Global gene expression changes caused by neurotoxins

SUMMARY OBJECTIVE: This study aims to explore the mechanisms of neuro-degeneration after exposure of different neuronal cell lines to candoxin PDB #1JGK and sarin GB, a three-finger neu

GLOBAL GENE EXPRESSION CHANGES CAUSED BY

NEUROTOXINS

PACHIAPPAN ARJUNAN (B Sc., B Ed., M Sc., M Phil.)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF ANATOMY

NATIONAL UNIVERSITY OF SINGAPORE

2006

ACKNOWLEDGEMENTS

I am greatly indebted to my supervisor Professor P Gopalakrishnakone for his

continuous guidance, abundant support, encouragement, effort and a touch of philosophy which directed my line of doctoral research that is of immense importance

to my academic career I have learnt so much working with him and am very grateful that he has allowed me to come into my own as a researcher I truly appreciate the fact that he shared his ideas with me and was always ready to hear mine

I also take this opportunity to thank Professor Ling Eng Ang, Head of the

department for his strong support and provision of excellent working facilities extended towards my candidature with kind encouragement to carry out this research

I would also like to extend my sincere thanks to Professor G Jayaraman, Madras

University, India, for the generous gift of his suggestions and critical improvement of this thesis

My special debt of gratitude is always due to Dr M M Thwin, (Research Fellow,

Department of Anatomy) for his consecutive discussion, fruitful teamwork and insightful suggestions that have greatly improved the contents of this thesis as well as

of my research publications I also wish to thank Mr J Manikandan, Department of

Physiology, for his great help on Gene Chip data analysis

I continue to be indebted to the academic, technical and administrative staff of the department of Anatomy, BFIG core lab (Clinical Research Center, Faculty of Medicine) and the protein and proteomics center (Department of Biological Sciences) for their assistance

It has indeed been my very good fortune to work with members of the Venom and

Toxin Research Programme (VTRP), both present and past, Doctors S Nirthanan,

K N Srinivasan, K Subramaniyan, R Saminathan, Le Van Dong, Le Khac Quyen, R Perumal Samy, Zhong Shuang and Ms J Hema, Mr Feng Luo, Mrs Ler Siok Ghee, Ms Xiao Jun, from whose experience and expertise I have

benefited The friendly atmosphere they created has been and will always be unforgettable They are the greatest bunch of colleagues and have helped me to maintain my sanity till today!

I could not have come this far without the constant support and encouragement of all

my friends, in particular Dr V Sivakumar, Dr B Susithra and Mr R Sriram

I am also thankful to Mr Yick Tuck Yong and Mr Gobalakrishnan, Multimedia

Unit, and all those at the Operation Theater, Histology, and Neurobiology Laboratories, Anatomy department, for their technical assistance and continuous co-operation Thanks are also due to the general office staff of the department of Anatomy for the support and co-operation given to me, especially during the final days of thesis preparation

Indebted and duty-bound as I am, I acknowledge with immense gratitiudem the support and co-operation extended to me by the staff of the department of Biological

Sciences, especially to Professor R.M Kini, and my colleagues Dr R

Lakshminarayanan, Mr N Kishore, and Dr Vivek

I would like to acknowledge the National University of Singapore for awarding me

a research scholarship which made possible for me to complete my research work without much of the hurdles

Finally, I gratefully acknowledge the support and encouragement of my parents throughout the endeavor, and for their pivotal role in my progress The completion of this thesis is really the culmination of years of sacrifice, unconditional love, quiet support and constant prayers

Pachiappan Arjunan Singapore 2006

Chapter 1: INTRODUCTION

1.5.1 The categorization of neurotoxins 11

1.9 Biochemical and pharmacological properties of candoxin 19

1.11 The Structure of the nicotinic acetylcholine receptor 22

1.12 Nerve Agents

1.12.3 General mechanism of action of chemical nerve agents 28

1.12.5 Effects of nerve agent poisoning on blood ChE and others 30

1.13 Properties and pathogenesis of sarin

1.13c Toxicodynamics of acetylcholinesterase inhibtion by sarin 35

1.14 The gene expression study using microarray technology

1.14d Microarray data validation and statistical analysis 43

1.15 Real-time qRT-PCR detection methods and applications 46

1.16 Applications of gene expression studies on snake

1.18 Recent advances in microarray technology 54

Chapter 2: MATERIALS AND METHODS

2.1d Sodium dodecyl sulphate-polyacrylamide gel electrophoresis 60

2.1.2 Experimental groups and candoxin treatment 62

2.1.4 TUNEL-Histochemistry assay 63

2.1.6 Antineurotoxic effects of CDX inhibitor (P-NT.II) 65

2.1.8e Hs 683 cells-stock preparation and preservation 69

2.1.9 Total RNA Isolation and Quantification

2.1.10 Gene expression studies using oligonucleotide microarrays

2.1.10.5 Cleaning up and quantifying IVT products

2.1.10.6 Fragmenting the cRNA for target preparation 78

2.1.10.7 Eukaryotic target hybridization 79

2.1.11 Washing, Staining and Scanning of Probe Arrays

2.1.14 Quantitative Real Time–PCR (LightCycler) 88

2.2.2 Microarray GeneChip TM Analysis and Clustering Algorithm

2.2.2a Categorization and Criteria for Gene Selection Using Software’s 96

2.2.2b Gene Expression Profiles (URL Links) and Clustering Methods 97

2.2.3 Quantitative Real Time–PCR (qRT-PCR) 97

2.2.4 Protein Isolation 100

2.2.5 Western Blot Analysis 100

2.2.6 Analysis of immunoflurescence 101

2.2.7 Human Brain cDNA library 2.2.7a Chemicals 102

2.2.7b Polymerase Chain Reaction for Human Brain cDNA library 102

Chapter 3: RESULTS AND DISCUSSION–CDX 3.1 Results

3.1.1 Isolation and Purification of Candoxin 104

3.1.2 Determination of the Amino Acid Sequence of Candoxin 106

3.1.3 SDS-PAGE Profiles 108

3.1.4 Cell Propagation and EC50 Determination 110

3.1.5 Distribution of TUNEL Positivity 111

3.1.6 Cytotoxic Brain Edema 113

3.1.7 Differentially expressed gene profiles 114

3.1.8 Toxicofunctional genomics gene expression of CDX 117

3.1.9 Genes involved in signal transduction and internalization 120

3.1.10 Metabolic Pathways 121

3.1.11 Gene-specific correlation between RT-PCR and microarray results 122 3.1.12 Selective Inhibition of CDX-induced neurotoxicity by P-NT.II 125 3.1.13 Suppression of gene expression by a selective Inhibitor of CDX 127 3.1.14 Molecular Pathway and Target Identification 128

3.2 Discussion 130

3.3 Conclusion 145

Chapter 4: RESULTS AND DISCUSSION–GB 4.1 Results 4.1.1 Cell Viability and EC50Determination 146

4.1.2 Cell lysate AChE assay 148

4.1.3 Microarray Gene ChipTM Analysis and data mining 148

4.1.4 Tree view and clustering analysis 156

4.1.5 Principal component analysis (PCA) 158

4.1.9 Comparison of Microarray data with qRT-PCR, Western blot and

Chapter 5: GENERAL CONCLUSION AND FUTURE DIRECTIONS

5.1 General Conclusion and Future Directions 202

SUMMARY

OBJECTIVE: This study aims to explore the mechanism(s) of neuro-degeneration

after exposure of different neuronal cell lines to candoxin (PDB #1JGK) and sarin

(GB), a three-finger neurotoxin from Bungarus candidus venom, and a chemically

modified (O-isopropyl methylphosphonofluoridate) neurotoxic agent, respectively

The former induces delayed glial cell-death (EC50 ∼1µM) by inhibiting postsynaptic neuromuscular and neuronal 7nACh-receptors, while the latter exerts a delayed

neurotoxic effect [EC50 ~375.7µM)] through inhibition of acetylcholinesterase

activity

METHODS: Gene expression profiles of cultured human neuronal cells exposed to

candoxin (CDX) and sarin (GB), respectively were studied using Affymetrix

GeneChips (HG-U133A) A single dose of CDX was used on Hs 683 glial cell line,

while a single (3h-acute or 24h-intermediate) as well as repeated (48h-delayed) doses

of sarin (5ppm) were used on SH-SY5Y cells Genes altered by ≥ 3-fold (105 for CDX and 370 for GB treatments) were selected by GeneSpring (v7.0) analyses

Quantitative Real-time PCR (qRT-PCR) and western blot analysis were used to

confirm the results obtained with the expression profiling studies

RESULTS & DISCUSSION: The in-vivo results from TUNEL-histochemistry of

the whole brain regions after intracerebroventricular (i.c.v) administration of CDX

combined with the in-vitro data obtained from exposure of Hs 683 glial cells to CDX

confirmed the nuclear fragmentation of neuronal cells, which might lead to brain

damage (hippocampus, frontal cortex, and temporal regions in particular) CDX

caused alteration of genes involved in signal transduction, ubiquitin-inflammation,

mitochondrial-dysfunction, and DNA-damage-response pathways Using qRT-PCR

and specific CDX-binding peptide P-NT.II, the following genes - IL7R, IL13RA2,

IL-1β, TNFRSF12A, GADD45A, CD44 and IFI44- were identified as important genes in playing a role in CDX-induced glial inflammation The results obtained with sarin

(GB) indicate that the low-level single dose exposure do not always parallel acute

toxicity, but can cause a reversible down-regulation of genes and a range of

anti-cholinesterase effects In contrast, repeated doses produced persistent irreversible

down-regulation of genes related to neurodegenerative mechanism at 48h

Quantitative Real-time PCR, western blot and confocal microscopic analysis

confirmed the increased expression of apolipoproteinE (ApoE), V-ets

erythroblastosis-2 (Ets-2), and reduced expression of presenilin1 (PSEN1),

presenilin2 (PSEN2), -2-Microglobulin ( 2M), CD9 molecule (CD9),

dopa-decarboxylase (DDC) mRNAs and proteins

CONCLUSION: The present findings reveal new insight into the mechanisms of

neuro-degeneration after exposure to animal and/or chemical neurotoxins Besides

providing an in-vitro experimental model for studies on the neuropathophysiology of

brain cells, this investigation further yields the molecular mechanism of glial-driven

neuro-degeneration and provides a clue by which nerve agents such as sarin could

mediate neuro-degeneration

PUBLICATIONS FULL PAPERS IN INTERNATIONALLY REFEREED JOURNALS

1 A Pachiappan, M M Thwin, J Manikandan and P Gopalakrishnakone

(2005) Glial inflammation and neurodegeneration induced by candoxin, a

novel neurotoxin from Bungarus candidus venom: Global gene expression

analysis using microarray Toxicon, 46(8): 883-899

2 Kellathur N Srinivasan, Pachiappan Arjunan, Mer Lin DH, Lim Jim Jim, Wei-Yi Ong, Loke Weng Keong, Ho Diana SC, Tang Jing Ping, Raghavendra Prasad HS, Lee Fook Kay & Gopalakrishnakone Ponnampalam (2006) Global Gene Expression Profile, Immunocytochemical and Biochemical

Studies following Chronic Nerve Agent Toxicity Chemical Research in

Toxicology (Under revision)

MANUSCRIPT IN PREPARATION

3 A Pachiappan, P Gopalakrishnakone, Loke Weng Keong, Lee Fook Kay, J Manikandan and M.M Thwin Molecular neuropathogenesis of human neuronal (SH-SY5Y) cell lines exposed to a single and repeated low-dose of

sarin

CONFERENCE ABSTRACTS AND POSTERS

Conference Abstracts (Singapore)

1 A Pachiappan, S Nirthanan, K N Srinivasan and P Gopalakrishnakone

The use of oligonulceotide microarray to analyze the gene expression in

human brain cells exposed to a novel neurotoxin, candoxin

The First Bilateral Symposium on Advances in Molecular Biotechnology and Biomedicine between NUS and University of Sydney, Singapore (May 2002)

2 A Pachiappan, K N Srinivasan, S Nirthanan and P Gopalakrishnakone Functional Genomics Study of Human Brain Cells Exposed to a novel three

finger neurotoxin, Candoxin, from Bangarus candidus

6 th NUS-NUH Annual Scientific Meeting “from laboratory to clinic”, Singapore (Aug 2002)

3 A Pachiappan, S Nirthanan, K N Srinivasan, and P Gopalakrishnakone

Toxicogenomics Studies on Human Brain Cells Exposed to a Novel Neurotoxin, Candoxin, using High-Throughput Oligonucleotide Microarray

Technology

Life Sciences in Singapore: Integrating Multidimensional perspectives; Fourth combined scientific meeting (Incorporating the Second Singapore microarray meeting) Singapore (January 2003)

4 H S Raghavendra Prasad, K N Srinivasan, A Pachiappan, S Zhong, Z Qi and P Gopalakrishnakone Identifying potential biomarker genes specific for

neurotoxins based on gene expression profiles

Life Sciences in Singapore: Integrating Multidimensional perspectives; Fourth combined scientific meeting (Incorporating the Second Singapore microarray meeting) Singapore (January 2003)

5 A Pachiappan, K N Srinivasan, S Nirthanan, H S Raghavendra Prasad,

and P Gopalakrishnakone Global gene expression Study of Human Brain

Cells Exposed to a novel alpha neurotoxin, Candoxin, from Malayan krait

7 th NUS-NUH Annual Scientific Meeting, Singapore (October 2003)

Conference Abstracts (International)

1 P Gopalakrishnakone, K N Srinivasan, S Zhong, and A Pachiappan, C Ananth and Z Qi Global gene expression profiling of Human Genome

following exposure to toxins-emerging field of TOXINOGENOMICS

6 th Asia-pacific congress on animal, plant and microbial toxins and 11 th Annual scientific meeting of the Australasian College of tropical medicine, Cairns, Australia (July 2002)

2 P Gopalakrishnakone, K N Srinivasan, S Zhong and A Pachiappan Gene

Expression Studies of Human Brain and Liver Cells after Exposure to Toxins

Using Microarray Technology

HUGO’s Seventh International Human Genome Meeting (HGM2002), Shanghai, China (April 2002)

3 A Pachiappan and P Gopalakrishnakone Brain edema and neuro-glial damage induced by a neurotoxin candoxin, differential gene expression

analysis using microarray

International Biomedical Science Conference, Kunming, China (December 2004)

4 Gopalakrishnakone P and Pachiappan A Gene expression profiling and

fingerprinting of Human Genome following exposure to toxins/nerve agents –

emerging field of “TOXINOGENOMICS”

IST-15 th World Congress on Toxinology on Animal, Plant and Microbial Toxins Glasgow, Scotland, United Kingdom, (July 2006)

Oral Presentation:

5 Pachiappan K N Srinivasan, H S Raghavendra Prasad, and P

Gopalakrishnakone Toxicofunctional genomics of human brain cells exposed

to candoxin, a novel alpha neurotoxin of Bungarus candidus venom and its

implication in nAChR-mediated neurotransmission

IST-14 th World Congress on Toxinology on Animal, Plant & Microbial Toxins Convention Centre, Adelaide, Australia, (September 2003)

6 Pachiappan Arjunan, Loke Weng Keong, Lee Fook Kay and Gopalakrishnakone Ponnampalam Gene expression profiles of human neuroblastoma (SH-SY5Y) cell lines exposed to a single and repeated low-

dose of nerve-agent ‘sarin’

“Symposium on chemical, biological, nuclear and radiological threats” Finland, (June 18-21, NBC 2006)

7 Pachiappan A, Gopalakrishnakone P, Thwin M.M, Weng Keong L, and Lee

FOLLOWING EXPOSURE TO A SINGLE AND REPEATED LOW DOSE

NERVE AGENT SARIN AND CANDOXIN (SNAKE NEUROTOXIN)

“The 5 th Singapore International Symposium on Protection Against Toxic Substances” Singapore, (November 27-30, SISPAT-2006)

LIST OF FIGURES

Chapter 1

1.1 The sites of action by major classes of animal venom neurotoxin 9

1.4 Spatial structure of candoxin (PDB accession code (1JGK) 21

1.5 The Structure of the nACh-Receptor and Its Interaction 23

Chapter 2

2.1 Monitoring of cocktail preparation by agarose gel electrophoreses 72

2.2 Electropherogram for Human Neuronal (SH-SY5Y) cell total RNA 95

Chapter 3

3.2 Reverse-phase HPLC of peak 5 from Gel filtration Chromatography 105

3.6 SDS-PAGE Profiles of Bungarus candidus Venom and Candoxin 108 3.7 Densitometric tracing curves of Bungarus candidus Venom and CDX 109

3.10 Brain wet-to-dry Weight Analysis after Injection of Candoxin 113

3.13 Differential Gene Expression of Human Glial Cells to CDX-treatment 117

3.15 Semi-Quantitative (qualitative) RT-PCR for up-regulated genes 123

3.17 Inhibition of CDX (20µg/ml) and Erabutoxin-b (2 µg/ml) – induced

Neuromuscular Blockade in the Mouse Hemidiaphragm by P-NT.II 126 3.18 Changes of Gene Expression by a Selective Inhibitor (P-NT.II) of CDX 127

Chapter 4

4.1 Cell Viability Assay-XTT values for effective concentration (EC50) 146 4.2 Mortality rate of neuroblastoma cell lines exposed to sarin 147 4.3 The effects of sarin treatment on AChE activity in neuronal cell lines 148

4.6 Principle component analysis (PCA) 158

4.16 Schematic Pathway and Possible Mechanisms of Sarin-induced

LIST OF TABLES

Chapter 1

1.2 Classifications of Bungarotoxins and characterisation of AChRs 5

Chapter 2

2.2 Reverse transcriptase volumes for first strand cDNA synthesis 74

2.4 Primer sequences used in semi and quantitative RT-PCR analysis 88 2.5 Primer sequences used in qRT-PCR analysis of SH-SY5Y cell lines 98 2.6 Antibodies used for immunohistochemistry and immunofluorescence 101

Chapter 3

3.1 Differentially expressed genes in signal transduction and ubiquitin-

inflammation linking with neuro-glial pathogenesis in glial

3.2 Genes belonging to different metabolic and regulatory pathways

inter-relation according to KEGG and GenMAPP pathway databases 122

Chapter 3

4.1 Differentially expressed genes involved in in neurodegenration and

neuropathogenesis of human neuronal cells induced by sarin 149

ATCC american type culture collection

BLAST basic local alignment search tool

cAMP cyclic adenosine monophosphate

cDNA complementary deoxyribonucleic acid

cRNA complementary ribonucleic acid

DD-H2O double distilled water

DMSO dimethyl sulfoxide

DTNB dithiobisnitrobenzoic acid

EDTA ethylene diaminetetra acetic acid

ELISA enzyme-linked immunosorbent assay

ESTs expressed sequence tags

MALDI-TOF matrix-assisted laser desorption ionization−time of flight

MAS microarray analysis suit

MEME minimum essential modified eagle’s

MES 2-(N-morpholino) ethanesulfonic acid

MIAME minimum information about a microarray experiment

PAGE polyacrylamide gel electrophoresis

qRT-PCR quantitative real-time reverse transcriptase−polymerase chain reaction Q-TOF quadruple−time of flight

RP-HPLC reverse phase –high performance liquid chromatography

RT-PCR reverse transcriptase - polymerase chain reaction

s seconds

SEM standard error of the mean

SPSS statistical package for social sciences

TEMED N,N,N’,N’-tetramethylethylene

TUNEL terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling

UPGMA unweighted pair-group method with arithmetic mean

CHAPTER 1

Introduction

CHAPTER 1

INTRODUCTION

1 1 Venomous Snakes

Snakes, a remarkable and intriguing group of animals, are successful not only on land, but

also in the seas The first snakes probably came into existence during the period of

Cretaceous, between 136 to 64 million years ago (Phelps, 1989) They can be grooved

(cobra and krait) or canalized (viper) (Jackson, 2002) Snake fangs, the curved teeth

situated on the maxillary bone, are bigger than other teeth and vary from family to family

These fangs are connected to venom sacks in the roof of the snake's mouth During the

bite, fang marks left at the bite site on the victim’s body are different among snake

families and can be used as signs in the diagnosis of snakebites (Nishioka et al., 1995)

However, identification of the fang mark is not always possible and needs experts in

snake taxonomy

Venomous snakebites, although uncommon, are a potentially deadly emergency in some

parts of the rural tropics, and are ranked in the top ten causes of death (White, 2000;

White et al, 2003; White, 2005) Snake venom (modified form of saliva) is a combination

of numerous substances, which induce diverse clinical features that require different

antidotes for neutralizing their activities The potential extent and implications of snake

venom variability from the individual level to that between geographical populations and

species have been well reported in the literature (Chippaux et al., 1991) The

pathophysiologic basis for morbidity and mortality is the disruption of normal cellular

functions by these venom enzymes and toxins In simple terms, venom proteins can be

divided into 5 arbitrary categories: (1) Cytotoxins that cause local tissue damage, (2)

Hemotoxins that cause internal bleeding, (3) Cardiotoxins that act directly on the heart,

(4) Myotoxins that cause skeletal muscle damage and (5) Neurotoxins that affect the

nervous system (see below for details in section 1.5)

1 2 Classification of Venomous Snakes and Their Distribution

Snakes belong to the kingdom animalia, phylum chordate, order squamata, and suborder

serpents (Table 1.1) There are about 3000 species of snakes, comprising 438 genera and

15 families that are widely distributed all over the world, especially in tropical and

sub-tropical areas, except the Arctic regions (Halliday and Adler, 2002) Of which, more than

400 (~14%) species are venomous and might cause fatalities in human (Vidal, 2002)

Venomous snakes are considered as most fearsome animals which possess one of the

most sophisticated integrated weapon systems in the nature world with awesome striking

power combined with lethal venoms (Fry, 2005) The venoms are secretory products of

venom glands and are used for both offence (to immobilize prey) and defense (Hider et

al., 1991) Venomous snakes are classified into four families viz Colubridae, Elapidae, Viperidae and Atractaspididae (Mattison, 1995) or five families if Elapidae is subdivided into one more family as Hydrophiidae (Vidal, 2002) as shown in Table 1.1 The members

of these snake families are often the cause of envenomation in humans (Ismail and

Memish, 2003)

1 3 Value of Snake Toxins in Science and Medicine

Despite its harmful effects, snake venom has significance as a highly specific research

tool for neurobiologist in scientific discovery and medicine (i.e., drug discovery) (Mebs,

1989)

Table 1.1 Venomous snakes classification and their distribution

Kingdom Phylum Class Order Suborder Family Subfamily Genera Species Distribution

Animalia Chordata Reptilia Squamata Serpentes -COLUBRINAE

-CALAMARINAE -HOMALOPSINAE -LAMPROPHINAE -NATRICINAE -PAREATINAE -PSAMMOPHINAE -PSEUDOXENNDONTINAE -XENODERMINAE

-XENODONTINAE

300 1850 -In most parts of the world, but

poorly represented in Australia.

-CALLIOPHEINAE -ELAPINAE (cobras, mambas)

-LATICAUDINAE -MATICORINAE -HYDROPHEINAE (or Hydrophiinae - 2 species, marine and Australian terrestrial)

65 270 -Almost global but more in the

southern than the northern: Southern North America, whole in the Southern and South-East Asia and Australia.

-Sea snakes present in most oceans.

-CAUSINAE (night adders) -CROTALINAE (pit vipers) -VIPERINAE (true vipers)

33 240 -Found throughout the world

except Madagascar and Australia.

Adapted from Mattison, 1995 andVidal, 2002

In addition, -neurotoxins, toxins that cause paralysis by binding to the nicotinic

receptors at the postsynaptic region of the neuromuscular junction have been widely

studied in terms of their structure-function relationships as well as gene structure,

organization and expression The value of -neurotoxins in therapy and research has also

been discussed to highlight their potential applications especially in the area of drug

discovery (Phui Yee et al., 2004) The discovery of α-bungarotoxin has led directly to the

characterisation of acetylcholine receptors (AChR), and has been proven useful for

receptor studies as shown in Table 1.2 Other toxins have also been found useful as ion

channel probes and in the study of inflammation Phospholipase A2 (PLA2) enzymes,

present in most venom, are involved in many inflammatory disorders such as asthma,

oedema, allergic rhinitis, acute pancreatitis and autoimmune diseases Hence, venom

PLA2 offers an opportunity to investigate the mechanism of PLA2 action and molecular

interaction at gene level (Cher et al., 2003) The myriad of proteins found in venoms has

an important place in the treatment of thrombosis, arthritis, cancer and many other

diseases The usefulness of venom toxins lies in their ability to evade regular control

mechanisms through their independence from co-factors and their non-recognition by

inhibitors Venom toxins have developed highly specific molecular targets, which make

them valuable for drug usage in terms of limiting potential side effects The considerable

discrepancy of snake toxins has therefore made them a valuable resource for potential

lead compounds

1 4 Snake Venoms and Components

Snake venom components are of considerable interest to researchers across a wide variety

of disciplines, including biochemistry, pharmacology, medicine, molecular biology, and

evolutionary genetics

Table 1.2 Classifications of Bungarotoxins and characterisation of acetylcholine receptors (AChR)

Neurotoxins Uniprot Entry Protein Name Organism Function

NXLA_BUNMU (P60615) -bungarotoxin, isoform A31

NXLV_BUNMU (P60616) -bungarotoxin, isoform V31

Neurotoxin, blocks neuromuscular transmission

NXL1_BUNMU (P01398) -bungarotoxin (Long neurotoxin 2)

NXL2_BUNMU (P15816) -neurotoxin (Long neurotoxin CB1)

NXL3_BUNMU (P15817) 3-bungarotoxin (Long neurotoxin CR1 )

NXL4_BUNMU (O12961) 4-bungarotoxin

NXL5_BUNMU (O12962) 5-bungarotoxin

NXL6_BUNMU (Q9W729) 6-bungarotoxin

Bungarus multicinctus Many-banded krait Neurotoxin, inhibits neuronal

nicotinic AChR

NXLH_BUNMU (P15818) Long neurotoxin homolog

NXSH_BUNMU (P43445) Short neurotoxin homolog

NXH1_BUNMU (Q9YGI0) Short neurotoxin homolog NTL1

NXH2_BUNMU (Q9YGH9) Long neurotoxin homolog NTL2

NXH3_BUNMU / NXG1_BUNMU (Q9YGJ0)

-bungarotoxin (Long neurotoxin homolog NTL2I)

NXH4_BUNMU (Q9YGI8) Short neurotoxinhomolog

NXH5_BUNMU / NXG2_BUNMU (Q9W796)

-bungarotoxin 2 (Long neurotoxin homolog)

NXH6_BUNMU (O12963) Long neurotoxin homolog TA-bm16

Possible neurotoxin

NGF_BUNMU (P34128) Nerve growth factor

Bungarus multicinctus Many-banded krait

Neurotoxin

IVB1_BUNMU (P00987) -bungarotoxin B1in, major component

IVB2_BUNMU (P00989) -bungarotoxin B2 chain

Bungarus multicinctus Many-banded krait

Presynaptic neurotoxin

PA20_BUNMU (P00606) Phospholipase A2 (EC 3.1.1.4)

PA21_BUNMU (P00617) Phospholipase A2, (EC 3.1.1.4)

Inhibits neuromuscular transmission by blocking ACh release from nerve termini

PA26_BUNMU (Q90251) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A6 chain

PA27_BUNMU (Q9PU97) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A7 chain

PA2A_BUNMU (Q9PTA1) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A-AL1 chain

PA2B_BUNMU (Q9PTA7) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A-AL2 chain

PA2C_BUNMU (Q9PTA6) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A-AL3 chain

PA2D_BUNMU (Q9PTA5) Phospholipase A2, (EC 3.1.1.4)

-bungarotoxin A-AL4 chain

TXML_BUNMU (Q9W727) Muscarinic toxin-like protein Inhibits muscarinic AChR

CXH_BUNMU (P79688) Cardiotoxin homolog TA-ctx-like Cardiotoxin

Other Bungarus

toxins CXL_BUNMU (Q9YGH0) Cytotoxin-like protein TA-BMBGT3

Bungarus multicinctus Many-banded krait Toxin

Neurotoxin, inhibits irreversible 7nAChR & reversible mAChR Bungarus

candidus CDX_(P81783)

Candoxin (Three-finger protein)

Bungarus candidus

(Malayan krait) Neurotoxin, Neuro-glial

degeneration (Current Study)

Snake venoms are biochemically complex mixtures of pharmacologically active enzymes,

proteins (~90%), and peptides Many have neuro-active properties (Stocker, 1990; Kini,

2002) that target a variety of vital physiological functions in mammals (Phui Yee at al.,

2004) Several of them also exhibit lethal and unbearable effects as a consequence of their

neurotoxic, cardiotoxic and tissue necrotizing effects, whereas others induce various

pharmacological effects but are of a lower order of toxicity (Kini, 2005b) All these

protein toxins isolated from the venom either inhibit or activate a vast number of targets

such as ion channels, acetylcholine receptors, acetylcholinesterase, membranes,

coagulant/anticoagulant pathways, and metalloproteinase, with high selectivity and

affinity, and they also attack various physiological processes at specific sites (see reviews

Nirthanan et al., 2004 and Kini, 2005a)

One of the primary targets of snake venom is the peripheral nervous system, the skeletal

muscle neuromuscular junction in particular, where neurotransmission is inhibited,

leading to paralysis of skeletal muscles including those of respiration (Chang, 1979;

Warrell et al., 1983) Venoms of the Elapidae family of snakes (kraits, cobras, coral

snakes and mambas - the world’s most dangerous snakes) are a cocktail of

pharmacologically active protein toxins that kill primarily through neuromuscular

paralysis (Warrell, 1999) In contrast to the deadly toxins present in snake venoms, a

large number of venom components may possibly serve as drug libraries, diagnostic tools,

and as target specific research tools Additionally, minor non-protein components

(biomolecules) such as metal ions, lipids, nucleic acids, carbohydrates and amines are

also present (Hider et al., 1991) and play a functional role in the venom Some snake

venoms are also rich in bioactive amines and acetylcholine Mamba (Dendroaspis)

venoms in particular, have a high content of acetylcholine that is postulated to play a

defensive role as a potent analgesic agent (Welsh, 1967) Citrate is another important

constituent of several snake venoms, presumably serving a self-protective function by

inhibiting the deleterious activity of venom enzymes within the venom gland (Freitas et

al., 1992; Odell et al., 1999)

1 5 Neurotoxinology

The current study will reveal differential expression of genes after induction with snake

venom toxins like candoxin in particular, and to their effects on nervous system or ion

channels/receptors related genes as well as their interactions This makes it necessary to

provide a brief overview of neurotoxins from other species, which represent a rich

combinatorial-like library of evolutionarily selected, neuropharmacologically active

proteins/toxins Neurotoxins are found in a wide variety of animals, covering a great

diversity from arachnids to amphibians to mollusks to snakes (Figure 1.1) In the most

dangerous species of venomous snakes and other animals, the most significant action of

the venom lies in its effect upon the victim's nervous system in either agonistic or

antagonistic manners, hindering the operation of muscles and causing paralysis leading to

death from respiratory failure As such, these potent molecules are useful for

demonstrating the structure-function relationships of toxins and also the tremendous

potential venoms have as a source of useful investigational ligands or even as

therapeutics

1 Amphibian Neurotoxins:- The frog toxins are not limited to presynaptic activity, with

postsynaptic toxins also isolated from the poison arrow frog Epipedobates tricolor

(Epibatidine) (Spande et al., 1992) The major dendrobatid alkaloids are batrachotoxin,

decahydroquinolines, histrionicotoxins, indolizidines, pumiliotoxins, pyrrolizidines,

quinolizidines and tetrodotoxin Pumiliotoxins, the widespread alkaloids found in a

number of species, increase the rate of sodium channel opening and closing, producing

spontaneous activity of the nerve (Figure 1.1)

Adopted from Dr Bryan Grieg Fry, www.venomdoc.com

Figure 1.1 The Sites of Action by Major Classes of Animal Venom Neurotoxins The

distribution of neurotoxins found in a wide variety of animals, covering a great diversity from arachnids to amphibians to mollusks to snakes (see the text for details in section 1.5)

2 Cone Snail Neurotoxins:- Cone snail venoms contain a tremendously diverse natural

pharmacology Conotoxins, typically composed of 12-30 amino acid residues in length,

are highly constrained peptides due to their high density of disulfide bonds Three main

classes of paralytic toxins, which interfere with neuronal communication but with

different targets, have been the focus of intense investigation They are: -conotoxins that

bind to and inhibit the nicotinic acetylcholine receptor (Loughman et al., 1998);

mu-conotoxins, that directly abolish muscle action potential by binding to the postsynaptic sodium channels; and -conotoxins, that decimate the release of acetylcholine through

the prevention of voltage activated entry of calcium into the nerve terminal (Figure 1.1)

3 Scorpion Neurotoxins:- The nomenclature of scorpion toxins recognizes two general

classes, -and -toxins These toxins are sometimes called 'Androctonus' and

'Centruroides-like' toxins after the genera from which they were first isolated; these two

genera being representative of Old and New World species, respectively Tityustoxin-VII

from Tityus serrulatus venom is a peptide that binds to both insect and mammalian

nervous tissues (Figure 1.1)

4 Spider Neurotoxins:- Despite the vast majority of spiders being harmless to humans,

many spider venoms contain in their venoms potent small molecules called polyamines

and they are often very effective and specific blockers of ion channels or of receptors

Their target receptors are those which recognize excitatory amino acids in the mammalian

central nervous system and are classified into three major subtypes, ones which prefer

N-methyl-D-aspartate (NMDA), quisqualate (QA), or kainate (KA) as type agonists,

respectively Jorotoxin has been shown to be a specific blocker of the postsynaptic

glutamate receptors, in contrast to bacterial pertuissus toxin that blocks the presynaptic

glutamate receptors (Figure 1.1)

5 Tetrodotoxin:- It is a bacteria-derived organic molecule assimilated into the tissues of

the puffer fish or into the modified salivary glands of the blue-ringed octopus

Envenomation causes acute respiratory failure through paralysis of the respiratory

musculature due to tetrodotoxin being a highly specific Na+ channel blocker in excitable

tissues (Figure 1.1) This affects only the nerves of the voluntary muscles such as eyelids

or lungs, leaving the involuntary muscles such as cardiac muscles unaffected

1 5.1 The Categorization of Snake Neurotoxins

Venoms often contain different neurotoxins that work synergistically to cripple the

nervous system Especially, snakes belonging to the families Elapidae and Hydrophidae

have typical neurotoxic venoms with a high neurotoxin contents According to their site

of action, neurotoxins can be classified as: (i) pre-synaptic neurotoxins that block

neurotransmission by affecting acetylcholine transmitter release (Fletcher and Rosenberg,

1997) and (ii) post-synaptic neurotoxins that antagonize the acetylcholine receptor

(Nirthanan et al., 2002), thereby preventing the depolarizing action of acetylcholine They

are referred to as curaremimetic, curariform or simply neurotoxins Together, these

neurotoxins effectively block skeletal neuromuscular transmission by crippling receptors,

while at the same time acting to destroy any neurotransmitter that might compete with the

toxin for receptor binding Venoms often contain several post-synaptic neurotoxins, each

with a high affinity for a nicotinic receptor subtype, and as a result the venom can cripple

as many receptors as possible Post-synaptic neurotoxins are found only in elapids and sea

(Hydrophidae) snakes In the many-banded krait (Bungarus multicinctus), a post-synaptic

toxin is α-bungarotoxin, while pre-synaptic toxins are β- and -bungarotoxins (see Table 1.2) Interestingly, the venom of a single snake may contain a cocktail of over a few

hundred different protein toxins (Kini, 2002; Cher et al., 2003) which can, however, be

classified into a small number of structural superfamilies (Menez, 1998; Kordis and

Gubensek, 2000) Very broadly, this superfamily can be further grouped into two

categories: enzymatic or non-enzymatic proteins The predominant enzyme groups found

in snake venoms are: (i) phospholipase A2 enzymes (Kini, 2005b); (ii) serine proetinases

(Serrano and Maroun, 2005); (iii) metalloproteinases (Teixeira et al., 2005; Fox and

Serrano, 2005); (iv) acetylcholinesterase (Cousin and Bon, 1999); (v) L-amino oxidases

(Du and Clemetson, 2002); and (vi) nucleases (Bailey, 1998; Kini, 2002) Although

many snake venoms contain a number of active components, it is safe to say that no

single venom contains all the active components Numerous venom enzymes have been

used clinically as anticoagulants while other venom components are used in pre-clinical

research to examine their possible therapeutic potential (Markland, 1998) Some of the

well recognized families of non-enzymatic proteins include the three-finger toxins

(Nirthanan et al., 2003; Kini, 2002), lectin-related proteins (Morita, 2004), serine

proteinase inhibitors (Kolodzeiskaia, 2000) and disintegrins (Calvete et al., 2005a;

Calvete, 2005b) The scope of this thesis limits this review to just a description of the

three-finger neurotoxin family

1 6 The Three-finger Neurotoxins

The three-finger family (MW range 6000−8000 Da) of snake venom toxins is a particularly interesting and biochemically complex group of venom peptides, since they

are encoded by a large multigene family and display a diverse array of functional

activities (Fry et al., 2003) The three-finger toxin is a family of non-enzymatic

polypeptides containing 60-74 amino acid residues (Harvey, 1991; Fry et al., 2003) They

are found only in the venom of Elapidae (Australian elapids, cobras, coral snakes, kraits,

and mambas) and Hydrophiidae (sea snakes), but not in those of vipers and crotalids

(rattlesnakes) (Warrell, 1993; Nirthanan et al., 2003) Evidence indicates that the

systemic poisoning by elapid venoms is primarily characterized by neurotoxicity and/or

cardiotoxicity (Gopalakrishnakone et al., 1990) The neurotoxic manifestations,

especially of elapids include ptosis and opthalmoplegia with blurred vision or diplopia,

difficulty in swallowing with inability to handle oral secretions, slurred speech, weakness

of facial muscles and occasionally, the loss of the sense of taste and smell

To date, more than a hundred three-finger -neurotoxins ( -NTXs) have been isolated and

sequenced from Elapidae and Hydrophiidae snakes (see Nirthanan et al., 2004 for

review) A large number of the family members are relatively small water-soluble

proteins possessing different biological activities, and they interfere with cholinergic

transmission at various post-synaptic sites in the peripheral (PNS) and central nervous

(CNS) systems (Kini, 2002; White et al., 2003; Nirthanan et al., 2004; Osipov et al.,

2004) Due to their high affinity binding to specific receptors, three-finger neurotoxins

can affect nervous system at various stages (Dufton, 1993; Nirthanan et al., 2002; 2003;

Tsetlin and Hucho, 2004) Snake venom -neurotoxins bind selectively to nicotinic

acetylcholine receptor (nAChR) with high affinity (Tsetlin and Hucho, 2004), and almost

irreversibly block Torpedo ( ) receptor, and chick or rat neuronal 7-nAChR (Servent

et al., 1997; Servent and Menez, 2001)

1 6a The Structure of Three-finger Toxins

Three-finger toxins have been widely studied in terms of their structure-function

relationships as well as gene structure, organization and expression (Phui Yee et al.,

2004) The characteristic feature of all three-finger toxins is their distinctive structure of

leaf-like shape formed by three adjacent loops that protrude from a small, globular,

hydrophobic core containing the four conserved disulfide bridges (Menez, 1998; Tsetlin,

1999; Servent and Menez, 2001; Kini, 2002; Nirthanan et al., 2003; Osipov et al., 2004)

The three loops (fingers) that project from the core region are said to resemble, with some

imagination, three outstretched fingers of the hand; because of this appearance, this

family of proteins is called the three-finger toxins or 3FTx (Figure 1.2) The three-finger

fold is agreeable to a variety of overt and subtle deviations such as the number of

-strands present, size of the loops and C-terminal tail as well as twists and turns of various

loops which have great significance with respect to function and selectivity of molecular

targets (Servent and Menez, 2001) Therefore, despite the similar overall fold,

three-finger toxins demonstrate an assorted range of pharmacological activities including, but

not limited to, peripheral and central neurotoxicity, cardiotoxicity (cyotoxicity), inhibition

of enzymes such as acetylcholinesterase, hypotensive effect and platelet aggregation

(Tsetlin, 1999; Kini, 2002; Paaventhan et al., 2003)

Figure 1.2 The Structure of Three-Finger toxins Representative examples of: (A)

NT II, neurotoxin II Naja oxiana, (1NOR); (B) CTX, cobratoxin Naja siamensis,

(2CTX); (C) Fasciculin 1 (1FAS) The protein database accession codes are stated in parentheses The disulfide-confined loops are marked as I, II, III in the structures that is with the N-terminal loop I to the right from the central loop II Blue ribbons depict the β -structures, disulfide bridges are shown in light yellow The structures presented here are modified from Victor Tsetlin, 1999

1 6b Non-conventional Three-finger Proteins

The Weak Toxins (WTX) or non-conventional toxins isolated, so far, exclusively from

Elapidae venoms, constitute another class of three-finger toxins, (Servent and Menez, 2001; Utkin et al., 2001; Nirthanan et al., 2003) On the other hand, unlike / –

neurotoxins and –neurotoxins, the fifth disulfide bridge in WTXs is located in

N-terminus loop I To date, four non-conventional (three-finger) toxins have been isolated

from the venom of Bungarus species that consist of 62-68 amino acid residues and five

disulfide bridges They are, -bungarotoxin from B multicinctus (Aird et al., 1999), and

bucandin (Torres et al., 2001), bucain (Watanabe et al., 2002) and candoxin (Nirthanan et

al., 2002), from Bungarus candidus In addition, four other isoforms of weak neurotoxins have been isolated from the venom of Naja sputatrix The genes contained three exons

interrupted by two introns, a structure similar to other members of the three-finger toxin

family (Jeyaseelan et al., 2003) In light of the increased diversity of the snake

three-finger neurotoxins, recent evidence suggests that the candoxin (CADO_BUNCA:-

P81783) be categorized into orphan group IV, and thus may ultimately be designated the

Type IV neurotoxins (Fry et al., 2003) Apart from toxicity studies, the non-conventional

toxin, candoxin from Bungarus candidus (see below) has been poorly investigated in

terms of their cellular function or molecular target using gene expression

(toxicogenomics) technology, which is the focus of the present study, and will be

discussed in greater detail

1 6c Three-finger Toxins from Other Species

A group of other three-finger toxins and polypeptides are structurally similar to

short-chain neurotoxins, containing 60 amino acid residues and four conserved disulphide

bridges The similar overall fold notwithstanding, three-finger toxins exhibit various

range of pharmacological activities Curaremimetic neurotoxins ( bungarotoxin,

-cobratoxin) and / -neurotoxins (erabutoxin-b) belong to the family of three-finger toxins

(Servent and Menez, 2001; Kini, 2002) Other members of this family include

-bungarotoxins which recognize various subtypes of neuronal nicotinic receptors (Grant

and Chiapinelli, 1985; Fiordalisi et al., 1994), muscarinic toxins with selectivity towards

distinct types of muscarinic receptors (Jerusalinsky et al., 2000), fasciculins that inhibit

acetylcholinesterase (Falkenstein et al., 2004), calciseptins that block the L-type calcium

channels (De Weille et al., 1991, Albrand et al., 1995), and cardiotoxins (cytotoxins) that

exert their toxicity by forming pores in the cell membrane (Bilwes et al., 1994; Wang and

Wu, 2005) In addition, the cardiotoxins are basic -sheet polypeptides containing ~60

amino acid residues, and the three-dimensional characterization of the toxin has shown

that it belongs to the family of finger toxin (Tan, 1982) Interestingly, the

three-fingered fold is not restricted to snake venom toxins because several other non-venom

proteins and polypeptides also belong to this superfamily of proteins For example, the

three-finger fold is adopted by proteins from non-venom sources like Ly-6 family of

cell-surface accessory proteins expressed on immune system cells (Kieffer et al., 1994;

Gumley et al., 1995; Tsetlin, 1999), wheat germ agglutinin (Drenth et al., 1980) and

peptides (xenoxins) from frog (Xenopus laevis) skin secretions (Kolbe at al., 1993) In

addition, gene lynx 1, which is highly expressed in rat brain, has been found to encode for

a three-finger protein (TFP) that is a novel modulator of neuronal nAChRs in vitro (Miwa

et al., 1999) Very recently, a novel PATE gene has been identified which has a structural

similarity to three-finger toxins It is highly expressed in human prostate cancer, normal

prostate, and testis (Bera et al., 2002)

1 7 The Malayan krait (Bungarus candidus)

The Malayan krait, Bungarus candidus (Figure 1.3) is one of the most medically

significant snakes, belonging to the most widely distributed krait species (Kuch et al.,

2003) It is nocturnal in habit and feeds on other snakes, reptiles and rodents (Lim and

Ibrahim, 1970), and is widespread in Southeast Asia extending from Thailand to

Malaysia, Indonesian islands of Bali and Java across Sumatra, Cambodia and Southern

Vietnam (Lim, 1990; Kuch et al., 2003)

Figure 1.3 The Malayan krait (Bungarus candidus) The Malayan krait (Bungarus

candidus) is one of the most widely distributed (in Southeast Asia including Thailand,

Malaysia, Burma and Indonesia) krait species It is black with white cross-bands which partially encircle the body and tail tapering to a point It is often mistakenly identified as

the commoner species, Bungarus multicinctus or Bungarus fasciatus It can grow as long

as 1.6 m It is nocturnal in habit and feeds on other snakes, reptiles and reportedly very frightened Its venom is predominantly neurotoxic and as lethal as those of other kraits and cobras Candoxin was isolated and purified from its venom

It is morphologically similar to the many-banded krait (Taiwan banded krait) Bungarus

multicinctus and the banded krait Bungarus candidus, and is hence often misidentified

during encounters with human This has led to significant underestimation of the clinical

significance of the nocturnal elapid snake B candidus bites (Looareesuwan et al., 1988;

Virvan et al., 1992) Fatalities caused by B candidus envenomation are probably

underreported because most occur in rural areas and time to death is very short (Kuch et

al., 2003) Clinical signs following envenomation by B candidus is characterized by

generalized paralysis and occasionally by decreased parasympathetic activity; local signs

at the site of bite are minimal or absent (Warrell et al., 1983; Loathing and Sitprija, 2001)

Interestingly, Thai B fasciatus and Taiwan B multicinctus antivenom were ineffective in

protecting mice experimentally envenomed with B candidus venom whereas, the

Australian tiger snake (Notchis scutatus scutatus) antivenom was very effective (Warrel

et al., 1983)

1 8 Pharmacological Properties of Bungarus candidus

Many snake venoms, especially those of elapids, contain a variety of chemically similar

basic proteins which exhibit different types of pharmacological activities (including

cardiotoxicity, cytotoxicty and potent neurotoxic activity) by interfering with normal

physiological process (Tu, 1977 and 1996) Few biochemical and pharmacological studies

have been carried out on B candidus venom or its components A high content of

hyaluronidase is reportedly present, almost exclusively, in B candidus venom when

compared with other Southeast Asian elapids (including other Bungarus species) and

vipers (Pukrittayakamee et al., 1988; Tan et al., 1989) This is surprising since

hyaluronidase is known to contribute to local inflammation by disrupting connective

tissue and potentiating necrosis, thereby accelerating the systemic absorption of

polypeptide toxins (Tu and Hendon, 1983) It is, therefore, difficult to reconcile the

minimal local effects seen following B candidus bites with the reported high

hyaluronidase activity of its venom It has also been reported that B candidus venom

exhibits high acetylcholinesterase and phospholipase A2 and moderate L-amino acid

oxidase activity (Tan et al., 1989) The major lethal fraction in the venom is a basic lethal

phospholipase A2 (MW ~21 KDa) that accounts for ~13% of the venom protein and has a

LD50 of ~0.02mg/kg This description suggests that the lethal phospholipase A2

component may be a β-bungarotoxin isomer (Lee, 1972; Chang, 1979) More recently, B

candidus venom has been fractionated and a nontoxic, non-conventional toxin (bucandin) has been isolated and its structure determined by X-ray diffraction and NMR (Kuhn et al.,

2000; Torres et al., 2001)

1 9 Biochemical and Pharmacological Properties of Candoxin

Candoxin (MW 7334.46), a neurotoxin isolated from the venom of the Malayan krait,

belongs to the poorly characterized subfamily of nonconventional three-finger toxins

present in Elapid family The pharmacological study of candoxin at the neuromuscular

junction revealed a novel pattern of neuromuscular blockade in isolated nerve-muscle

preparations and the tibialis anterior muscle of anaesthetized rats The biochemical as

well as pharmacological assays of candoxin on acetylcholinesterase demonstrated that

candoxin faild to produce any effect on the response of the anococcygeus muscle to

cholinergic agonist implying the absence of inherent antocholinesterase activity even at

higher concentration (100µM) In addition, the electrophysiological experiments

demonstrated that candoxin produced a readily reversible blockade of oocyte-expressed

muscle ( ) nAChRs (Nirthanan et al, 2002) Besides, a significant train-of-four fade

has been reported during the onset of and recovery from neuromuscular blockade, both, in

vitro and in vivo (Nirthanan et al, 2003) Interestingly, it is also a potent antagonist of

neuronal α7 nicotinic acetylcholine receptors, but in this case is poorly reversible Due to the intensive use of the snake venom three-finger toxins as investigational ligands in

biomedical, pharmacological and biochemical research, a large number have been

characterized and sequenced, making this class of toxins particularly valuable for

molecular evolutionary studies