummifer myotoxin

and B. asper myotoxin II cross-reacted immunologically with both polyclonal

and monoclonal antibodies raised against B. asper myotoxin I, a phospholi¬

pase A 2 22 - 35 ' 36 ' 39 . Moreover, the myotoxin from B. jararacussu, lacking this

enzymatic activity, showed conspicuous homology in the amino acid se-

quence of the amino terminal region with toxic phospholipases A 2 from

crotaline and elapid venoms 29 . Thus, all myotoxins isolated from Bothrops

venoms may be tentatively classified in the group of "toxins with phospholi¬

pase A 2 structure" 31 on the basis of their evident biochemical similarities

with toxic phospholipases.

Immunochemical data evidenced the presence of components that cross-

react with B. asper myotoxin I in the venoms of B. atrox 18 , B. schlegelli 37 ' 39

B. godmani, B. nummifer, B. picadoi and Agkistrodon bilineatus 39 .

Moreover, recent unpublished results obtained at the Instituto Clodomiro

Picado indicated that there are cross-reacting proteins in the venoms of B.

jararacussu, B. neuwiedi, B. jararaca, B. moojeni, B. colombiensis, B. pic-

tus, B. bilineatus, B. xanthograma and B. brazili. Therefore, there seems to

exist a "family" of closely related components in a variety of Bothrops ven¬

oms having a leading role in the development of muscle tissue damage in

these envenomations.

Upon intramuscular injection in mice these myotoxins induced rapid de-

generative changes in muscle cells leading to necrosis 18 , 19 , 27 , 29,35 | n the

case of B. asper and B. nummifer myotoxins, the cells underwent early

changes characterized by the presence of ''delta lesions", followed by clump-

ing of myofibrillar material into amorphous, dense masses which alternat-

ed with spaces in the cytoplasm devoid of myofilaments 18 , 19.27 Afterwards,

myofilaments redistributed in the cellular space and necrotic cells became

more hyaline and homogeneous in appearance. An inflammatory infiltrate

was observed after the sixth hour, reaching maximum leveis by 48-72

hr 19.27,29 interestingly, after removal of necrotic debris by phagocytes, there

was a normal and successful muscle regenerative process, with the forma-

tion of myotubes and regenerative muscle fibers 2 °.29. The only morpho-

logical feature that distinguished these regenerative muscle cells from adult

normal muscle cells was the presence of centrally-located nuclei 20 ' 29 .

Otherwise, there was no fibrosis nor proliferation of adipose cells substitu-

ting necrotic muscle fibers 20 ' 29 . This pattern of regeneration contrasted

with the one observed after myonecrosis induced by crude venoms which,

by affecting muscle microvasculature, severely hampered the regenerative

response 20 , 21,58

The mechanism of action has been studied in the cases of B. asper my¬

otoxin I and B. nummifer myotoxin. The following experimental findings

strongly indicate that the first site of action of these toxins is the skeletal

muscle plasma membrane: (a) The first morphological lesions detected were

focal, wedgeshaped areas of degeneration in the periphery of muscle

fibers 18,19,27, ver y similar to the "delta lesions" described in biopsy material

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Mem. Inst. Butantan, 51(41:211-223, 1989.

from patients with Duchenne muscular dystrophy 46 . Ultrastructural obser-

vations corroborated the fact that the plasma membrane was discontinu-

ous or absent in these early lesions, 19 (b) Both in vivo and in vitro there was

a rapid and drastic release of intracellular markers, such as creatine kinase

and creatine, after addition of the toxins 18,22,23,27, concomitantly with

a rapid influx of calcium 18 ’ 27 (c) Immunohistochemical findings corrobo¬

rated the binding of B. asper myotoxin I to skeletal muscle plasma

membrane 7 (d) B. nummifer myotoxin disrupted multilamellar liposomes 27 .

Interestingly, although B. asper myotoxin I exerted its phospholipolytic

activity in muscle tissue 18 , it induced the release of creatine kinase and

creatine in vitro in conditions where phospholipase A 2 activity was inhibit-

ed, i.e. when calcium was eliminated and EDTA added to the bathing

solution 23 . Similar observations were made with a myotoxic phospholipase

fraction from the venom of B. jararacussu 62 . These findings pointed

towards a dissociation between enzymatic and toxic activities in these tox¬

ins, as has been shown for other toxic phospholipases A 2 9 ' 64 . Kini and

Iwanaga 32 studied the amino acid sequences of several myotoxic phospholi¬

pases and concluded that a variety of myotoxins possess characteristic ca-

tionic and hydrophobic domains which are different from the catalytic site.

Structural studies suggested that the "myotoxic segment" of these

molecules is amphiphilic in nature and readily availabe for interaction with

membranes 32 . In this regard, it might be relevant the observation that B.

nummifer myotoxin behaves as an amphiphilic protein when tested by

charge-shift electrophoresis 27 .

In view of these findings, it is tempting to propose that the myotoxins

isolated from Bothrops venoms form a group with strong homologies. If

indeed myotoxicity depends on a molecular region different from the ca¬

talytic site, then the preservation of the catalytic domain is irrelevant from

the point of view of myotoxicity. This hypothesis requires support from com-

parative studies on the amino acid sequences of these "myotoxins with

phospholipase A 2 structure".

If enzymatic degradation of membrane phospholipids is not the basis

of the myotoxic activity of these toxins, an alternative mechanism has to

be proposed. On the basis of the findings discussed above, it is possible

that they exert their myotoxicity by an interaction of the amphiphilic por-

tion of the molecule with the phospholipid bilayer, inducing membrane lea-

kiness that eventually would lead to irreversible muscle cell injury. The nature

of the binding site, as well as the mechanism of membrane disorganiza-

tion, remain unknown.

How important are these "myotoxins with phospholipase A 2 structu¬

re" in the total myonecrosis induced by crude Bothrops venoms? In the

case of B. asper venom, where four myotoxin variants have been

described 36 , Lomonte eia/. 37 ' 38 observed that incubation of crude venom

with horse and rabbit polyclonal antibodies neutralized approximately 75%

of the myotoxic effect induced by the venom, a demonstration that this group

of toxins are responsible for the major part of muscle damage induced by

B. asper venom. This conclusion was supported by recent unpublished da¬

ta in which a monoclonal antibody against myotoxin was able to fully neu¬

tralize myotoxicity induced by this venom.

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Mem. Inst. Butantan, 51(41:211-223, 1989.

MUSCLE DAMAGE DUE TO ISCHEMIA

Besides myotoxicity caused by the direct action of tdxins on muscle

cells, there is also muscle damage due to ischemia in bothropic

envenomations 19 ' 57 . Although there are no studies describing biochemi-

cal evidences of ischemia in muscle affected by venoms, it is likely that

there is ischemia due to (a) drastic damage to the microvasculature lea-

ding to hemorrhage, and (b) alterations in larger vessels, particularly in in¬

tramuscular arteries.

Damage to the capillary network is due to the action of hemorrhagic

components (see below). As a consequence of their action, capillaries col-

lapse and blood leaks to the extravascular compartment 48 . This phenome-

non causes an immediate effect on the blood supply to muscle cells, inducing

ischemia. In this regard, it is relevant to point out that experimental inocu-

lations of hemorrhagic components from the venom of B. jararaca resulted

in immediate hemorrhage, followed by widespread muscle damage 59 .

Queiroz et al , 59 suggested that damage to muscle fibers after injection of

hemorrhagic toxins might be a consequence of the ischemia which deve-

loped after the drastic hemorrhagic effect. However, it is also possible that

some hemorrhagic components exerted a direct myotoxic activity, as has

been proposed for viriditoxin and hemorrhagic toxin b, from the venoms

of Crotalus wr/cf/s 10 - 13 and Crotalus atrox 53 respectively. Studies on the ac¬

tion of hemorrhagic toxins on skeletal muscle in vitro are required in order

to discriminate between direct myotoxicity and myotoxicity due to ische¬

mia. Evidently, there is an urgent need of pathological studies with purified

hemorrhagic components from Bothrops venoms.

Ischemic muscle damage can also occur as a consequence of altera¬

tions in intramuscular arteries. Homma and Tu 28 described the occurren-

ce of arterial lesions in mice injected intramuscularly with the venoms of

B. atrox, B. nasuta, B. nummifer, B. picadoi and B. schlegelii. These le¬

sions included disintegration of endothelial cells, leucocytic infiltration be-

neath the endothelium, necrosis and disappearance of the smooth muscle

fibers in the media, hemorrhage and insudation of a fibrin-like substance

into the subendothelial and medial layers, and formation of mural thrombi.

Queiroz and Petta 57 described hyaline necrosis of the media in arteries of

muscle tissue obtained from mice injected with B. alternatus venom. Mo-

reover, inoculation of high doses of B. jararacussu venom induced hyalini-

zation of the media of some arteries, as well as loss of endothelium 58 . Little

effort has been made on the isolation and characterization of factors affec-

ting large vessels. However, it is interesting that the hemorrhagic compo-

nent HF 2 of B. jararaca venom induced necrosis of intramuscular arteries

59 . Arterial necrosis and thrombosis may have dramatic effects in the blood

supply to muscle tissue, originating ischemia in groups of muscle cells. This,

in turn, might induce irreversible cell damage, also affecting the process

of muscle regeneration 57 .

An additional element which may impair the blood supply to muscle

tissue is the elevation in the interstitial pressure of some muscle compart-

ments, due to the accumulation of large volumes of fluid after the action

of hemorrhagic and edemaforming toxins on the microvasculature. Although

this phenomenon has received little attention at the experimental levei, so-

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Mem. Inst. Butantan, 51(41:211-223, 1989.-

me studies suggested that it may be a relevant component in the pathoge-

nesis of local tissue damage in crotaline envenomations 12 .

HEMORRHAGE INDUCED BY BOTHROPS VENOMS

Hemorrhage is one of the most characteristic effects induced by crota¬

line venoms (see reviews by Ohsaka 48 and Ownby 50 ). The pioneer work

made by Ohsaka and his group on the nature and mechanism of action of

hemorrhagic components purified from the venom of Trimeresurus flavo-

viriridis, a close relative of Bothrops 6 , led to the proposal that these toxins

induce hemorrhage by "diapedesis", i.e. by opening the intercellular junc-

tions between endothelial cells in capillaries, with the consequent

extravasation 48 . In contrast, Ownby et a/. 52 ' 53 presented strong evidence

indicating that several hemorrhagic toxins from the venoms of Crotalus atrox

and Crotalus horridus induce hemorrhage "per rhexis", i.e. by disrupting

the integrity of endothelial cells, escaping the erythrocytes through gaps

formed within these cells.

Many hemorrhagic components isolated from crotaline venoms are

proteases 67 , and this enzymatic activity may be relevant to the process of

capillary damage. Several hemorrhagic toxins degrade collagen, and it is

known that collagen type IV is a component of the basal lamina that sur-

rounds endothelial cells in the capillaries 66 Ohsaka et a/. 49 demonstrated

that several hemorrhagic components from Trimeresurus flavoviridis venom

released proteins and carbohydrates from isolated glomerular basement

membrane. Thus, it might be that hemorrhagic toxins induce damage to

the microvasculature by first affecting the integrity of the basal lamina, in-

ducing the collapse of the capillary structure. However, a direct cytotoxic

effect on endothelial cells cannot be ruled out at the present time. Further

work is required to elucidate the mechanism of action of hemorrhagic toxins.

Five hemorrhagic toxins have been isolated and characterized from Both¬

rops venoms: HF 1( HF 2 and HF 3 from B. jararaca^' 43 and NFHF a and NHF b

from the venom of B. neuwiedi 42 ' 43 . These five toxins have similar mole¬

cular weights (46,000-62,000), being all of them proteins with acidic pl.

They were described as heat labile metalloproteins, inhibited by EDTA, EGTA

and 1.10 phenanthroline 41 ' 4 2,43 interestingly, many hemorrhagic compo¬

nents isolated from snake venoms were Zn 2+ — containing proteases

which lost their activity upon Zn 2+ removal 4 - 67 .

The hemorrhagic toxins isolated from Bothrops venoms displayed pro-

teolytic activity on casein, with the exception of HF 3 from B. jararaca 43 .

However, all of them hydrolyzed fibrinogen and the B-chain of oxidized

insulin 43 . In this regard, there has been a controversy concerning the pro-

teolytic activity of hemorrhagic toxins 4 . Since casein is not the best subs-

trate for this enzyme, it is necessary to test this activity on other substrates

such as dimethylcasein and collagen. It is likely that the large majority of

hemorrhagic toxins are proteases with limited substrate specificity.

Besides these five bothropic hemorrhagic components, two additional

proteases, bothropasin from B. jararaca venom and moojeni protease A from

B. moojeni venom, induced hemorrhage, although at much higher

doses 40 ' 59 . These enzymes were highly active in terms of proteolysis, ha-

ving a low hemorrhagic effect. Immunologically, these two proteases clearly

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Mem. Inst. Butantan, 51(41:211-223, 1989.

differed from hemorrhagic components 40 . On the other hand, polyclonal

antibodies raised against hemorrhagic factors reacted with several Both¬

rops venoms, indicating that immunologically-related proteins are present

in a variety of these venoms.

EDEMA AND INFLAMMATION

Bothropic envenomations are also characterized by the rapid develop-

ment of edema and inflammation at the site of venom injection. 2 ' 5 ' 65 .

Despite its clinicai relevance, the study of venom-induced edema has received

little attention, as demonstrated by the scarcity of reports on the isolation

and characterization of edema-forming toxins.

When tested on the foot-pad assay described by Yamakawa et al. 70 ,

the venom of B. asper induced a rapid edema which remained at a high

levei even at 24 hours 16 . The edema induced by Bothrops snake venoms

is problably due to the action of a variety of substances: (a) Hemorrhagic

toxins which disrupt the microvasculature inducing extravasation 48 ' 5C) . (b)

Toxins acting directly on the endothelial cells of the capillaries and venules,

increasing their permeability 48 ' 50 . (c) Venom components (phospholipases

or cytotoxins) which induce the release of histamine from mast cells. (d)

Phospholipases A 2 that release arachidonic acid from phospholipids in cell

membranes, initiating the pathway leading to the synthesis of

prostaglandins 60 (e) Proteases that act on plasma kininogens, liberating ki-

nins (e.g. bradykinin), as was demonstrated by Rocha e Silva et a/. 61 back

in 1949. Moreover, kallikrein can be activated by factor XII of the coagula-

tion cascade, once this factor is activated after damage to the vasculature.

(f) components of the complement cascade, particularly C3a and C5a,

which participate in the inflammatory reaction 60 . Due to the presence of

many pharmacologically active proteins in Bothrops venoms, it is likely that

the development of edema is due to the combination of these elements.

Lomonte 34 , Gutiérrez et a1. 25 and Rojas et al. 63 observed that neutrali-

zation of edema-forming activity of Central American crotaline venoms by

a polyvalent antivenom is difficult to achieve. Due to the relevance of ede¬

ma in bothropic envenomations, it is necessary to identify and characterize

edema-forming components in these venoms, as well as to study their

mechanism of action, in order to design new therapeutic strategies to con-

front this significant local effect.

Besides edema, there is a conspicuous cellular component in the in¬

flammatory reaction in bothropic envenomations. In the case of intramus¬

cular injection of B. asper venom, inflammatory infiltrate started after the

sixth hour and reached its highest levei by 48-72 hours 24 . Initially, the

predominant cell type was the polymorphonuclear neutrophil but, at later

time periods, macrophages became more abundant. 24 It was proposed that

degradation of myofibrillar proteins in necrotic rnuscle cells after injection

of B. asper venom was accomplished by proteases from invading phago-

cytic cells.

FINAL REMARKS: THE NEED OF AN INTEGRATED VIEW

Muscle tissue, where local effects take place, is complex both anatomi-

cally and functionally. Bothrops venoms affect not only skeletal muscle cells,

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Mem. Inst. Butantan, 51(41:211-223, 1989.

but also intramuscular arteries, microvessels and nerves. Therefore, the study

of local effects must include an integrated analysis of the effect of these

venoms on the various cell types and extracellular structures forming a given

muscle.

Until now there have been few studies in this direction. For instance,

it is not known how relevant is the ischemia in the development of muscle

damage. Furthermore, it is necessary to determine which phenomenon is

more important in the development of ischemia, if damage to microvascula-

ture by hemorrhagic toxins or lesions in intramuscular arteries. This partic¬

ular point is especially relevant, since the design of new therapeutic avenues

will depend on this information. For instance, if ischemia is basically due

to microvessel damage, then there is a good point in using angiogenic sub-

stances to stimulate revascularization 11 which, in turn, would contribute to

a more successful skeletal muscle regeneration. If, on the other hand, ar¬

terial damage predominates, the approach must be different.

Two additional points that need to be investigated are: (a) The role of

axonal degeneration in local tissue damage, particularly in regard to the

process of muscle regeneration; and (b) the effect of Bothrops venoms on

the extracellular matrix, including the basal lamina surrounding muscle fibers

and capillaries. Since this matrix is determinant in the maintenance of an

adequate spatial relationship between cells, its alteration may have drastic

effects in the muscle as a whole.

In conclusion, it is necessary to isolate and characterize additional com-

ponents from Bothrops venoms which induce myonecrosis, hemorrhage and

edema. It is also important to study the immunological relationships of these

components, in order to have a more rational basis for selecting the ven¬

oms to be used in the production of antivenoms. Concomitantly, the integra-

tion of biochemical, physiological and pathological studies is essential, in

order to gain a comprehensive knowledge of these complex and challeng-

ing phenomena.

ACKNOWLEDGEMENTS

Some of the findings discussed in this work are the result of research

projects supported by Vicerrectoría de Investigación, Universidad de Costa

Rica and by the International Foundation for Science, projects F/883-1,

F/883-2 and F/1388-1. J.M. Gutiérrez and B. Lomonte are research fellows

of the Costa Rican National Council for Science and Technology (CONICIT).

RESUMO: A revisão focaliza os efeitos locais induzidos pelo veneno de

Bothrops caracterizados por mionecrose, hemorragia e edema, de evolu¬

ção rápida. As miotoxinas classificadas como fosfolipase A 2 pela estru¬

tura, embora algumas não possuam essa atividade enzimática; as

hemorraginas caracterizadas como metaloproteínas acídicas, e os fato¬

res edematogênicos, estão presentes nos venenos botrópicos. Várias mio¬

toxinas e hemorraginas foram purificadas de venenos botrópicos.

UNITERMOS: Venenos de Bothrops, miotoxinas, mionecrose, hemorra¬

gia, edema.

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GUTIÉRREZ, J. M. & LOMONTE, B. Local tissue damage induced by Bothrops snake venoms. A review.

Mem. Inst. Butantan, 51(41:211-223, 1989.

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Mem. Inst. Butantan

51 ( 41 : 225 - 237 , 1989 .

ISOLATION AND PROPERTIES OF A PHOSPHOLIPASE A2 FROM

THE VENOM OF THE SNAKE BOTHROPS MOOJENI (CAISSACA)

Antonia Paula REICHL

Solange Maria de Toledo SERRANO

Marina Tizuko ASSAKURA

Fajga Ruchla MANDELBAUM

ABSTRACT BM-PLA 2 , a phospholipase A2, was purified from the venom

of the snake Bothrops moojeni by chromatography on Sephadex G-100,

DEAE-Sephadex A-50 and rechromatography on DEAE-Sephadex A-50.

BM-PLA, is an acidic protein of pl 4.6. The enzyme is homogeneous in

polyacrylamide gel electrophoresis and ultracentrifugal analyses. The s 20w

and D° 0w are 2.08 S and 14.9 x 10' 7 cm 2 /sec., respectively. The molé-

cular vveight of 11,300 was calculated by s°/D° ratio. In SDS-

-polyacrylamide gel electrophoresis the denatured and reduced enzyme

exhibits a single polypeptide chain of molecular weight of 11,900. The

enzyme is cross-linked by four disulphide bridges and has histidine as N-

-terminal amino acid. Chemical modifications of BM-PLA 2 with p-

-bromophenacylbromide and N-bromosuccinimide cause complete loss

of enzymatic activity. The purified phospholipase A2 is nontoxic, nonhe-

morrhagic and no edema forming.

KEY WORDS: Venom, Bothrops moojeni, phospholipase A2.

INTRODUCTION

Snake, bee and scorpion venoms and mammalian pancreas are rich

sources of phospholipase A2. Snake venoms contain several phospholipase

A2 enzymes which differ in enzymatic and pharmacological features. A great

number of phospholipases A2 have already been purified from the venoms

of snakes of the Elapidae and Viperidae families. However very little is known

about phospholipases A2 from venoms of Bothrops species. The venoms

of Bothrops species were considered by Marinetti 23 and Mebs 24 as poor

sources of phospholipase A2. Vidal and Stoppani 34 and Vidal et al 33 , on

Serviço de Bioquímica. Instituto Butantan. C.P. 65 — 01051 — São Paulo — Brasil.

Dedicated to Dr. Saul Schenberg's 70th birthday.

Received 07/6/1989: accepted 11/9/1989.

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REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M, T.; MANDELBAUM, F. R. Isolation and proper-

tíes of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissacal. Mem. Inst.

Butantan, 51(41:225-237, 1989.

the contrary, observed a high phospholipase A2 activity in various venoms

of Bothrops species, such as Bothrops jararaca, Bothrops jararacussu and

Bothrops atrox. Until now phospholipases A2 of venoms of Bothrops spe¬

cies were isolated only from B. neuwiedi (Vidal and Stoppani 34 ), B. asper

(Alagon et al Gutierrez et a/ 11 ) and B. alternatus (Nisenbon et al 25 ).

In the venom of Bothrops moojeni was found a high phospholipase A2

activity associated to high blood-clotting, high proteolytic and low hemor-

rhagic activities. From this venom was isolated the coagulant enzyme batro-

xobin (Stocker and Barlow 30 ). Also the major proteolytic enzyme, moojeni

protease A, was already isolated (Assakura et al 2 ).

In the present study, a phospholipase A2 from the venom of Bothrops

moojeni was isolated to homogeneity and some biochemical, biophysical

and physiopathological properties were characterized.

MATERIALS AND METHODS

Purification of phospholipase A2

Dried crude venom (1.8g) of Bothrops moojeni was dissolved in 0.05

M Tris-HCI buffer (pH 7.5) containing 0.5 mM CaCI 2 and 0.02% sodium

azide (buffer A) and clarified by centrifugation. The supernatant solution

containing 1.62g of protein in 12.5 ml was chromatographed on a column

(2.6 x 80 cm) of Sephadex G-100 previously equilibrated in buffer A under

the conditions described by Assakura et al 2 for the isolation of moojeni pro¬

tease A. The fractions from peak II containing the phospholipase A2 activi¬

ty were combined and lyophilized. A solution of 530 mg of protein in 17

ml, dialyzed against buffer A, was applied to a 2.5 x 50 cm column of DEAE-

Sephadex A-50 equilibrated with buffer A. After washing the non adsorbed

protein, a linear gradient up to 0.3 M NaCI in the same buffer (mixing ves-

sels 500 ml) was applied. The fractions in the protein peak, which contained

the phospholipase A2 activity, were combined, lyophilized and dialyzed

against 0.05 M Tris-HCI buffer (pH 7.5) containing 0.5 mM CaCI 2 , 0.02%

sodium azide and 0.075 M NaCI (buffer B), and applied to a 2.5 x 50 cm

column of DEAE-Sephadex A-50 previously equilibrated with buffer B. The

fractions (45 to 60) with constant specific activity were combined and

lyophilized.

Phospholipase A2 activity

This was determined using egg-yolk lecithin as substrate according to

Kornalik and Master 17 . Lysolecithin released as a result of enzyme activity

was assayed by the hemolytic effect on washed horse red blood cells. One

unit of phospholipase A2 activity was defined as the amount of protein which

causes 50% hemolysis corresponding to an increase in O.D. of 1.0 at 540

nm. Specific activity is expressed as Units per mg of protein.

Caseinolytic activity

Activity was determined by a modification of the method of Kunitz 18 ,

as described by Mandelbaum et al 22 . One unit of caseinase activity is de¬

fined as the amount of enzyme yielding an increase in O.D. of 1.0 per min

at 750 nm.

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ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

Blood-clotting activity

This activity was assayed on citrated horse plasma [1 ml of trisodium

citrate 3.8% (w/v) for 10 ml of blõod] and measured as described by Hen¬

riques et a/ 13 .

Hemorrhagic activity

The hemorrhagic activity was assayed by intradermal injection into the

backs of mice with doses varying from 2 to 20 ng of venom or isolated phos¬

pholipase A2 protein in a volume of 0.1 ml. Four Swiss mice (20-22g) weré

used for each dose. The mice were sacrificed 2 h later by ether inhalation,

the skins removed and the hemorrhagic lesions were evaluated on the visceral

side and also on the dorsal surface of the musculature.

Estima tion of protein

Protein concentration was measured by the method of Lowry et al 20

using bovine serum albumin as standard, or by spectrophotometric deter-

mination of absorption at 280 nm.

Polyacrylamide gel etectrophoresis

Polyacrylamide 10% gel electrophoresis in Tris-glycine buffer, pH 8.5 was

performed according to the method of Maizel 21 with a 2.5% gel spacer fol-

lowing the method of Davis 4 . Runs were of 3 h with 1.25-1.50 mA per tube

(0.5 x 8.0 cm), bromophenol blue being used as indicator. Gels were stained

for 16 h at room temperature with 0.05% Coomassie brilliant blue R in 12.5%

trichloroacetic acid, containing 40% methanol, and destained with several

changes of 7.5% acetic acid -5% methanol. For glycoprotein detection the

gels were stained with periodic acid-Schiff according to the procedure of

Zacharius et al 40 as modified by Glossmann and Neville 9 .

Molecular weight determinatlon

Sodium dodecyl sulfate (SDS) 10% (w/v)-polyacrylamide gel electropho¬

resis was performed according to the method of Weber and Osborn 36 . Gels

were cast in 0.5 cm i.d. tubes to a height of 8 cm, and the protein sample

contained 1% SDS (w/v) and 1% 2-mercaptoethanol (v/v) in 0.01 M phos-

phate buffer (pH 7.4). The mobilities of ovalbumin, bovine serum albumin,

ribonuclease and y -globulin (H-and L-chains) were measured in compari-

son to that of bromophenol blue.

Analytlcal isoelectric focusing

Isoelectric focusing was conducted in a Pharmacia Fiat Bed Apparatus

FBE 3000 in a layer of 5% polyacrylamide gel with 2% Ampholine (LKB)

in the pH range 3-6, at 200 V for 20 h and 4°C, according to the procedure

of Vesterberg 32 . Ovalbumin (pl 4.6) and methyl blue (pl 3.6) were run as

markers. The pH gradient in the layer was measured potenciometrically af-

ter 4 h diffusion of equal samples cut from the gel in 1 ml of water at 25°C.

Amino acid analyses

Amino acid analyses were carried out with a Technicon amino acid

analyzer (Spackman et al 28 : Hamilton 12 ). Lyophilized salt-free samples were

hydrolyzed in 3N p-toluene sulfonic acid containing 0.2% 3(2-aminoethyl)

indol at 110°C for 24,48 and 72 h according to the method of Liu and

Chang 19 . Half-cystine content was determined as cysteic acid after perfor-

mic acid oxidation (Hirs 14 ) prior to hydrolysis for 16 and 24 h in constant

227

REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M, T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

boiling HCI (6N) at 110°C.

spectrophotometrically in

Morton 8 .

Tryptophan and tyrosine were also determined

0.1N NaOH, as described by Goodwin and

Partial specific volume

V was calculated from the amino acid composition of the enzyme, ac-

cording to the procedure of Cohn and Edsall 3 .

Ultracentrifugal anatyses

These analyses were performed in a Spinco Model E ultracentrifuge

equipped with RTIC control unit and phase plate. Both Schlieren and inter-

ference optics were employed. All measurements of ultracentrifuge patterns

were made with a Zeiss small toolmaker's measuring microscope. Sedimen-

tation and diffusion coefficients were determined at 20°C in 0.1 M Tris-HCI

containing 0.1 M NaCI and 2 mM CaCI 2 buffer solution (pH 7.6), in double

sector, capillary type, synthetic boundary cells. Sample concentrations were

in the range of 0.9 to 3 mg per ml. Sedimentation velocity experiments were

performed with Schlieren optics at 56,000 revs/min (D rotor). The sedimen¬

tation coefficients were calculated according to the method of

Schachman 27 . Diffusion coefficients were determined at 6,000 revs/min (J

rotor), and the pictures analysed by the height-area method of Ehrenberg 6 .

Sedimentation and diffusion coefficients were corrected to standard condi-

tions of density and viscosity of water at 20°C. Low speed sedimentation

equilibrium experiments were performed at 20°C with double sector cells

having a 12mm light path, fitted with quartz Windows. The column heights

of enzyme solution were of 3 mm (Van Holde and Baldwin 31 ; Yphantis 39 ).

Fluorcarbon FC-43 was used as base fluid to give a transparent cell bot-

tom. The rotor speeds were sufficiently low for the solute concentration at

the meniscus not to be zero. Equilibrium was established when no meas-

urable change occurred in fringe displacement with time. Molecular weights

were determined with samples of 1.8 and 0.9 mg per ml.

Oxidation with N-bromosuccinimide (NBS)

The modification of the phospholipase A2 was perfomed essentially ac¬

cording to the procedure of Spande and Witkop 29 . The enzyme sample

(0.72 mg which corresponds to O.D. at 280 nm of 1.0) was dissolved in

1 ml of 0.1 M acetate buffer, pH 4.0. To this solution 5 mM NBS were ad-

ded in 10H portions. After each addition the change in absorbance at 280

nm was measured and the number of oxídized tryptophan residues was es-

timated from the decrease in the absorbance at 280 nm.

Modification with p-bromophenacylbromide

The enzyme (0.5 mg) was dissolved in 0.5 ml of 0.025 M Tris-HCI buffer,

pH 7.5 containing 0.2 mM EDTA. To this solution 25 p\ of 2 mM p-

bromophenacylbromide were added. After 4 and 6 h at room temperature,

phospholipase A2 activity was assayed in aliquots of the reaction mixture,

as described above.

Free sulphydryl group

Free sulphydryl group was measured according to the method of

Ellman 7 . The reactions were performed with 0.025 to 0.05 p moles of the

enzyme in 0.1 M phosphate buffer, pH 8.0 in presence of 1% SDS and 10' 4

228

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REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M. T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

M EDTA. The absorbance at 412 nm was measured 30 min and 1 h after

addition of 5,5'-dithiobis (2-nitrobenzoic acid).

N-terminal amino acid analysis

This was carried out using dansyl chloride as described by Gros and

Labouesse 10 with denatured and reduced enzyme according to the proce-

dure of Weber and Osborn 36 . The DNS-amino acids obtained after acid

hydrolysis were identified by the method of Woods and Wang 37 .

RESULTS

Isolation of phospholipase A2

A significant phospholipase A2 activity was observed in the venom of

Bothrops moojeni when it was fractionated by the procedure used for the

isolation of moojeni protease A (Assakura et a! 2 ). The phospholipase A2

activity was found in the second peak of the gel filtration on Sephadex G-100.

This fraction contained also various proteolytic enzymes, being however free

from the blood-clotting and hemorrhagic activities present in the venom

</>

Cvl

FIG. 1. ELUTION PROFILE OF BM-PLA, FROM RECHROMATOGRAPHY OF DEAE-

SEPHADEX A-50.

A sample of phospholipase A2 of 22 mg in 7 ml, previously dialyzed against 0.05 M Tris-

HCI — 0.5 mM CaCL buffer, pH 7.5 containing 0.02% sodium azide and 0.075 M NaCI

was applied to a DEAE-Sephadex A-50 column (2.5 x 50 cm) previously equilibrated with

the same buffer solution.

1, | SciELO

229

REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M. T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

TABLE 1

YIELD AND ACTIVITY OF PHOSPHOLIPASE A2 ISOLATED FROM THE VENOM

OF BOTHROPS MOOJENI.

Purification

steps

Protein

mg %

Phospholipase A2

specific total

10’ 6 x Units/mg xlO' 6

activity

purification

factor

Crude venom

1620

100

31.2

50544

100

1.0

Sephadex G-100

530

32.8

43.5

23098

1.4

DEAE-Sephadex A-50

(first chromatography)

1.37

185

4096

8.1

5.9

DEAE-Sephadex A-50

(rechromatography)

8.7

0.54

340

2958

5.8

Specific activity is expressed as described in "Materials and Methods"

A good separation of phospholipase A2,from the various proteolytic enzymes

contained in this fraction, was achieved by chromatography on DEAE-

Sephadex A-50 column. Proteins with phospholipase A2 activity were eluted

in a narrow peak with a NaCI gradient added to the initial buffer. The pat-

terns of these chromatographies were similar to those already published

by Assakura et a! 2 . In the present work, the material with phospholipase

A2 activity from the DEAE-Sephadex A-50 column was rechromatographed

under the same conditions (figure 1). The elution of the major protein peak

was followed by the phospholipase A2 activity, with a constant specific ac¬

tivity of 340 x 10 6 Units/mg. With this procedure a purification of 11-fold

was achieved, with a recovery of 5.8% of the total phospholipase A2 ac¬

tivity present in crude venom. The activities and yields of the various steps

of the purification of phospholipase A2 are shown in table 1.

The isolated phospholipase A2 shows an indirect hemolytic activity on

horse red cells. An amount of 3 j^g of this enzyme causes 50% hemolysis

of intact erythrocytes in the test. This enzyme does not exhibit direct hemo¬

lytic activity on washed horse red cells.

Homogeneity of phospholipase A2

Analyses in polyacrylamide gel electrophoresis of various steps of the

purification of phospholipase A2 are shown in figure 2. The isolated phos¬

pholipase A2 showed a single protein band in polyacrylamide gel electropho¬

resis (figure 20. This protein, after denaturation and reduction by SDS and

2-mercaptoethanol, presents in SDS-polyacrylamide gel electrophoresis,

a single polypeptide chain (figure 2D). The molecular weight for this en¬

zyme calculated using bovine serum albumin, ovalbumin, human Y-globulin

(H and L-chains) and ribonuclease as protein standards gave the value of

11,900.

Analytical ultracentrifugation using sedimentation velocity experiments

yielded symmetrical single schlieren patterns. The values of the sedimen¬

tation and diffusion coefficients varied with the protein concentration. When

concentration of phospholipase A2 varied from 3.6 to 0.9 mg/ml, the values

for s 20w varied from 2.99 to 2.53, while D 20w x io 7 varied from 12.55 to

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ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

TABLE 2

BIOPHYSICAL PROPERTIES OF BOTHROPS MOOJENI PHOSPHOLIPASE A2 (BM-PLA 2 )

Parameter

Value

Sedimentation coefficient (s 20w S)

protein (mg/ml)

3.6

2.99

1.8

2.99

1.5

2.85

0.9

2.53

2-08 (s°)

Diffusion coefficient (D 20w x10 7 , cm z /sec)

protein (nhg/ml)

3.6

12.55

1.8

12.70

1.5

13.99

0.9

14.21

14.90 (D° 0w x10 7 )

Partial specific volume (v, ml/g)

0.704

Isoelectric point (pl)

4.60

Extinction coefficient (Ey 7 ° m , 280 nm)

13.96

Molecular weight

Sedimentation-diffusion (s/D)

protein (mg/ml)

3.6

19,600

1.8

19,380

1.5

16,800

0.9

14,650

11,300 (s°/D°)

Sedimentation equilibrium

protein (mg/ml)

1.8

19,100

0.9

13,900

SDS-polyacrylamide electrophoresis

11,900

Values of s, D, v and molecular weights determined as described in Materials and Methods.

14.21. At infinite dilution, values of 2.08 S and 14.90 x 10' 7 cm 2 /sec were

obtained for the sedimentation and diffusion coefficients, respectively. The

molecular weights determined from sedimentation and diffusion values, with

these protein concentrations, gave values varying from 19,600 to 14,600.

The extrapolated value of 11,300 was obtained at zero protein concentra-

tion. Molecular weights were also calculated from sedimentation equilibri-

um experiments. By this method the molecular weights of phospholipase

A2 in the concentrations of 1.8 and 0.9 mg/ml, gave values of 19,000 and

13,900, respectively.

By isoelectric focusing in thin layer of polyacrylamide gel, phospholi¬

pase A2 showed to be an acidic protein with a pl of 4.60.

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lies of a phospholipase A2 from the venom of the snake Bothrops mocjeni (Caissaca), Mem. Inst.

Butantan, 51(41:225-237, 1989,

This homogeneous phospholipase A2 isolated from B. moojeni venom,

was named BM-PLA 2 . The biophysical constants of BM-PLA 2 are shown

in table 2.

D E

FIG. 2. POLYACRYLAMIDE GEL ELECTROPHORESIS.

(A) Crude venom (80 \a g), (B) Proteins from peak II of Sephadex G-100 column (30 pg).

ture of reduced bovine serum albumin, ovalbumin, y -globulin (H and L-chains) and

ribonuclease used as standards for molecular weight measures.

Amino acid composition

The amino acid composition of BM-PLA 2 is shown in table 3. The pro-

tein has a high content of acidic amino acids, 11 Asp and 8 Glu residues.

BM-PLA 2 presents also high contents of 1/2 Cys, Gly and Lys. Tryptophan

and tyrosine were also determined spectrophotometrically. The number of

residues given by this procedure were the same as those obtained by hydrol-

ysis with p-toluene sulfonic acid, The amino acid analyses showed that BM-

PLA 2 contained 8 cysteine residues. Free sulfhydryl groups were not found.

The method of Ellman 7 indicated less than 0.1 residues per mole of pro-

232

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REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M. T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

TABLE 3

AMINO ACID COMPOSITION OE BOTHROPS MOOJENI PHOSPHOLIPASE A2

(BM-PLAJ. HYDROLYSIS WITH 3 N p-TOLUENE SULFONIC ACID.

Amino Acid

Residues/mole of

enzyme

nearest

integer

ASP

11.32

THR a

3.98

SER a

4.75

GLU

7.91

PRO

3.74

GLY

7.64

ALA

3.62

VAL b

3.96

1/2CYS 0

7.76

MET

1.15

ILEU b

2.45

LEU

2.12

TYR

4.00

PHE

4.38

LYS

5.80

HIS

1.68

TRP

1.01

ARG

3.61

NH 3 a

12.21

TOTAL

Average values of duplicate analyses of 24,48 and 72 h acid hydrolysis. Number of

residues based on molecular weight of 11,900.

a — extrapolated to zero time hydrolysis.

b — values of 72 h hydrolysis.

c — determined as cysteic acid after perfomic acid oxidation.

tein. Based on amino acid composition, the minimum molecular weight of

BM-PLA 2 corresponds to 9,400. This value is 20% lower than those esti-

mated by ultracentrifugal analyses and SDS-polyacrylamide gel electropho-

resis. Considering that this low value given by the amino acid composition

could be due to the presence of carbohydrates, BM-PLA 2 was assayed for

glycoprotein. However, no Schiffs stain was observed.

The N-terminal amino acid of BM-PLA 2 was found to be histidine.

Stability of BM-PLA 2 at pH variation

The effect of pH on the enzyme was tested with Solutions adjusted with

dilute NAOH or HCI to various pHs. After 6 h at room temperature, the phos¬

pholipase A2 activity was measured. The enzyme is stable, as no signifi¬

cam alteration in enzymatic activity was observed at pHs ranging from 4

to 8. However, an increase of 30% of its activity was observed when the

enzyme was submitted to pHs 8 to 10 and loss of 25% of the activity was

observed at pHs below 4.

Chemical modifications

BM-PLA 2 lost completely its activity after oxidation with N-

bromosuccinimide. The tryptophan residue was modified without denatu-

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tios of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

ration of BM-PLA 2 with urea. The modified enzyme gave a single band on

SDS-polyacrylamide gel electrophoresis, with the same mobility as the na-

tive enzyme, excluding the possibility of a split in the polypeptide backbone.

Treatment for 4 h of BM-PLA 2 with p-bromophenacylbromide resulted also

in total loss of activity. Only one of the two histidine residues of the enzyme

was alkylated. The other histidine was not modified and was found as N-

terminal amino acid.

Biological activities

Some biological tests were assayed with the isolated BM-PLA 2 in mice.

BM-PLA 2 was not toxic when injected intraperitonéally. No deaths after 24

h were observed in the injected mice with various doses up to 1 piglg body

weight (a minimum of 6 animais were used per dose). However crude ven¬

om in dose of 7.5 ^g/g body weight, was enough to kill mice at the end

of 3 h. Also no edema was observed when injected in the foot pads of the

hind limbs with doses up to 25 M g of BM-PLA 2 following the method of

Yamakawa et a! 38 . Hemorrhage was also not detected even with doses of

20-25 ng of BM-PLA 2 injected intradermally, intraperitonéally or into the

foot pads. With crude venom hemorrhage and edema were already observed

with doses of 2 ng. This shows that BM-PLA 2 possesses no lethal, hemor-

rhagic or edema-forming activities.

DISCUSSION

The isolated BM-PLA 2 showed to be homogeneous by polyacrylamide

gel electrophoresis, SDS-polyacrylamide gel electrophoresis, isoelectrofocus-

ing, ultracentrifugation and N-terminal amino acid analysis.

BM-PLA 2 in ultracentrifugal analyses showed that both sedimentation

and diffusion coefficients varied with the protein concentration. The decreas-

ing values of the molecular weight calculated by s/D ratio or sedimentation

equilibrium with the decreasing of the protein concentration indicate that

BM-PLA 2 is found in reversible associated form. The extrapolated value of

11,300 daltons at zero protein concentration for the molecular weight of BM-

PLA 2 is close to the molecular weight of 11,900 determined for the single

polypeptide chain in SDS-polyacrylamide gel electrophoresis. Concentration-

dependent aggregation was also observed in phospholipases A2 from the

venoms of Agkistrodon halys blomhoffii (Kawauchi et a/ 16 ) and of Naja naja

naja (Deems and Dennis 5 ).

BM-PLA 2 is an acidic protein of pl 4.6, with predominance of Asp and

Glu residues over the basic amino acids. BM-PLA 2 contains 8 half-cystine

residues, a lower content when compared to the phospholipases A2 isolat¬

ed from elapidic venoms, which contain, in general 12 to 14 residues. Our

results are close to the half-cystine content of phospholipases Pt and P 2

isolated from the venom of B. asper (Alagon et aP). Compared to the phos¬

pholipases A2 of the elapidic venoms, the low content of half-cystine residues

of phospholipases A2 of venoms of Bothrops species suggests a different

cross-linking.

The loss of activity by Chemical modification of BM-PLA 2 by N-

bromosuccinimide is correlated to the destruction of the single tryptophan

residue of the enzyme molecule. The oxidation of this tryptophan without

234

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REICHL, A.; SERRANO, S. M. deT.; ASSAKURA, M. T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

prior denaturation of the enzyme suggests that this amino acid must be on

the surface of the native enzyme. Modification of the histidine residues of

BM-PLA 2 with p-bromophenacylbromide also caused complete loss of en¬

zyme activity. Volwerk et a/ 35 'demonstrated that the histidine residue lo-

cated at the active site of pancreatic phospholipase A2 is modified by

p-bromophenacylbromide. Considering that one histidine of the modified

BM-PLA 2 was found as the N-terminal amino acid we concluded that the

histidine essential for the catalytic activity was alkylated by p-

bromophenacylbromide.

The acidic phospholipase A2 isolated in this study, although having a

high indirect hemolytic activity, is nonhemorrhagic, nontoxic and no ede¬

ma forming.

Acid phospholipases A2 from snake venoms are usually less toxic in con-

trast to the basic ones (Rosenberg 26 ). In spite of this fact, a phospholipase

A2 with lethal activity was isolated from the venom of Bothrops alternatus

(Nisenbon et a/) 25 . On the other hand, not all basic phospholipases A2 are

highly toxic. One of the phospholipases A2, VRV PL-Vllla, isolated from the

venom of Vipera russelli, with a low indirect hemolytic activity, is less toxic

compared to the whole venom (Kanturi and Gowda 15 ).

ACKNOWLEDGMENTS

We are very grateful to Neusa de Lima and Maria Aparecida Siqueira

for skillful technical help.

RESUMO: Do veneno de Bothrops moojeni foi isolada uma fosfolipase A2,

BM-PLA 2 , por cromatografia em Sephadex G-100, DEAE-Sephadex A-50

e recromatografia em DEAE-Sephadex A-50. BM-PLA 2 é uma proteína

ácida de pl 4,6. A enzima mostrou ser homogenea por eletroforese em

gel de poliacrilamida e análises de ultracentrifugação. Os valores para

s° 20w e D° 20w são de 2,08 S e 14,90 x 10' 7 cm 2 / seg, respectivamente.

O peso molecular calculado da relação s°/D° é de 11.300 daltons. A en¬

zima desnaturada e reduzida apresenta, por eletroforese em SDS-gel de

poliacrilamida, uma única banda de peso molecular 11.900. BM-PLA 2

possui quatro pontes dissulfeto e seu N-terminal é histidina. Modificações

químicas da enzima com p-bromofenacilbrometo e N-bromosuccinimida

resultaram na perda total da atividade enzimática. A fosfolipase A2 isola¬

da não causa hemorragia, não é tóxica e não é edematogênica.

UNITERMOS: Veneno, Bothrops moojeni, fosfolipase A2.

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KAPLAN, N.O. eds. Methods in Enzymology, vol. 4. New York, Academic Press

1957. p. 32-103.

28. SPACKMAN, D.H.; STEIN W.H.; MOORE, S. Automatic recording apparatus for use in

the chromatography of amino acids. Analyt. Chem., 30: 1190-1206, 1958.

29. SPANDE, T.F. WITKOP, B. Determination of the Tryptophan content of proteins with N-

bromosuccinimide. In: HIRS, C.H.W. ed Methods in Enzymology, vol. 11. New York,

Academic Press, 1967, p. 498-506.

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REICHL, A.; SERRANO, S. M. de T.; ASSAKURA, M. T.; MANDELBAUM, F. R. Isolation and proper-

ties of a phospholipase A2 from the venom of the snake Bothrops moojeni (Caissaca). Mem. Inst.

Butantan, 51(41:225-237, 1989.

30. STOCKER, K.F. & BARLOW, G.H. The coagulant enzyme from Bothrops atrox venom

(Batroxobin). In: COLOWICK, S.P. & KAPLAN, N.O. eds. Methods in Enzymology

Vol. 45, part B. New York, Academic Press, 1976, p. 214-223.

31. VAN HOLDE, K.E. & BALDWIN, R.L. Rapid attainment of sedimentation equilibrium.

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32. VESTERBERG, O. Isoelectric focusing of protein in thin layers of polyacrylamide gel.

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33. VIDAL, J.C.; MOLINA, H.; STOPPANI, A.O.M. A general procedure for the isolation and

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Latino Am., 23: 91-109, 1972.

34. VIDAL, J.C. & STOPPANI, A.O.M. Isolation and purification of two phospholipases a from

Bothrops venoms. Arch. Biochem. Biophys., 145: 543-556, 1971.

35. VOLWERK, J. J.; PIETERSON, W. A.; DE HAAS, G. H. Histidine at the active site of phos¬

pholipase A. Biochemistry, 13: 1446-1454, 1974.

36. WEBER, K. & OSBORN, M. The reliability of molecular weight determinations by dodecyl

sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244: 4406-4412, 1969.

37. WOODS, K.R. & WANG, K.T. Separation of dansyl-amino acids by polyamide layer chro-

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39. YPHANTIS, D.A. Rapid determination of molecular weights of peptides and proteins.

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40. ZACHARIUS, R.M.; ZELL, T.E.; MORRISSON, J.H.; WOODLOCK, J.J. Glycoprotein staining

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Mem. Inst. Butantan

51 (41:239-254, 1989.

COLETANEA DE RESUMOS DE TRABALHOS

PUBLICADOS PELOS PESQUISADORES DO

INSTITUTO BUTANTAN

(1988)

1. ALMEIDA, M.E.S. de; MARTINI, M.H.; PORTO, E.; CAMARGO, A.M.M. de; RIZZO, E. de; LACAZ,

C.S.

Identificação da microbiota fúngica de ambientes considerados assépticos.

Rev. Saúde púbi, S. Paulo, 22 (3): 201-6, 1988.

Resumo: Objetivou-se isolar e identificar a microbiota fúngica em ambientes considerados assépticos,

através de exposições com meios de cultivo adequados, em três épocas distintas do ano, antes

e imediatamente após as manobras técnicas realizadas em três áreas de trabalho: ambiente aberto,

ambiente fechado sem filtração de ar e ambiente fechado com filtração de ar, utilizadas em produção

de imunobiológicos. Os meios ágar-Sabouraud e ágar-soja, enriquecidos com 0,2% de extrato de

levedura e sem cloranfenicol, foram estudados quanto à sua eficácia no isolamento de bolores e

leveduras, considerando-se o número de colônias desenvolvidas e a frequência dos diversos fungos

isolados. Isolaram-se 67 espécimens, sendo 64 fungos filamentosos (bolores) e três leveduras. Dos

bolores, 54 pertenciam a 22 gêneros da divisão Deuteromycota , famílias Moniliaceae e Dematiaceae,

cinco amostras filamentosas foram incluídas na ordem Agonomycetales (Mycelia Sterilia), e uma

amostra foi classificada na divisão Deuteromycota, ordem Sphaeropsidales, classe Coelomycetes.

Da divisão Zygomycota, ordem Mucorales, família Mucoraceae, um único mucoráceo foi identificado

até gênero. As três leveduras pertenciam também à divisão Deuteromycota (Fugi Imperfecti), família

Cryptococcaceae, e foram identificadas como sendo duas Rhodotoruta rubra e uma Torulopsis candida.

Comprovou-se que o número de colônias isoladas aumentou após a realização das manobras técnicas

e que a filtração de ar através de filtros tipo HEPA, reduzindo o número de colônias isoladas nos

ambientes fechados, aumenta a segurança do trabalho; comumente é recomendada para áreas de

atividade técnica cujos resultados satisfatórios estão diretamente relacionados com uma baixa

incidência de contaminantes.

Abstract: The isolation and identification of the fungic microbiota present in areas considered aseptic

(open and/or restricted, with or without air filtration (HEPA filters) where production of

immunobiologicals is carried out) were investigated. Three exposures were made with appropriate

culture media, in different seasons of the year, before and immediately after the performance of the

technical work. Agar-Sabouraud and agar-soy media supplemented with 0.2% yeast extract and

with-out chloranphenicol had their efficacy tests in isolating the filamentous fungiae (molds) and

yeasts in the light of the number of cultures grown and the frequency exhibited by the various

specimens isolated. Sixty-seven specimens (64 of filamentous fungiae (molds) and three yeasts)

havc been identified. Of the filamentous fungiae (molds), 54 belonged to 22 genera of the division

Deuteromycota, families Moniliaceae and Dematiaceae, five were included in the order

Agonomycetales (Mycelia Sterilia), and one was classified in the order Sphaeropsidales, class

Coelomycetes. Of the division Sygomycota, order Mucorales, family Mucoraceae, only one specimen

could be identified as to genus. The three yeasts, belonging also to the division Deuteromycota (Fungi

Imperfecti), family Cryptococcaceae, were identified as two Rhodotorula rubra and one Torulopsis

candida. It was demonstrated that the number of colonies isolated in the areas studied increased

after the performance of technical maneuvers and that air filtration by means of HEPA filters, reducing

to number of colonies isolated in restricted areas, increased safety and is consequently recommended

for working areas where successful or unsuccessful results depend on a low incidence of

contamination.

2. BEÇAK, M.L.; SANTOS, R.C.S. dos; SOARES-SCOTT, M.D.; BATISTIC, R.F.; COSTA, H.

Chromosome structure in man and amphibia-anura, restriction enzymes.

Rev. bras. Genet., II (4): 939-48, 1988.

Abstract: Fixed metaphase chromosomes from human and from polyploid anurans (Odontophrynus

americanus, 4n = 44), were treated with Mspl, Hpa II, Eco RI, Bgl II, Dra I and Hae III, restriction

enzymes, followed by Giemsa staining. Mspl (C f CGG) and Hae III (GG 1 CC) produced G-like bands

in human chromosomes indicating that the bands are caused by the removal of CG-rich specific

DNA classes. However, comparable G-band patterns were also obtained in human chromosomes

with Eco Ri (G f AATTO.Bgl II (A I GATCT) and Dra I (TTT | AAA). Whose digesting sites do

not reveal a particular CG-richness. In the 4n anurans, Mspl-removed chromosomal DNA from the

long arm of two to three homologues of group I (1q), from the secondary constriction of group 11

as well as from centromeric, telomeric and intercalary C bands, These C bands were not removed

with Hpa II (C I CGG), which does not cut methylated DNA. Eco RI caused bands on 2p and

3q. The results suggest that: 1. the action of these enzymes depends on the accessibility of the

enzymes to chromosome regions oí different DNA packaging. 2. The difficulty in obtaining interstitial

bands in anurans may result from the smaller amount of CG-rich sequences probably arranged in

small clusters or from the higher levei of chromatin packaging in the chromorneres.

Resumo: Cromossomos metafásicos fixados de humano e de anuros poliplóides (Odontophrynus

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americanus, 4n =44) foram tratados com enzimas de restrição Mspl, Hpa II, Eco RI, Bgl II, Dra

I e Hae III, seguidos pela coloração Giemsa. Mspl (C CGG) e Hae III (GG 4 CC) produziram bandas

similares a G nos cromossomos humanos, indicando que as bandas são causadas pelas remoções

de classes específicas de DNA ricas em CG. Contudo, padrões comparáveis de banda G foram também

obtidos nos cromossomos humanos com Eco RI IG \ AATTC), Bgl II (A + GATCT) e Dra I

(TTT i AAA), cujos sítios de digestão não revelam abundância particular em CG. Nos anuros 4n,

Mspl removeu DNA cromossômico do braço longo de dois a três homólogos do Grupo I (1q), da

constricção secundária do grupo 11 bem como as bandas centroméricas, teloméricas e intercalares.

Essas bandas não foram removidas pela Hpa II (C + CGG), a qual não corta DNA metilado. Eco

RI causou bandas 2q e 3q. Os resultados sugerem que: 1. a ação dessas enzimas depende da

acessibilidade da enzima a regiões cromossômicas com compactações diferentes do DNA; 2. a

obtenção difícil de bandas intersticiais em Anura pode resultar da menor quantidade de seqüências

ricas em CG provavelmente arranjadas em cachos menores, ou da maior condensação cromatínica

nos cromômeros.

BRODSKYN, C.I.; SILVA, A.M.M. da; TAKEHARA, H.A.; MOTA, I.

Characterization of antibody isotype responsible for immune clearance in mice infect with Trypano-

soma cruzi.

Immunology Letters, 18: 255-58, 1988.

Abstract: Humans and mice chronically infected with Trypanosoma cruzi present a strong humoral

immune response mediated by specific antibodies. Passive transfer of homologous immune serum

to normal mice containing circulating bloodstream trypomastigotes (Bts) induces a very fast clearance

of the parasites. In order to find out the role of the different immunoglobulin classes in the clearance

mice containing a known number of these parasite forms in circulation were injected with total immune

serurn, IgG-free serum, IgGI, or lgG2 fractions and the speed of removal of the parasites from circulation

was determined. The results of these experiments suggest that the immune clearance of T. cruzi

is due to antibodies located in the IgG isotype, particularly in the lgG2 subclass.

Resumo: Homens e camundongos com infecção crônica com T. cruzi apresentam uma forte resposta

humoral com níveis elevados de anticorpos anti-T cruzi. Transferência passiva de soro imune homólogo

para camundongos normais contendo tripomastigotas sanguíneos circulantes causa uma rápida

remoção dos parasitas da circulação. Com a finalidade de verificar a importância das diferentes classes

de imunoglobulinas neste fenômeno, camundongos com um número conhecido de tripomastigotas

na circulação foram com soro imune total ou com as frações IgGI ou lgG2 e a velocidade de remoção

dos parasitas circulantes foi determinada e comparada em cada situação. Os resultados destes

experimentos sugerem que a remoção imune do T. cruzi é devido a anticorpos localizados na classe

IgG, particularmente na subclasse lgG2.

4. CASTRO, N.H.C.; WALTER, J.; SANTOS, R.C.S.; D'ANGELINO, J.L.; BENESI, E; BIRGEL, E.H.; BEÇAK, W.

Cytogenetic study of cattle affected by persistem lymphocytosis.

JVet.Med. A, 35: 380-4, 1988.

Abstract: Eight BLV-seropositive female cattle presenting persistem lymphocytosis associated with

BLV-infection were submitted to cytogenetic analysis. As a control group, six BLV-seronegative fomales,

from the same herd, with no haematological or clinicai changes were used. The affected group revealed

a significam increase in the proportion of aneuploid cells with chromosome aberrations. An

immunological assay revealed that the cells presenting the chromosomal aberrations were from the

B cell population. The question of a direct or indirect action of the virus on the chromosomes in

causing these changes is discussed.

Resumo: Foram analisadas citogeneticamente 8 fêmeas bovinas soropositivas quanto ao vírus da

leucose bovina IBLV), que apresentavam quadro de leucocitose persistente. Constituíram o grupo

controle 6 fêmeas soronegativas quanto ao BLV, da mesma fazenda, sem alterações clínicas ou

hematológicas. A proporção do cólulas aneuplóides com aberrações cromossômicas encontrada

nos animais infectados pelo BLV foi significantemente maior que no grupo controle. Através de teste

imunológico verificou-se que as células portadoras de aberrações cromossômicas pertenciam a

população de células B. A ação direta ou indireta do vírus, como agente causador das alterações

cromossômicas detectadas, é discutida.

CIPOLLA-NETO, J.; AFECHE, S.C.; MENNA-BARRETO, L; MARQUES, N.; BENEDITO-SILVA, A.A.; FOR-

TUNATO, G.; RECINE, E.G.I.G.; SCHOTT, C.

Lack of similarity between the effect of lesions of the suprachiasmatic nucleus and subparaventricu-

lar hypothalamic zone on behavioral circadian rhythms.

Brazilian J. Med. Biol.Res., 21: 653-4, 1988.

Abstract: Rats were submitted to eletrolytic lesion of either the suprachiasmatic nucleus ISCN) or

the subparaventricular hypothalamic zone ISPVH) and the effects on circadian behavioral rhythms

were compared. While the SCN lesion abolished the circadian rythmicity of all behavioral patterns,

the SPVII lesion only abolished that of the eating and drinking behavior and reduced the amplitude

of a behaviora item usuaily associated with REM sleep.

Resumo: Foram realizadas lesões eletrolíticas bilaterais dos núcleos supraquiasmáticos (NSQ) ou

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da região hipotalâmica subparaventricular (RHSP) em ratos e comparados os efeitos destas lesões

sobre os ritmos circadianos comportamentais. A lesão dos NSQ aboliu a ritmicidade circadiana de

todos os padrões comportamentais, enquanto a lesão da RHSP aboliu apenas a ritmicidade circa¬

diana dos comportamentos de comer ç beber e reduziu a amplitude de um item comportamental

geralmente associado ao sono paradoxal.

6 .CIPOLLA-NETO, J.; NEGRÃO, N.; AFECHE, S.C.; PALUDETTI, L.A.; BENEDITO-SILVA, A.A.; MARQUES,

N.; MENNA-BARRETO, L.

Remarkable similarities between the temporal organization of neocortical electrographic sleep pat-

terns of rats and humans.

Brazilian J.Med.Biol.Res., 21: 599-601, 1988.

Abstract: Electrocorticographic activity wasautomatically recorded in albino rats fôr 72 consecu-

tive h and analyzed by procedures suitable to detect 24-h rhythms. Beta (alert wakefulness), theta

(somnolencel, delta (slow wave sleep) and sigma I (superficial synchronized sleep) activities showed

a robust circadian rhythmic distribution. The acrophases (maxima of the adjusted cosine curve) oc-

cured at 23:39,07:59, 08:37 and 13:25h, respectively. EMG atonia and extreme hypotonia (less than

10% of mean EMG levei) episodes showed a 24-h rhythm peaking at 14:18 h. The temporal sequence

within the circadian rest period, i.e., somnolence, show wave sleep, superficial synchronized sleep

and paradoxical sleep, is very similar to that known to occur during the nocturnal sleep of humans.

Resumo: A atividade eletrocorticográfica de ratos albinos foi registrada automaticamente por 72h

consecutivas e, posteriormente, analisada por procedimentos adequados para detectar a presença

de ritmos de 24 h. As atividades beta (vigilia com alerta), teta (sonolência), delta (sono de ondas

lentas) e sigma I (sono sincronizado superficial) apresentaram uma distribuição circadiana evidente.

As acrofases (ponto máximo da curva cosseno ajustada) ocorreram às 23:39; 07:59; 08.39 e 13:25h,

respectivamente. Os episódios de atonia e hipotonia extrema (menos do que 10% do nível médio

do EMG) apresentaram um ritmo de 24h com um pico às 14:18h. A sequência temporal das diver¬

sas fases do sono dentro do período circadiano de repouso, isto é, sonolência, sono de ondas len¬

tas, sono sincronizado superficial e sono paradoxal, é muito semelhante à que ocorre durante o

sono noturno em humanos.

7. CONSALES, C.A; VALENTINI, E.J.G.; ALBAS, A., MENDONÇA, R.M.Z.; FUCHES, R.M.M.; SOARES,

M.A.; PEREIRA, C.A.

The preparation of cultured rabies virus and the production of antiserum for human use.

J.biol.Stand., 16: 27-32, 1988.

Abstract: In this paper we describe a methodology for the preparation of the Pasteur strain of fixed

rabies virus in BHK-21 clone 13 cells and also its use for the production of antisera in horses. The

methodology showed here is simple, rapid, facilitates the attainment of high protective titers, and

the antisera produced are of high quality.

Resumo: Descrevemos uma metodologia para o preparo da cepa Pasteur de virus rábico em células

BHK-21 e o seu uso na preparação de antisoro em cavalos. A metodologia é simples, rápida, permi¬

te a obtenção de altos títulos de anticorpos e um soro de alta qualidade.

8. CORTADAS, J. & RUIZ, I.R.G.

The organization of ribosomal genes in diploid and tetraploid species of the genus Odontophrynus

(Amphibia, Anura)

Chromosoma, 96: 437-42, 1988.

Abstract: The organization of the repeat unit of the ribosomal genes was determined in populations

of Odontophrynus americanas 2n, 4n, O. occidentalis 2n and O. barrioi 2n (Amphibia, Anura) from

South America. Hindlll, EcoRI and BamHI restriction patterns of rDNA from single individuais were

probed with two rDNA clones from Xenopus laevis. Variations in length and sequence were found

among different species, populations and specimens. A general pattern could be established from

the most frequent type of repeat observed in each case, so that diploid populations could be distin-

guished from tetraploid ones. The molecular evolution of variant repats is discussed. It is suggested

that the reduced size of the non-transcribed spacers in the tetraploids from São Paulo could play

a role in the control of rDNA transcription. Distinct restriction maps were tentatively correlated to

the varying localization of active rDNA clusters on metaphase chromosomes of corresponding popu¬

lations.

Resumo: A organização da unidade repetitiva dos genes ribossômicos foi determinada em popula¬

ções de O. americanus 2n e 4n, O. occidentalis 2n, e O. barrioi 2n (Amphibia, Anura) da América

do Sul. Os padrões de restrição obtidos com as enzimas Hind III, Eco Ri, e Bam Hl a partir de DNA

total de cada indivíduo foram sondados com dois clones de rDNA de Xenopus laevis. Encontramos

variações no comprimento e seqüôncia, das unidades repetidas entre diferentes espécies, popula¬

ções e espócimens. Um modelo geral pôde ser estabelecido baseado no tipo de maior freqüência

de repetição observada em cada caso, de modo que as populações diplóides podiam ser distingui¬

das das tetraplóides. Discute-se a evolução molecular da variante repetitiva. Sugere-se que a redu¬

ção de tamanho do espaçador INTS) nos tetraplóides de São Paulo poderia desempenhar um papel

no controle da transcrição do rDNA. Correlacionamos os diferentes mapas de restrição com as lo-

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calizações variadas de agrupamentos de rDNA ativos em cromossomos metafâsicos de populações

correspondentes.

9. DENARO-MACHADO, L; MACHADO, J.C.; LONGO, M.A; OGATA, T.R.P.; DAVID, M.L.V.M.G.; KFOURI,

S.A.; CARNEIRO, S.M.

Heterotransplante de células KB em ratos nude atímicos. II. Estudos morfológicos.'

Acta Oncol. Bras., 8: 21-5, 1988.

Resumo: Estudos morfológicos de microscopia óptica e eletrônica foram realizados em tumores cres¬

cidos em ratos nude atímicos, pós-transplantes de linhagem celular tumoral humana de carcinoma

epidermóide de boca (KB), desde a 1 ? inoculação realizada, até a 8 a passagem seriada do tumor

nesses animais. Os autores descrevem o aumento do volume celular e nuclear, bem como a eleva¬

ção do número de mitoses e uma maior velocidade de crescimento da massa tumoral, à medida

que as passagens seriadas do tumor se sucedem. Também foi observado que, com o progredir des¬

sas passagens, a reação inflamatória por parte do hospedeiro aumenta.

Abstract: Morphological studies of optical and electronic microscopy were done in tumors grown

in athymic nude rats transplanted with human tumor cell line derived from epidermoid mouth carci¬

noma (KB), since the first inoculation till the 8th serial passage of the tumor in these animais. The

authors describe the increase of the cellular and nuclear volume, as well as the increase of the num-

ber of mitoses and a greater speed of growth of the tumoral mass throughout the serial passages.

Also, with the progress of the passages, it has an increase of the inflammatory reaction of the host.

10. DIAS, R.M.D.S.; MANGINI, A.C.S.; TORRES, P.M.A.G.V.; CORRÊA, M.O.A.; LUPETTI, N.; CORRÊA,

EM.A.; CHIEFFI, P.P.

Cryptosporidiosis among patients with acquired immunodeficiency syndrome (AIDS) in the country

of São Paulo, Brazil.

Rev. Inst. Med. trop. São Paulo, 30 (4): 310-2, 1988.

Abstract: Stool samples of 157 patients with AIDS, living in the country of São Paulo, were submit-

ted to several techniques in the search for Cryptosporidium sp. Among the various techniques test-

ed for slide preparation (direct smear, spontaneous sedimentation method, and formol-ether

concentration), the latter, formol-ether concentration, offered the best results, clearly outdoing all

the others. Nineteen samples out of 157 prepared by this technique, after dyeing by the Kinyoun

method or by carbol fuchsin dimethyl sulfoxide, were found to be positive for Cryptosporidium sp.

Resumo: Amostras de fezes de 157 pacientes acometidos pela síndrome de imunodeficiência ad¬

quirida, residentes no município de São Paulo, foram submetidas a diversas técnicas para pesqui¬

sa de Cryptosporidium sp. Entre as diversas técnicas testadas para o preparo das lâminas (esfregaço

direto, método de sedimentação espontânea e concentração pelo formol-éter) a concentração pe¬

lo formol-éter foi a que forneceu melhores resultados, superando nitidamente as demais. Nas 157

amostras preparadas por esta técnica, após coloração pelo método de Kinyoun ou pela fucsina-

carbólica dimetilsufóxido, encontraram-se 19 (12,1%) positivas para oocistos de Cryptosporidium sp.

11. DORCE, V.A.C. & PALERMO-NETO, J.

Effects of ovariectomy and of estrogen administration on some dopamine-related behaviors in the rat.

Brazilian J. Med. Biol. Res., 21: 313-320, 1988.

Abstract: 1. The effects of ovariectomy and of estrogen administration were determined on some

dopamine-related behaviors.

2. Haloperidol withdrawal induced a Progressive increase in locomotion and rearing frequencies

of female rats observed in an open-field. This increase started earlier and was greater in ovariec-

tomized than in sham-operated animais.

3. Repeated administration of increasing doses of 17- -estradiol (from 0.05 to 0.3mg/kg once

daily, for 28 days) significantly decreased apomorphine-induced stereotyped bchavior in ovariec-

tomized rats, 30 min after the last hormone injection.

4. Withdrawal from long-term 17- -estradiol administration increased the sensitivity of the animais

to apomorphine.

5. These results suggest not only an antidopaminergic effect for estrogen but also a possible sum-

mation of estrogen and haloperidol withdrawal effects.

Resumo: 1. Os efeitos da castração e da administração do estrógeno foram determinados em com¬

portamentos relacionados aos sistemas dopaminérgicos.

2. A retirada do haloperidol induziu um aumento progressivo na frequência de locomoção e de

levantar de ratas observadas no campo-aberto. Esse aumento começou logo após a retirada da

droga e foi mais intenso em ratas ovariectomizadas que em ratas falso-operadas.

3. A administração de drogas crescentes de 17- -estradiol (de 0,05 a 0,3 mg/kg uma vez ao

dia, por 28 dias) diminui significativamente o comportamento esteriotipado induzido pela apomor-

fina em ratas ovariectomizadas, 30 minutos após a última injeção do hormônio.

4. A retirada da administração prolongada do 17- -estradiol aumenta a sensibilidade dos ani¬

mais à apomorfina.

5. Esses resultados sugerem não só um efeito dopaminérgico para o estrógeno mas também uma

possível somação dos efeitos da retirada do estrógeno e do haloperidol.

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12. KIKUCHI, O.K.; OKAZAKI, K.; KAWANO, T.; RIBEIRO, A.A.G.F.C.

Estudos ultra-estruturais de embrião de Biomphalaria glabrata Say, 1818.

Publicação IPEN, (203): 1-10, 1988.

Resumo: Os estudos ultra-estruturais 'de embriões de Biomphalaria glabrata (Mollusca: Gastropo-

da), um importante caramujo vetor da esquistossomose, não têm sido explorados. No presente

trabalho foi avaliada a técnica mais adequada para o processamento dos embriões para a micros-

copia eletrônica. A técnica que forneceu resultados bastante promissores foi a fixação dupla em

glutaraldeído 1% mais tetróxido de ósmio 1% em tampão cacodilato 0,05 M (pH 7,4) a 4°C, pré-

-contraste em acetato de uranila 1% durante uma noite e a embebição tanto em resina EPON co¬

mo Polylite sob vácuo. Foram utilizados embriões no estádio de trocófora jovem que se caracteriza

pela intensa organogênese. Alguns aspectos da ultra-estrutura de células embrionárias de B. gla¬

brata são apresentados.

Abstract: Ultrastructural studies of Biomphalaria glabrata embryos (Mollusca: Gastropoda), an im-

portant snail vector of schistosomiasis, has not been explored. In the present work it was evaluted

a suitable electron microscopical technique for embryos Processing. Promising results was obtained

with double fixation in 1% glutaraldehyde plus 1% osmium tetroxide in 0.05 M cacodylate buffer

(pH 7.4), preliminary staining overnight in 1% uranyl acetate and embedding in EPON or Polylite

under vacuum. It was used embryos at young trochophore stage wich is characterized by active

organogenesis. Some ultrastructural aspects of B. glabrata embryos cells are presented.

13. LEBRUN, I.; CAMARGO, A.C.M.; CORRÊA, F.M.A.

Pharmacological effects and metabolism of neurotensin and bradykinin in the isolated rat uterus.

Europ. J. Pharmacol., 148: 231-7, 1988.

Abstract: Neurotensin (NT) and bradykinin (BK) were found to cause contractions of isolated rat

uterus preparations. In 97% of the experiments, acute tachyphylaxis followed soon after the initial

administration of NT. Interrelation between the oxytocic effects of NT and BK was not observed.

Among the NT fragments studied, only NT-19-13) had an oxytocic effect ( 1.0%). All NT fragments

tested induced tachyphylaxis to NT regardless of their efficacy. Using HPLC analysis, NT but not

BK was found to be degraded by the intact rat uterus. A major involvement of a carboxydipeptidase

cleaving at Tyr^-lle 12 is suggested. Carboxyl-blocked neurotensinamide (NT-NH 2 ) was found to be

resistent to proteolysis and not develop tachyphylaxis. No cross-tachyphylaxis was observed be¬

tween NT and NT-NH 2 . The oxytocic effects of NT-NH 2 were markedly potentiated by pre-incubation

with NT or NT-d-11). The results suggest the existence of different receptors for NT and BK in the

uterus, as well as the existence of different receptors or receptor States that interact with NT or

NT-NH 2 in the rat uterus.

Resumo: Tanto a Neurotensina (NT) quanto a Bradicinina (BK) induzem contração em preparações

de útero isolado de rata. Verificou-se que em 97% dos experimentos houve o desenvolvimento

de uma taquifilaxia aguda após a administração de NT. Não se verificou uma interrelaçâo entre

os efeitos acitócicos da NT e BK. Estudando-se fragmentos da NT, somente NT 9.13 possui um efeito

acitócico (1%). Todos outros fragmentos testados também induziram taquifilaxia, utilizando-se análise

em HPLC verificou-se que a NT mas não a BK é degradada pela preparação intacta do útero isola¬

do de rata. Sugere-se um maior envolvimento de uma carboxidipeptidase clivando o peptídeo em

Tir 11 — lie 12 . A neurotensinamida, um análogo carboxil-bloqueado, foi resistente à proteólise e

não desenvolveu taquifilaxia. Não se observou taquifilaxia-cruzada entre NT e NT-NH 2 . Os efeitos

acitócicos da NT-NH 2 foram marcadamente potenciados pela pré-incubação com NT ou NT (1-11).

Os resultados sugerem a existência de diferentes receptores para a BK e NT no útero, bem como

a existência de diferentes conformações de receptores que interagem com NT ou NT-NH 2 .

14. LEBRUN, I.; LEBRUN, F.L.A.S.; AIZENSTEIN, M.L.

Evidence for a role of B-Endorphin in activity of nigrostriatal neurons in the rat.

Brazilian J. Med. Biol. Res., 21: 645-7, 1988.

Abstract: The effects of icv administration of (3 -endorphin on secretory activity of dopaminergic

neurons is described. Homovanilic and dihydroxyphenyl acetic acid leveis in cerebrospinal fluid and

extracts of brain tissue were determined after administration of [3 -endorphin to animais pretreat-

ed or not with naloxone. The results suggest that |3 -endorphin interferes with formation of dopaminer¬

gic metabolites by acting on opioid receptors.

Resumo: Foram estudados os efeitos da administração i.c.v. de/3-endorfina na atividade secretória

dos neurônios dopnérgicos.

Os níveis dos ácidos homovanflico e dihidroxifenilacético foram determinados após a administra¬

ção de /3-Endorfina no fluido cérebro-espinhal e extratos de tecido cerebral em animais pré-tratados

ou não com naloxone. Os resultados sugerem que a /3-Endorfina interfere com a formação dos

metabólitos da dopamina através da ação em receptores apióides.

15. LUCAS, S.

Spiders in Brazil.

Toxicon, 26 (9): 759-72, 1988.

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Abstract: Descriptions of the principal venomous spiders in Brazil, of the genera Phoneutria, Lox-

oscetes, Latrodectus and Scaptocosa, are given, together with a list of species and their geographical

distribution. Detailed information on their habitat, behaviour and venom quantity, symptomatology

of human accidents and epidemiology is given and recommendations are made for the prevention

of accidents. General observations are made on the most common mygalomorph spiders.

Resumo: Sâo fornecidas descrições das principais aranhas peçonhentas brasileiras pertencentes

ao gênero Phoneutria, Loxosceles, Latrodectus e Scaptocosa: uma lista das especies e sua dis¬

tribuição geográfica. Informamos sobre o habitat, comportamento, quantidade de veneno, sin¬

tomatologia dos acidentes humanos, dados epidemiológicos. São dadas recomendações sobre

prevenção dos acidentes e informações gerais sobre as aranhas MYGALOMORPHAE mais comuns.

16. MANCINI, D.A.P.; GERALDES, E.A.; PINTO, J.R.; SOARES, M.A.

Ocorrência de influenza eqüina na Fazenda São Joaquim, São Roque-SP, do Instituto Butantan.

Rev.Fac.Med.Vet.Zootec.Univ.S.Paulo, 25 III: 93-100, 1988.

Resumo: Foi verificada a incidência de influenza eqüina através da positividade de respostas de an¬

ticorpos contra o vírus influenza, cepa A/Eq 2 , em soros de cavalos pertencentes ao Setor de Imu¬

nizações da Fazenda São Joaquim, do Instituto Butantan. Foi observada pequena diferença nas

respostas de anticorpos, tanto no rebanho de produção, como de controle de imunoterápicos; ambos

tiveram decréscimo de títulos de anticorpos no período de 80 dias após o surto. A queda de nível

de anticorpos protetores verificada na análise sorológica dos cavalos indica a baixa proteção que

possuem contra a infecção ou reinfecção.

Abstract: The prevalence of equine influenza was verified by means of the positivity of the antibody

responses to the influenza virus strain A/Eq 2 , on sera taken from horses belonging to São Joa¬

quim Farm, Immunizations Section, of the Instituto Butantan, placed in the State of São Paulo. Lit-

tle differences were observed in antibody responses between horses designated for production and

control of immunotherapeutic agents there was, however a decrease of antibody titles in the peri-

od of 80 days after the outbreak. The drop in the levei of the antibodies observed by serologic anal-

ysis of those horses, indicates the low protection conferred by the antibodies against infection or

reinfection.

17.

MANDELBAUM, F.R. & ASSAKURA, M.T.

Antigenic relationship of hemorrhagic factors and proteases isolated from the venoms of three species

of Bothrops snakes.

Toxicon, 26 141: 379-85, 1988.

Abstract: By comparative studies of the immunological properties of the metalloproteins (hemor¬

rhagic factors and proteases) isolated from the venoms of Bothrops jararaca, Bothrops neuwiedi

and Bothrops moojeni, it was found that the hemorrhagic factors contain common antigenic de-

terminants and the proteases were immunologically distinct entities. The rabbit antisera raised for

the hemorrhagic factors not only neutralized the hemorrhagic activities of the respective factors

but also activities of the other hemorrhagic factors. Although the homology among these proteins

are not yet known, these studies have shown that the hemorrhagic factors must have a similar

partial structure which includes the catalytic hemorrhagic active site.

Resumo: Estudos comparativos das propriedades imunológicas de metaloproteínas (fatores he¬

morrágicos e proteases) isolados dos venenos de Bothrops jararaca, Bothrops neuwiedi e Both¬

rops moojeni, mostraram que os fatores hemorrágicos contêm determinantes comuns enquanto

que as proteases são imunologicamente distintas. Os antissoros específicos produzidos em coe¬

lhos contra os fatores hemorrágicos, foram capazes de neutralizar tanto a atividade hemorrágica

dos respectivos fatores como as atividades dos outros fatores hemorrágicos. Embora a estrutura

molecular destas proteínas ainda não seja conhecida, estes estudos mostraram que os fatores he¬

morrágicos devem apresentar homologia parcial correspondente ao sítio catalítico da molécula.

18. MANDELBAUM, F.R.; REICHL, A.P.; ASSAKURA, M.T.

Hemorrhagic factors from the venoms of two species of Bothrops snake.

SIMPÓSIO ANUAL DA ACIESP SOBRE TOXINAS PROTEICAS, 12. 1988, v.1, p. 9-24, 1988.

Abstract: From two venoms of Bothrops species were isolated five hemorrhagic factors, NHF a and

NHF b from the venom of 6 . neuwiedi and HF,, HF 2 and HF 3 from the venom of B. jararaca. Like

the other hemorrhagic principies isolated from snake venoms, they are heat labile metalloproteins,

unstable at acidic médium and inhibited by metal chelator compounds such as EDTA, EGTA and

1.10-phenanthroline. They have no action on arginine synthetic substrates and they are not affect-

ed by inhibitors of serine proteases such as DFP, TLCK and TPCK. They are acidic proteins whose

pis range from 3.9 to 4.3 and the molecular weights range from 46,000 to 62,000. The minimum

hemorrhagic dose (MHD), the amount of protein which produces a hemorrhagic spot of 1 square

cm on rabbit skin was used to compare activities of these factors. NHF b , NHF,, HF 3

also diftei

required 0.2, 4.8, 15,20 and 100ng of protein, respectively. There are also differences concerning

the action on casein. The proteolytic action of HF, and NHF a were higher than the others. The

hemorrhagic factors hydrolyze similarly the B-chain of oxidized insulin. The hydrolysis occurs preferen-

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10 11 12 13 14 15 16

cially, in bonds where leucine and phenylalanine contribute with the amino group. Comparative

studies of these isolated hemorrhagic factors with horse monovalent antivenoms revealed that they

were not identical but immunologically related proteins. This relation ship was confirmed with rab-

bit specific antisera raised to these hemorrhagic factors. Also the specific antisera not only neutral-

ized the hemorrhagic activities of the homologous proteins, but also the hemorrhagic activities of

the heterologous isolated factors. These specific sera were also able to recognize hemorrhagic proteins

and neutralize partially the hemorrhagic activity in several venoms of the Crotalinae subfamily.

Resumo: Do veneno de B. jararaca foram isolados os fatores hemorrágicos HF!. HF 2 e HF 3 e do

veneno de B. neuwiedi NHF a e NHF b . Semelhantemente a outros fatores hemorrágicos isolados

de venenos de serpentes, estes são metaloproteases instáveis a pHs ácidos e inibidas por com¬

postos quelantes tais como EDTA, EGTA e o-fenantrolina. Essas proteases não agem sobre subs¬

tratos sintéticos da arginina e não são afetadas por inibidores de serina proteases tais como DFP,

TLCK e TPCK. São proteínas ácidas cujos pis variam de 3,9 a 4,3 e seus pesos moleculares são

da ordem de 46.000 a 62.000. A D.M.H. (dose mínima hemorrágica), que corresponde à quanti¬

dade de proteína necessária para produzir uma área hemorrágica de Icm 2 na derme de coelho,

foi de 0,2; 4,8; 15; 20 e 100 ng para NHF b , NHF a , HF 3 , HF 2 e HF,, respectivamente. Igualmente

na atividade sobre a caseína foram detectadas algumas diferenças. A ação proteolítica de HF 2 e

NHF a foi maior do que os outros fatores. Esses fatores hemorrágicos hidrolisam semelhantemen¬

te a cadeia B de insulina oxidada. A hidrólise ocorre preferencialmente nas ligações onde leucina

e fenilalanina participam com o grupo amino. Estudos comparativos desses fatores hemorrágicos

usando antivenenos produzidos em cavalos mostraram que não são proteínas idênticas mas imu-

nologicamente relacionadas. Estes dados foram confirmados por soros anti-hemorrágicos especí¬

ficos produzidos em coelhos. Esses antissoros neutralizaram a atividade hemorrágica do fator

homólogo, bem como dos outros fatores hemorrágicos isolados. Estes soros específicos ainda fo¬

ram capazes de reconhecer proteínas hemorrágicas e também neutralizar parcialmente a ativida¬

de hemorrágica de vários venenos da subfamília Crotalinae.

19. MARQUES, N.; AFECHE, S.C.; BENEDITO-SILVA, A.A.; CIPOLLA-NETO, S.; FORTUNATO, G.; PALUDET-

n, L.A.; RECINE, E.G.I.G.;SCHOTT, C.; MENN A-BAR RETO, L.S.

Chronobiology courses at the University of São Paulo/Brasil; a four-year experience.

Chronobiologia, 15: 235-42, 1988.

Abstract: The Grupo Multidisciplinar de Desenvolvimento e Ritmos Biológicos — GMDRB (Mul-

tidisciplinary Group of Development and Biological Rhythms) of the University of São Paulo (USP)

— is the first group in Brazil to have Chronobiology as its central topic of research.

Besides research activities the group are involved with the spread of basic information on Chronobi¬

ology through graduate and post — graduate courses.

This article describes the didatic experience on graduate and post — graduate courses on Chronobi¬

ology in São Paulo, Brazil.

Resumo: O Grupo Multidisciplinar de Desenvolvimento e Ritmos Biológicos (GMDRB) do Instituto

de Ciências Biomédicas da USP é o.primeiro Grupo brasileiro que tem como tema central de pes¬

quisa a Cronobiologia. Além dos trabalhos de pesquisa o grupo desenvolve também atividades

de divulgação de informações básicas sobre cronobiologia através da realização de cursos a nfvel

de graduação e pós-graduação.

Este artigo descreve a experiência didática de um curso de Cronobiologia, para alunos já gradua¬

dos e pós-graduandos, realizado anualmente em São Paulo, Brasil.

20. MOURA, J.W.; STOCCO DOS SANTOS, R.C; DAGLI, M.L.Z.; D'ANGELINO, J.L.; BIRGEL, E.H.; BE-

ÇAK, W.

Chromosome aberrations in cattle raised on bracken fern pas*”'"

Experientia, 44: 785-8, 1988.

Abstract: Thirteen cows maintained on natural bracken fern IPteridium aquilinum) were analyzed

cytogenetically. The frequency of structural chromosome aberrations detected in peripheral blood

cells was significantly higher when compared to that detected in animais raised on pasture con-

taining no bracken fern. We discuss the clastogenic action of fern and its synergistic action with

infection by type 2 and 4 papilloma virus in the same animais.

Resumo: Analisamos citogeneticamente 13 bovinos mantidos em pastagem natural de samam¬

baia Pteridium aquilinum. A freqüência de aberrações cromossômicas estruturais encontrada, a

nível de sangue periférico nesses animais, é altamente significante, quando comparada a de ani¬

mais de áreas sem samambaia. Discute-se a ação clastogênica da samambaia e seu efeito sinergi -

camente à infecção, desses animais, pelo vírus do papiloma bovino tipo 2 e 4.

21. SALOMÃO, M. da G. & SAWAYA, P.

Morphology and regeneration of the gonopodium in poecilíd fish.

Zooi Anz., 220 (3/4) S.135-143, 1988.

Abstract: In the present study the development of the gonopodium was accompanied and its histology

245

in adult of Poecilia reticulata and Phalloceros caudimaculatus was described. Regeneralion ot the

gonopodium in fresh water and sea water was examined. The gonopodium of P. reticulata terminates

in a globular expansion while in P. caudimaculatus the gonopodial extremity is bifurcated. Variation

with age in terminal portion of the gonopodium is marked in P. caudimaculatus. In both species

the organ is composed of hyaline conjuctive tissue, the regeneration after sectioning is not com¬

plete and the gonopodium is not functional in the transmission of sperm.

Resumo: No presente estudo o desenvolvimento do gonopódio de Poecilia reticulata e Phalloceros

caudimaculatus foi acompanhado, a histologia do órgão em animais adultos foi descrita. A regene¬

ração do gonopódio em água doce e água do mar foi examinada. 0 gonopódio de P. reticulata

tem sua parte terminal numa expansão globular, enquanto em P. caudimaculatus essa extremida¬

de é bifurcada. A variação dessa terminalização em função da idade é marcante em P. caudimacu¬

latus. Em ambas as espécies o órgão é composto de tecido conjuntivo hialino, a regeneração após

secção não é completa e o gonopódio não é funcional na transmissão do esperma.

22. SALVADORI, D.M.F.; RIBEIRO, L.R.; PEREIRA, C.A.B.; BEÇAK, W.

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice.

Mutation Research, 204: 283-7, 1988.

Abstract: Male mice dermally exposed to single or multiple treatment (5 days/2 weeksl showed

that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ

cells (primary spermatocytes) was related to the type of treatment and dose used.

Statiscally significam increases of chromosome aberrations in bone marrow cells occurred after

single treatment 1500 and 2000 mg/kg body wtl when chromatid gaps were included and after

multiple treatment 1250 and 500 mg/kg) when they were excluded. No dose-response relation-

ships were observed for either treatment. In germ cells, malathion induced a significam increase

of univalents in both types of treatment but structural chromosome aberrations were induced only

by multiple treatment.

Malathion induced a significat decrease of the mitotic Índices in the bone marrow.

Resumo: Camundongos machos expostos na derme a tratamentos simples ou múltiplos 15 dias/2

semanasl mostraram que a habilidade do malation em induzir aberrações cromossômicas em cé¬

lulas somáticas (medula óssea) e germinativas (espermatócitos primários) está relacionada ao tipo

de tratamento e dose usada.

Aumentos estatísticos significantes de aberrações cromossômicas em células da medula óssea

ocorreram após tratamento único (500 e 2000 mg/kg peso) quando as falhas cromatídicas foram

incluídas e após tratamento múltiplo (250 e 500 mg/kg) quando elas eram excluídas. Não foram

encontradas relações dose-resposta para qualquer tratamento. Em células germinativas o mala¬

tion induziu um aumento significante de univalentes em ambos tipos de tratamento mas aberra¬

ções cromossômicas estruturais foram induzidas somente por tratamento múltiplo.

O malation induziu um decréscimo significante dos índices mitóticos na medula óssea.

23. SERTIÉ, J.A.A.; BASILE, A.C.; PANIZZA, S.; M ATI DA, A.K.; ZELNIK, R.

Pharmalogical assay of Cordia verbenacea; Part 1. Anti-inflammatory activity and toxicity of the

crude extract of the leaves.

Planta médica, (1): 7-10, 1988.

Abstract: Anti-inflammatory effects and toxicity of lyophilized extracts of the leaves of Cordia ver¬

benacea DC (Boraginaceae) were investigated through various experimental models in rats. The

crude drug significantly inhibited the carrageenin-induced edema proportionally to doses ranging

from 0.59 to 2.98 mg/kg.Repeated administration of the extract at doses of 1.24 mg/kg for 6-d

period similarly reduced the granuloma formation. This response appears indentical to the one from

calcium phenylbutazone. The same dose of the crude extract reduced the vascular permeability

response to histamine. Toxicological experiments indicated a very low toxicity, confirmed by sub-acu-

te tests, the LD 50 value (LD 60 = 131.6 mg/kg) being 100 times that of the ED 50 (ED 50 = 1.24

mg/Kg).

Resumo: Os efeitos anti-inflamatório e toxicidade dos extratos brutos das folhas de Cordia verbe¬

nacea DC (Boraginacea) foram investigados por vários modelos experimentais em ratos. O extrato

bruto inibiu, significativamente, o edema induzido por carrageenina. A administração repetida do

extrato reduziu a formação de granuloma e resposta da permeabilidade vascular à histamina. Os

ensaios toxicológicos indicaram uma baixa toxicidade.

24. SILVA, A.M.M. da; COSTA, H.H.; TAKEHARA, H.A.;MOTA, I.

Trypanosoma cruzi: advantagens of isolating bloodstream trypomastigotes by the carboxy methyl

cellulose method.

Trans. roy Soc. trop. Med. Hyg., 82: 715-8, 1988.

Abstract: Bloodstream trypomastigotes were isolated from blood of A/Sn mice 7 d after infection

with 10 5 Trypanosoma cruzi Y strain. Red blood cells were removed by centrifugation and hypo-

tonic shock and platelets and leucocytes by passage through a carboxy methyl cellulose column.

Binding of trypomastigotes to the resin was prevented by including 10% normal mouse serum in

the eluting buffer. Im such conditions, more than 90% of the parasites applied to the columns were

246

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10 11 12 13 14 15 16

recovered, free of white blood cells and platelets. A comparative study of the pre and post-separation

trypomastigotes showed that both bad the same infecting capacity, ability to evade destruction by

the complement system, and antigenic profile.

Resumo: Tripomastigotas sanguíneos fo.ram isolados do sangue de camundongos A/Sn infectados

com T. cruzi 7 dias antes. As hemácias foram removidas por centrifugação e choque hipotônico

e as plaquetas e leucócitos por passagem através de uma coluna de carboximethil celulose. A ade¬

são dos tripomastigotas à resina foi impedida pela inclusão de 10% de soro normal homólogo no

tampão de eluição. Nestas condições mais de 90% dos parasitas colocados na coluna foram recu¬

perados, isentos de hemácias e plaquetas. Um estudo comparativo dos tripomastigotas antes e

depois da separação mostrou que ambos possuíam a mesma capacidade infectante, a mesma ca¬

pacidade de resistência à lise mediada pelo Ceo mesmo perfil antigênico.

25. SILVA, A.M.M. da; BRODSKYN, C.I.; TAKEHARA, H.A.; MOTA, I.

Comparison between the antigenic composítion of bloodstream and cell culture-derived

trypomastigotes of Trypanosoma cruzi.

Brazilian J.Med.Biol.Res., 21: 991-3, 1988.

Abstract: Antigens of bloodstream and cell culture-derived trypomastigotes of T cruzi were com-

pared by western blotting using sera of chronic chagasic patients as a source of antibodies. The

ímmunoblots demonstrated that the two forms display extensive homology except for the 85-and

52 kDa bands. These antigens were more strongly stained in culture-derived trypomastigotes.

Although the reported differences are not related to major antigens, these results might offer an

explanation for previous results showing that culture derived trypomastigotes are more antigenic

and infective in vitro than blodstream trypomastigotes.

Resumo: Foi feito um estudo comparativo dos antígenos obtidos de tripomastigotas sanguíneos ou

de tripomastigotas de cultura por meio de "immunobloting" usando-se soros de pacientes chagá-

sicos como fonte de anticorpos. Os "Ímmunoblots" mostraram que as duas formas apresentam

homologia intensa exceto nas bandas de 85 e 52kDa, Estes antígenos coravam-se mais fortemen¬

te nos tripomastigotas obtidos de cultura. Embora estas diferenças não sejam nos antígenos prin¬

cipais, estes resultados talvez ofereçam uma explicação para estudos anteriores mostrando que

os tripomastigotas derivados de cultura são mais antigênicos e infectantes in vitro do que os tripo¬

mastigotas sanguíneos.

26. SOARES, M.F.M.; OLIVEIRA, E.B.; MOTA, I.; MACEDO, M.S.

Suppresion of IgE antibody production by Ascaris suum extract: characterization of suppressive

componentls).

Brazilian J.Med.Biol.Res., 21: 527-9, 1988

Abstract: Partíal characterization of the suppressive componentls) of A. suum extract that is (are)

responsible for damping production of IgE antibody to ovalbumin was performed by physical and

Chemical methods. Digestion of the whole extract with trypsin and chymotrypsin completely abolished

the suppressive activity. Oxidation with sodium metaperiodate or heating at 56°C, however, had

no effect. These results indicate that the integrity of heat-stable protein(s) present in the crude

extract is essential for its suppressive effect. In addition, the carbohydrate moiety does not seem

to play an important role in this effect.

Resumo: Os componentes supressores do extrato de A. suum que agem sobre a produção de an¬

ticorpos IgE foram caracterizados por técnicas físicas e químicas. A digestão do extrato total com

tripsina e quimotripsina aboliu completamente a atividade supressora. Oxidação por metaperioda-

to ou aquecimento a 56°C não tiveram efeito. Estes resultados indicaram que a integridade das

proteínas presentes no extrato bruto é essencial para o efeito supressor e que os componentes

carboidratos não parecem ter uma função importante no efeito supressor.

27. SOARES-SCOTT, MD.; TRAJTENGERTZ, I.; SOMA, M.; BEÇAK, M.L.

C. and AgAs bands of the octaploid untanha frog Ceratophrys dorsata (C. aurita) (8n=104, Am-

phibia. Anura).

Rev. bras.Genet., 11 (3): 625-31, 1988.

Abstract: Ceratophrys aurita IC. dorsata), a rare octaploid species of Brazilian anuran (8n = 104),

has 9 NORs distributed among distinct chromosome groups 16 and 11) with centromeric and telo-

meric C bands, as analyzed in one male.

This species also presents interpopulational variability of DNA content. The NOR banding pattern

shows the occurrence of interspecific polymorphism as compared to C. ornata. The number and

distribution of NORs, as well as the variability in DNA content are caused by postploidy rear-

rangements.

Resumo: Ceratophrys aurita (C. dorsata), uma espécie octaplóide de anuros brasileiros, tem 9 NORs

distribuídos entre dois grupos distintos de cromossomos (6 e 11) com bandas C centroméricas

e teloméricas. Esta espécie apresenta variabilidade interpopulacional do conteúdo total de DNA.

Seu padrão de bandamento NOR indica haver polimorfismo interespecífico, quando comparado

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10 11 12 13 14 15 16

com C. ornata. O número de distribuição de NORs, bem como a variabilidade do conteúdo total

de DNA foram explicados como causados por rearranjos postpoliploidia.

28. TAKEHARA, H.A.; CARDOSO, D.F.; SILVA, A.M.M. da; MOTA, I.

Lytic antibodies elicited by Trypanosoma cruzi infection recognize epitopes present on both blood-

stream trypomastigote and epimastigote forms of parasite.

Rev.lnst.Med.trop.São Paulo,30 15): 351-6, 1988

Abstract: Sera of Chagas'disease patients containing anti-T cruzi lytic antibodies were submitted

to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimastigote

or trypomastigote forms of the parasite. Epimastigotes were obtained from culture at the exponen-

tial growth phase and the trypomastigotes from blood of infected and immunosuppressed mice.

Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifuga-

tion. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was

performed by passing sera from chagasic patients through an immunoadsorbent column contain¬

ing either epimastigotes or trypomastigotes antigens. Antibodies bound to the column were eluted

with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype

and lytic activity. The results showed that anti-I cruzi lytic antibodies present in sera from chagas¬

ic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomastigote

and epimastigote forms.

Resumo: Soro de pacientes com doença de Chagas na fase crônica foram submetidos à cromato-

grafia de afinidade com Sepharose 4B conjugada com um extrato antigênico obtido de formas

epimastigotas ou tripomastigotas de T.cruzi: os epimastigotas foram obtidos de cultura na fase

exponencial de crescimento e os tripomastigotas de sangue de camundongos infectados e imu-

nossuprimidos. Os antígenos de ambas formas parasitárias foram obtidos por'tratamento dos pa¬

rasitas por ultra-som, seguido de centrifugação.

A cromatografia de afinidade foi feita passando-se os soros chagásicos através de uma coluna

de imunoadsorvente contendo antígenos de epimastigotas ou tripomastigotas. Os anticorpos fo¬

ram eluldos da coluna com tampão glicina 0,2 M pH 2,8 a 4°C. Os anticorpos eluídos foram anali¬

sados quanto ao seu isótipo e atividade lítica. Os resultados mostraram que os anticorpos anti-T

cruzi com atividade lítica presentes em soros chagásicos estão localizados no isótipo IgG e reco¬

nhecem epitopos presentes tanto nos tripomastigotas quanto nos epimastigotas.

29. TOFFOLETTO, O.; CAMARGO, A.C.M.; OLIVEIRA, E.B.; METTERS, K.M.; ROSSIER, J.

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A.

Biochimie, 70: 47-56, 1988.

Abstract: Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by im-

munoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known

to hydrolyse the Phe 5 — Ser 6 — bond of bradykinin and the Arg 8 -Arg 9 bond of neurotensin has

been shown to produce, by a single cleavage. [Leulenkephalin or [Metlenkephalin from small

enkephalin-containing peptides. Enkephalim formation could be inhibited in a concentration de¬

pendem manner by the alternative substrate bradykinin. The optimal substrate size was found to

be 8-13 amino acids, with enkephalin the only product released from precursors in which this se-

quence is immediately followed by a pair of basic residues. However, the specificity constants

IK ca /K m ) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin

B are of the same order. Taken together, these results indicate that the substrate amino acid se-

quence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase

A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liber-

ate enkephalins from metorphamide and dynorphin V8 .

Resumo: Endooligopeptidase A altarnonte purificada, por cromatografia de imunoafinidade, a par¬

tir da fração citosólica do cérebro de boi, foi caracterizada como uma tiol endopeptidase. Mostrou-se

que esta enzima, que hidrolisa a ligação Phe 5 -Ser 6 da bradicinina e a ligação Arg 8 -Arg 9 da neuro-

tensina, produz [Leu] encefalina ou [Met] encefalina por hidrólise de uma única ligação peptídica

em pequenos peptídeos contendo encefalina. A formação de encefalina pode ser inibida pelo subs¬

trato alternativo bradicinina de forma dependente da concentração. Foi achado que o tamanho

ótimo do seu substrato é 8-13 aminoácidos, sendo a encefalina o único produto liberado de pre¬

cursores nos quais essa sequência é imediatamente seguida por um par de resíduos básicos. To¬

davia, as constantes de especificidade (Kcat/Km) obtidas para a hidrólise de bradicinina, neurotensina

e dinorfina B pela endooligopeptidase A são da mesma ordem. Juntos, esses resultados indicam

que a sequência de aminoácidos do substrato não é o único fator que determina o sitio de cliva-

gen dessa enzima. Finalmente, endooligopeptidase A e metaloendopeptidase EC 3.4.24.15 são

duas enzimas diferentes. A última não é capaz de liberar encefalinas de metofarmida e dinorfina 1-8.

30. TOFFOLETTO, O.; METTERS, K.M.; OLIVEIRA, E.B.; CAMARGO, A.C.M; ROSSIER, J.

Enkephalin is liberated from metorphamide and dynorphin A,_ 8 by endo-oligopeptidase A, but not

by metalloendopeptidase EC 3.4.24.15.

Biochem.J., 252: 35-8, 1988.

Abstract: lt has been previously reported that both the cysteinyl-endo-oligopeptidase A and the

248

í, | SciELO

metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-

containing peptides, including dynorphin A,_ 8 . The present study shows that only endo-

oligopeptidase A is able to generate [Leu 5 ]enkephalin and [Met 5 ]enkephalin from dynorphin A^g

and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydro-

lyses the specific EC 3.4.24.15 substratd aTM-benzoyl-GlyAla-Ala-Phe p-aminobenzoate, nor isinhibited

by thespecificEC 3.4.24.15 inhibitor N-f 1 ffíSTcarboxy-2-phenylethylLo-Ala-Ala-Phe-p-aminobenzoate.

Resumo: Foi relatado previamente que tanto a cisteinil-endo-oligopeptidase A como a metaloen-

dopeptidase EC 3.4.24.15 são capazes de gerar encefalina de uma série de peptídeos que contém

encefalina, inclusive de dinorfina A^g. O presente trabalho mostra que somente a endooligopep-

tidase A é capaz de gerar [Leu 6 ] encefalina e [Met 5 ] encefalina a partir de dinorfina A,. 8 e de me-

torfamida, respectivamente. Ele mostra também que a endo-oligopeptidase A nem hidrolisa o

substrato a N-benzoil-Gly-Ala-Ala-Phe p-aminobenzoato, específico paraEC3.4.24.15,nem é inibi¬

da porN-[1 (RS)-carboxi-2-feniletil]ci'-Ala-Ala-Phe p-aminobenzoato,inibidor específico de EC 3.4.24.15.

31. TRONCONE, L.R.P.; FERREIRA, T.M.S.; BRAZ, S.; SILVEIRA FILHO, N.G.; TUFIK, S.

Reversal of the increase in apomorphine-induced stereotypy and aggression in REM sleep deprived

rats by dopamine agonist pretreatments.

Psychopharmacology 94: 79-83, 1988.

Abstract: REM, sleep deprivation (REMSD) induces augmented responses to dopaminergic agonits.

Prolonged administration of neuroleptics induces a similar State, probably by the production of su-

persensitivity of dopaminergic receptors. Such a supersensitive State could be induced by REMSD

as a result of impairment of dopamine neurotransmission. In order to test this hypothesis, bromocrip-

tine, nomifensine, amphetamine, L-dopa, imipramine and electroconvulsive shock (ECS) were ad-

ministered to rats during REMSD, and aggressive and stereotyped behaviors were measured.

Amphetamine and L-dopa pretreatment attenuated the increases in apomorphine-induced stereotypy

and aggression in REMSD rats, but ECS selectively reduced apomorphine-induced aggression. The

orther drugs tested were ineffective on both behavioral tests. Such a selective action may reflect

different effects of ECS on different dopaminergic systems such as those involved with stereotypy

and aggression. The results suggest that REMSD induces an increase in dopaminergic sensitivity

which may be reversed by pretreatment with some dopaminergic agonists.

Resumo: A privação do sono REM (PSREM) induz a um aumento da resposta comportamental a

agonistas dopaminergicos. A administração prolongada de neurolépticos leva a efeitos análogos

provavelmente por causar supersensibilidade de receptores, sendo que o mesmo efeito pode estar

ocorrendo pela PSREM. Para se testar esta hipótese administramos bromocriptina, nomifensina,

anfetamina, L-DOPA, imipramine e eletrochoque convulsivo (ECO aos ratos durante a PSREM e

avaliamos os comportamentos agressivo e estereotipado induzidos por apomorfina. Os resultados

mostraram que a anfetamina e a L-DOPA reverteram o aumento observado em ambos os compor¬

tamentos enquanto que o ECC reduziu apenas a agressividade. As outras drogas foram ineficazes

em alterar estes comportamentos aumentados. 0 efeito seletivo do ECC sobre a agressividade pode

refletir a ação seletiva deste tratamento sobre sistemas diferentes de neurotransmissâo. De forma

global os resultados também corroboram a hipótese de supersensibilidade dopamínergica decor¬

rente da PSREM.

32. UMEKITA, L.F.; TAKEHARA, H.A.; MOTA, I.

Role of the antibody Fc in the immune clearance of Trypanosoma cruzi.

Immunology Letters, 17: 85-9, 1988.

Abstract: Passive transver of immune serum obtained from mice chronically infected with Trypano¬

soma cruzi to mice containing circulating bloodstream trypomastigotes induces a very fast clear¬

ance of the parasites. Comparison of Tripomastigotes clearance in normocomplementemic and

C5-deficient mice showed no difference. IgG fraction obtained from immune serum was very effi-

cient at inducing complement-mediated lysis and immune clearance of bloodstream trypomastigotes,

whereas its Fc-missing F(ab') 2 fragments, although able to induce lysis, were unable to induce clear¬

ance. It is suggested that the immune clearance of bloodstream trypomastigotes is dependent on

the antibody Fc region and that complement-mediated lysis is not a prerequisite for elimination of

the parasites from circulation.

Resumo: Transferencia passiva de soro imune obtido de camundongos com infecção crônica com

T. cruzi para camundongos receptores com tripomastigotas na circulação resulta na rapida remo¬

ção dos parasitas da circulação. A velocidade da remoção dos parasitas da circulação foi igual

em camundongos normais ou deficientes em C5. A fração IgG obtida de soro imune foi muito efi¬

ciente para induzir lise mediada por Cea remoção dos parasitas da circulação. Os fragmentos

F(ab') 2 obtidos da IgG mostraram-se capazes de induzir lise porém incapazes de induzir a remo¬

ção dos parasitas no sangue periférico. Sugere-se que a remoção imune dos tripomastigotas san¬

guíneos depende do fragmento Fc e que a lise mediada pelo C não é um pré-requisito para a remoção

dos parasitas da circulação.

249

SciELO

10 11 12 13 14 15 16

(1987)"

33. ÁVILA, S.C.; ROHM ROSA, R.; IIZUKA, H.; VIEIRA, E.G.J.; FERNANDES, W.

Determinação da atividade letal do veneno de Crotalus durissus collilineatus (Amaral, 1926) em

Mus muscuius Linnaeus, 1758.

An.Farm.Quim. Dezembro — 1987.

Resumo: Foi determinada, em termos do DL50, a toxicidade do veneno de Crotalus durissus col¬

lilineatus, em camundongos, através da inoculação pelas vias intraperitoneal e intravenosa. Verificou-

se que a intraperitoneal é a mais sensível à ação tóxica da referida peçonha. Por outro lado, foi

também constatado que, sucessivas extrações podem provocar variação da atividade letal da mesma,

além de reduzir o índice de sobrevida das serpentes.

Abstract: The lethal activity, in terms of LD50, of the venoms of Crotalus durissus collilineatus was

determined in mice by intraperitoneal and intravenous route. It was found that both routs produce

satisfactory results, however it was observed that the intraperitoneal one is more effective.

By the other hand, sucessive venom extrations may provoke a variation in the toxicity leveis of the

venom, besides, redution of the snakes survival rate.

34. NAKAMURA, D.; WACHTEL, S.S.; LANCE, V; BEÇAK, W.

On the evolution of sex determination.

Proc.R.Soc.Lond. B, 232: 159-80, 1987.

Abstract: Female mice rejet skin grafts from intrastrain males because of the H-Y, transplantation

antigen. Those females produce antibodies that recognize a male-specific cell-surface antigen in

serological tests. The serologicaL antigen has also been called 'H-Y', but there is evidence that

the two antigens are distinct. We therefore refer to the transplantation antigen as H-Yt, or trans¬

plantation H-Y, and to the serological antigen as serological H-Y, or simply H-Y, without prejudice

whether these are the same or related or separate antigens.

In this study, sex-specific expression of serological H-Y antigen was found in 25 new vertebrate

species representíng each of seven major vertebrate classes. There was a strong correlation be-

tween expression of H-Y and occurrence of the heterogametic-type gonad, although unusual pat-

terns of H-Y expression were noted in cases of temperature-influenced sex determination and systems

representing possible transition from one mode of heterogamety to the other.

Male and female heterogamety are found side-by-side in certain freshwater toothed carps; and dis¬

tinct sex chromossomes have been recognized certain amphibians, even though they are not ap-

parent in certain reptiles and primitive birds. In seven pophidian species, in which the female is

the heterogametic sex, H-Y was detected in the female; and in three species of Ranidae in which

the male is heterogametic, it was detected in the male. In three species of cartilaginous fish and

in one of the cyclostomes, in which heterogamety has not been ascertained, H-Y was detected

in the male, suggesting that those primitive fishes are male-heterogametic. Evidently, then, heter¬

ogamety and sex-chromosome heteromorphism are polyphyletic, although certain sex-determining

genes may be held ín common among the diverse taxonomic groups.

Resumo: Camundongos fêmeas rejeitam transplante de pele de machos da mesma linhagem, de¬

vido ao antlgeno de transplante H-Y. Essas fêmeas produzem anticorpos que reconhecem um antí-

geno de superfície celular, especificamente masculino, em testes sorológicos. O antígeno sorológico

também foi denominado "H-Y" mas existem evidências de que os dois antígenos são distintos.

Nós, portanto, nos referimos ao antígeno de transplante como H-Yt ou H-Y de transplante e ao

antígeno sorológico como H-Y sorológico ou simplesmente H-Y, sem implicações de que eles se¬

jam iguais, relacionados ou antígenos separados.

Nesse estudo, a expressão do antígeno sexual H-Y sorológico, foi encontrado em 25 novas espé¬

cies de vertebrados, representando cada uma das sete grandes classes de vertebrados. Observou-

-se uma forte corrolação entre expressão do H-Y e ocorrência de gônada do tipo heterogamótico,

apesar de que padrões incomuns de expressão do H-Y foram notadas em casos de determinação

do sexo influenciado pela temperatura e em sistemas representando possível transição de um mo¬

do de heterogametia ao outro.

Heterogametia masculina e feminina foi encontrada lado a lado, em certas carpas dentadas de

água doce, e cromossomos sexuais distintos foram reconhecidos em certos anfíbios, apesar deles

não serem aparentes em certos répteis e aves primitivas. Em, sete espécies de ofídios, nos quais

a fêmea é o sexo heterogamético, o H-Y foi detectado na fêmea, e em três espécies de Ranidae

nas quais o macho é heterogamético ela foi detectada no macho. Em três espécies de peixes carti¬

laginosos e em um de ciclostomos nos quais a heterogametia não foi confirmada, o H-Y foi detec¬

tado no macho, sugerindo que esses peixes primitivos são de heterogametia masculina.

Evidentemente, portanto, a heterogametia e o heteromorfismo dos cromossomos sexuais são poli-

filéticos, apesar de que certos genes determinadores do sexo podem ser mantidos em comum en¬

tre os diversos grupos taxonômicos.

Trabalhos que não constaram do v 50(3): 103-110, 1988.

250

SciELO

10 11 12 13 14 15 16

35. RÉ, L. & KAWANO, T.

Effects of Laurus nobilis (Lauraceael on Biomphalaria glabrata (Say, 1818).

Mem.lnst.Oswaldo Cruz, 82 (supl.4): 315-20, 1987.

Abstract: Experiments were carried out using aqueous extracts from leaves and flowers of Laurus

nobilis on Biomphalaria glabrata. Treatments were performed on blastula stage (+ ou - 15h after

first cleavage) and on adult snails (11-18mm). In both instances they were exposed for 24h to differ-

ent concentrations of the extracts on snails (200 to 2500 ppm) and embryos (20 to 300 ppm)

at 25 + ou - 1°C. The embryos were observed for a period of 20 days after treatment and the

snails for 10 days.

Results obtained with leaf aqueous extracts have shown a degree of toxicity on embryos starting

at a concentration of 125 ppm, the flower extract being effective at,35 ppm. The malformation

obtained with the different concentrations falis into the unespecific type category, however some

cephalic and Shell malformations were found in embryos treated with concentrations over 50 ppm

(leaves) and 25 ppm (flowers).

The LD90 on adult snails obtained by treatments with flower and leaf extract was observed at con¬

centrations of 340 ppm and 1900 ppm respectively.

Resumo: Experimentos foram realizados utilizando-se extrato aquoso de folhas e flores de Laurus

nobilis (louro) em Biomphalaria glabrata. Embriões no estágio de blástula (+ ou - 15h, após a 1 a

clivagem) e adultos (11-18 mm de diâmetro) foram tratados com os extratos, durante 24 horas.

As concentrações dos extratos utilizados para os caramujos foram de 200 a 2.500pp, e para os

embriões foram de 200 a 300 ppm, à temperatura de 25 + ou - 1 °C. A observação diária dos

embriões foi realizada durante 20 dias e para os caramujos durante 10 dias.

Resultados obtidos com embriões demonstraram que o extrato aquoso das folhas mostrou um grau

de toxicidade a partir de 125 ppm, e das flores a partir de 35 ppm. A principal malformação obtida

foi o tipo inespecífico encontrado nas diferentes concentrações, entretanto algumas malformações

cefálica e de concha foram observadas nas concentrações acima de 50 ppm (folhas) e 25 ppm (flores)

O DL90 obtido em caramujo após o tratamento com os extratos de flores e folhas foi de respecti¬

vamente 340 ppm e 1900 ppm.

251

SciELO

10 11 12 13 14 15 16

ÍNDICE DO AUTOR/AUTHOR INDEX

AFECHE, S.C. 240, 241, 245

AIZENSTEIN, M.L.243

ALBAS, A. 241

ALMEIDA, M.E.S. de 239

ASSAKURA, M.T. 244

ÁVILA, S.C. 250

BASILE, A.C. 246

BATI STIC, R.F. 239

BEÇAK, M.L. 239, 247

BEÇAK, W. 240, 245, 246, 250

BENEDITO-SILVA, A.A. 240, 241, 245

BENESI, F. 240

BIRGEL, E.H. 240, 245

BRAZ, S. 249

BRODSKYN, C.l. 240, 247

CAMARGO, A.C.M. 243, 248

CAMARGO, A.M.M. de 239

CARDOSO, D.F. 248

CARNEIRO, S.M. 242

CASTRO, N.H.C. 240

CHIEFFI, P.P. 242

CIPOLLA-NETO, J. 240, 241, 245

CORRÊA, F.M.A. 242, 243

CORRÊA, M.O.A. 242

CONSALES, C.A. 241

COSTA, H. 239

COSTA, H.H. 246

CORTADAS, J. 241

DAG LI, M.L.Z. 245

DANGELINO, J.L. 240, 245

DAVI D, M.L.V.M.G. 242

DENARO-MACHADO, L 242

DIAS, R.M.D.S. 242

DORCE, V.A.C. 242

FERREIRA, T.M.S. 249

FERNANDES, W. 250

FORTUNATO, G. 240, 245

FUCHES, R.M.M. 241

GERALDES, E.A. 244

IIZUKA, H. 250

KAWANO, T. 243, 251

KFOURI, S.A. 242

KIKUCHI, O.K. 243

LACAZ, C.S. 239

LANCE, V. 250

LEBRUN, F.L.A.S. 243

LEBRUN, I. 243

LUCAS, S. 243

LONGO, M.A. 242

LUPETTI, N. 242

MACEDO, M.S. 247

MACHADO, J.C. 242

MANCINI, D.A.P. 244

MANDELBAUM, F.R. 244

MANGINI, A.C.S. 242

MARQUES, N. 240, 241, 245

MARTINI, M.H. 239

M ATI DA, A.K. 246

MENDONÇA, R.M.Z. 241

MENNA-BÁRRETO, L. 240, 241, 245

METTERS, K.M. 248

MOTA, I. 240, 246, 247, 248, 249

MOURA, J.W. 245

NAKAMURA, D. 250

NEGRÃO, N. 241

OGATA,T.R.P. 242

OKAZAKI, K. 243

OLIVEIRA, E.B. 247, 248

PALERMO-NETO, J. 242

PALUDETTI, L.A. 241, 245

PANIZZA, S. 246

PEREIRA, C.A. 241

PEREIRA, C.A.B. 246

PINTO, J.R. 244

PORTO, E. 239

RECINE, E.G.I.G. 240, 245

REICHL, A.P. 244

RÉ, L. 251

RIBEIRO, A.A.G.F.C. 243

RIBEIRO, L.R. 246

RIZZO, E. de 239

ROLIM ROSA, R. 250

ROSSIER, J. 248

RUIZ, I.R.G. 241

SALOMÃO, M. da G. 245

SALVADORI, D.M.F. 246

SAWAYA, P. 245

SCHOTT, C. 240, 245

SERTIÉ, J.A.A. 246

SILVA, A.M.M. da 240, 246, 247, 248

SILVEIRA FILHO, N.G. 249

SOARES, M.A. 241,244

SOARES, M.F.M. 247

SOARES-SCOTT, M.D. 239, 247

1, | SciELO

253

SOMA, M. 247

STOCCO DOS SANTOS, R.C. 239, 240, 245

TAKEHARA, H.A. 240, 246, 247, 248, 249

TOFFOLETTO, O. 248

TORRES, D.M.A.G.V. 242

TRAJTENGERTZ, I. 247

TRONCONE, L.R.P. 249

TUFIK, S. 249

UMEKITA, L.F. 249

VALENTINI, E.J.G. 241

VIEIRA, E.G.J. 250

WACHTEL, S.S. 250

WALTER, J. 240

ZELNIK, R. 246

254

SciELO

10 11 12 13 14 15 16

MEMÓRIAS

INSTITUTO BUTANTAN

VOLUME 51

1989

Mem. Inst. Butantan

V.51

n 1-4

p 1-254

1989

ÍNDICE DE AUTOR / AUTHOR INDEX

ALEXANDRE, S. R. 57

ARTIGAS, P. de T. 15

ASSAKURA, M. T. 225

BARBOSA, L.S . 169

BDOLAH, A. 205

BI AS I, P. De . 117

BRUNNER JÚNIOR, A. 133

CARDOSO, JÚNIOR, R. R .... 117

CARICATI, C. P.. 85, 153

CHUDZINSKI, A. M. 147

CIANCIARULLO, A. M. 133

DENARO-MACHADO, L. 169

DIAS da SILVA, W. 85,91,

107,153

DIAS, W. de 0. 195

EICKSTEDT, V. R. D. von. 43

ENOKI, N. 5

ESTEVES, M. 1. 195

FERREIRA, R. F. G. 169

FRATELLI, F. . 5

FURTADO, M. de F D. 25

FURUTA, J. A. 195

GRAUR, D. 205

GUIDOLIN, R. 85,91,

107,153

GUTIÉRREZ, J. M. 211

HAIDER, A. 141

IIIGASHI, H. G. 5,85,91,

107,153,195

IIZUKA, H. 5

ISHIZUKA, M.M. 33

JANEIRO-CINQUINI, T.R.F. ... 33

JOSÉ dos SANTOS, M. 91

KELEN, E. M. AM. 147

KFOURI, S.A. 169

KOCHVA, E. 205

KORNALIK, F. . 127

LANG, L. V. 33

LEINZ, F. F. . 33

LEVI, H. W. 43

LIMA, M. L. S. R. 85,107,153

LOMONTE, B. 211

LOPES, S. F. . 57

MACHADO, J. C. 169

MALUCELLI, M. I. C. 57

MANDELBAUM, F. R. 225

MARASSÁ, A. M. 79

MARCELINO, J. R. 85,153,

195

MARTELLINI-LANDSHOFF, F. . 101,141

MATTHIESEN, F. A. 63

MEBS, D. 127

MILLER, R. A. 177

MORAES, R. H. P. . 79

MORAIS, J. F.. 85,107,

153

MORENA, P.. 133

NISHIKAWA, A. K. 91,107,

153

OGATA, T. R. P.. 169

OISHI, N. Y. 195

OLIVEIRA, E. P. T. de . 5

PAULA, G. A. 169

PERRONE, M. A. L. 25

PETRELLA, S. M. C. N. 169

PINTO, J. R. 85,153

PRADO, S. M. A. 195

RABENHORST, E. 101

RAW, 1. 153,195

REICHL, A. P. . 225

RIGHETTI NETO, C. 57

ROLIM ROSA, R. 5

SANTOS, M. J. 91,153

SANTOS, S. M. de A. 117

SEELAENDER, M. C. L. 147

SERRANO, S. M. de T. 225

SILES VILLARROEL, M. 25

SILVA, M. A. da . 5

SILVA, M. F. A. da . 141

SPADACCI MORENA, D. D. ... 133

STEPHANO, M. A. 91,153

TANAKA, A. M. 195

TU, A. T. 177

UEDA, C. M. P. M. 91,195

UETA, M. T. . 15

VEIGA, R. M. de 0. 79

WOLLBERG, Z. 205

YAMAGUCHI, I. K. 91,107,

153

ZELNIK, R. 101,141

SciELO

11 12 13 14

CONTEÚDO/CONTENTS

VOLUME 51, 1989

N° 1

Preparação e teste da toxina botulínica tipo "E" e do seu anti-soro

específico.

Preparation and test of botulinum type "E" toxin and its specific

antiserum.

Hisako Gondo HIGASHI; Edison Paulo Tavares de OLIVEIRA;

Raymundo ROLIM ROSA; Maria Antonieta da SILVA; Fernando

FRATELLI; Naomi ENOKI; Hideyo IIZUKA .

5-13

[ Sobre a evolução de Ascaris lumbricoides Linnaeus, 1758, na fase

| larvar endovular.

j Larval development of Ascaris lumbricoides Linnaeus, 1758, within

j the egg.

Paulo de Toledo ARTIGAS; Marlene Tiduko UETA.

15-24

Estudo comparativo entre os venenos de serpentes do gênero

Bothrops, procedentes do Estado de São Paulo e do Estado do

1 Paraná com algumas espécies morfologicamente duvidosas.

Comparative study among snake venoms of Bothrops genus

including some morphologically dubious species from States of São

Paulo and Paraná.

Maria Amélia Lopes PERRONE; Medardo SILES VILLARROEL;

Maria de Fátima Domingues FURTADO .

25-32

Sobrevivência de Bothrops jararacussu (Serpentes, Viperidae,

Crotalinae) mantidas em cativeiro.

Bothrops jararacussu (Serpentes, Viperidae, Crotalinae) surviving

under captivity conditions.

Frederico Fontoura LEINZ; Thelia Rosana Forte JANEIRO-

-CINQUINI; Masaio Mizuno ISHIZUKA; Luíza Vasconcelos

LANG ..

33-38

N° 2

The Nephilinae spiders of the neotropics (Araneae: Tetragnathidae)

Aranhas Nephilinae da região neotropical (Araneae: Tetragnathidae)

Herbert W. LEVI & Vera Regina D. von EICKSTEDT

43-56

Isolamento e identificação de bactérias intestinais de camundongos

N:NIH(S), destinados a testes biológicos de vacinas bacterianas.

1 Isolation and identification of intestinal bactérias of the N:NIH(S)

1 mice destined for biological tests of bacterial vaccines.

Sandra Regina ALEXANDRE; Sandra Fernandes LOPES; Carlos

RIGHETTI NETO; Maria Ivette Carboni MALUCELLI .

57-62

SciELO

10 11 12 13 14 15 16

Anomalias morfológicas externas em escorpiões brasileiros.

Externai morphological abnormalities in Brazilian scorpions.

Fábio A. MATTHIESEN .

63-67

N° 3

Lauro Pereira Travassos Filho.

73-78

Adaptações em armadilha entomológica luminosa de sucção.

Adaptations in the entomological suction light trap.

Roberto Henrique Pinto MORAES; Rosa Maria de Oliveira

VEIGA; Ana Maria MARASSA .

79-84

Hiperimunização de cavalos soroprodutores com venenos

botrópicos e crotálico tratados por glutaraldeido.

Hiperimmunization of horses with Bothrops and Crotalus venoms

treated by glutaraldehyde.

Rosalvo GUIDOLIN; Wilmar DIAS da SILVA; Hisako Gondo

HIGASHI; Celso Pereira CARICATI; Maria Laura S. Rodrigues

LIMA; Josefina Farina MORAIS; José Ricardo PINTO; José

Roberto MARCELINO .

85-90

Produção de anticorpos antiveneno total de Crotalus durissus

terrificus em cavalos por fosfolipase A 2

Production of antibodies against Crotalus durissus terrificus whole

venom in horses by its phospholipase A 2 moiety.

Hisako Gondo HIGASHI; Rosalvo GUIDOLIN; Amélia Keiko

NISHIKAWA; Ivone Kazuko YAMAGUCHI; Marco Antonio

STEPHANO; Maristela JOSÉ dos SANTOS; Wilmar DIAS da

SILVA; Celina M. P. M. UEDA .

91-100

Chemistry of the brazilian Labiatae. Terpenoids constituents of

Lepechinia speciosa (St. Hil.) Epling.

Química das Labiatas brasileiras. Constituintes terpenoides de

Lepechinia speciosa (St. Hil.) Epling.

Raymond ZELNIK; Flávia MARTELLINI LANDSHOFF; Ema

RABENHORST .

101-106

Venenos botrópicos pré-tratados com inibidores ativos para os sítios

enzimáticos de proteases e com substância quelante preservam

seu poder imunogênico.

Pretreatment of Bothrops venoms with proteases active direct site

inhibitors or with cations chelator did not impairs their immunogenic

properties.

Hisako Gondo HIGASHI; Rosalvo GUIDOLIN; Amélia Keiko

NISHIKAWA; Ivone Kazuko YAMAGUCHI; Maria Laura S.

Rodrigues LIMA; Josefina Farina MORAIS; Wilmar DIAS da

SILVA.

107-115

Presença de Hepatozoon plimmeri (Sambon, 1909) Coccidia,

Haemogregarinidae — em exemplar de Bothrops jararaca (Wied,

1824) — Serpentes, Viperidae, Crotalinae — mantido em cativeiro.

Presence of Hepatozoon plimmeri (Sambon, 1909) Coccidia,

Haemogregarinidae — in a specimen of Bothrops jararaca (Wied,

1824) — Serpentes, Viperidae, Crotalinae — maintained incaptivity.

Pérsio De BIASI; Rubens Pinto CARDOSO JÚNIOR; Selma

Maria de Almeida SANTOS. 117-121

N° 4

Studies on the cross-reactivity of snake venom antisera.

Estudos sobre reatividade cruzada de antivenenos de serpentes.

D. MEBS & F. KORNALIK .127-132

Hemoglobin biosynthesis in bone marrow and peripheral blood

erythroid cells of the snake Waglerophis merremii (Reptilia, Ophidia,

Colubridae)

Biossíntese de hemoglobina nas células eritróides de medula óssea

e do sangue periférico da serpente Waglerophis merremii (Reptilia,

Ophidia, Colubridae)

Diva Denelle SPADACCI MORENA; Pasquale MORENA; Aurora

Marques CIANCIARULLO; Adolpho BRUNNER JÚNIOR . . 133-139

The occurrence of N-methyltaurine in the sea anemone

Bunodosoma caissarum

A ocorrência de N-metiltaurina na anémona do mar Bunodosoma

caissarum

Raymond ZELNIK; Maria de Fátima Alves da SILVA; Flávia

MARTELLINI-LANDSHOFF; Akhtar HAIDER .141-146

Standardization of anesthesia with pentobarbital in the snake

Bothrops jararaca.

Padronização de anestesia com pentobarbital em serpente Bothrops

jararaca.

Ana Marisa CHUDZINSKI; Marília C. L. SEELAENDER; Eva M.

A. KELEN .147-152

Cross-reactivity of horse monovalent antivenoms to venoms of ten

Bothrops species.

Produção de soros hiperimunes monovalentes para venenos de

espécies de serpentes do gênero Bothrops.

Wilmar DIAS da SILVA; Rosalvo GUIDOLIN; Isaías RAW; Hisako

Gondo HIGASHI; Celso Pereira CARICATI; Josefina Farina

MORAIS; Maria Laura S. R. LIMA; Ivone Kazuko YAMAGUCHI;

Amélia Keiko NISHIKAWA; Marco Antonio STEPHANO; José

Roberto MARCELINO; José Ricardo PINTO; Maristela José

SANTOS .153-168

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10 11 12 13 14 15 16

Variação do comportamento biológico do tumor humano KB,

heterotransplantado seriadamente em ratos "nude" atímicos.

Variation on biological behavior of human KB tumor serially

heterotransplanted into athymic nude rats.

Leonor DENARO-MACHADO; Suely A. KFOURI; Terezinha R.

P. OGATA; Jesus Carlos MACHADO; Gilberto Alvarenga PAULA;

Lúcia Silva BARBOSA; Regina F. G. FERREIRA; Selma M. C.

N. PETRELLA.169-176

Biochemical studies of the sea snake neurotoxins.

Estudos bioquímicos de neurotoxinas de serpente marinha.

Anthony T. TU & Roger A. MILLER .177-193

Chromatographic purification of antivenoms and antitoxins.

Purificação cromatográfica de antivenenos e antitoxinas.

Waldely de Oliveira DIAS; Maria Izabel ESTEVES; Joana Akiro

FURUTA; Hisako Gondo HIGASHI; José Roberto MARCELINO;

Nilza Yurimi OISHI; Sally Muller Affonso PRADO; Anita Mitico

TANAKA; Celina Maria Pompeo Mome UEDA; Isaías RAW 195-203

Sarafotoxins, a new group of cardiovascular modulators from snake

venom

Sarafotoxinas, um novo grupo de moduladores cardiovasculares

a partir do veneno de serpente.

Elazar KOCHVA; Avner BDOLAH; Dan GRAUR; Zvi

WOLLBERG . 205-210

Local tissue damage induced by Bothrops snake venoms. A revievy

Lesão local tissular induzida pelos venenos de serpente Bothrops.

Uma revisão.

José Maria GUTIÉRREZ & Bruno LOMONTE .211-223

Isolation and properties of a phospholipase A 2 from the venom of

the snake Bothrops moojeni (Caissaca)

Isolamento e propriedades da fosfolipase A 2 do veneno de

Bothrops moojeni (Caissaca)

Antonia Paula REICHL; Solange de Toledo SERRANO; Marina

Tizuko ASSAKURA; Fajga Ruchla MANDELBAUM .225-237

Coletânea de resumos de trabalhos publicados pelos pesquisadores

do Instituto Butantan (1988).

Collection of sumaries of articles published by the scientific staff

of Instituto Butantan (1988). 239-254

SciELO

10 11 12 13 14 15 16

COMPOSIÇÃO, FOTOLITO E IMPRESSÃO

IMPRENSA OFICIAL

DO ESTADO SAIMESP

Rua da Mooca, 1921 Fone: 291-3344

Vendas, ramais: 257 e 325

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Caixa Postal: 8231 — São Paulo

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TRABALHO E DESIHVOLVIMEHTO

SciELO

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Exemplos;

Para livros: autor, título, edição, local de publicação, editor, ano, páginas.

7. BIEK, O. Microbíologia e imunologia. 24.ed. São Paulo. Melhoramentos, 1983. 1234p.

Para artigos: autor, título do artigo, título do periódico, volume, página inicial e final, ano.

8. MACHADO, J.C. & SILVEIRA F.", J.F. Obtenção experimental da pancreatite hemorrágica aguda no cão

por veneno escorpiônico. Mem. lnst. llutantan, 40/41: 1-9, 1976/77.

As citações no texto devem ser por números-índices correspondentes às respectivas referências bibliográficas.

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... método derivado de simplificação de armadilha de Disney 1

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... segundo vários autores*-*' 4

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7 SCÍEL0) 2.1 12 13 14 15 16