Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.717

Neurotoxins Affecting Neuroexocytosis

Micro total analysis systems. Recent developments.

Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

1.1. Discovery of the Phospholipase A2 Superfamily
Phospholipases represent one of the earliest enzyme activities to be identified and studied and the phospholipase A2 (PLA2) superfamily (see defining specificity1 in Figure 1) traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. The enzyme was first purified and characterized from cobra venom and later from rattlesnake venom. As protein sequencing methodologies advanced in the 1970’s, it became apparent that these enzymes had an unusually large number of cysteines (over 10% of the amino acids) and as secreted enzymes, that they were all in the form of disulfide bonds. It was further recognized that in the case of PLA2, cobras and rattlesnakes had six disulfides in common, but one disulfide bond is located in distinctly different locations. This led to the designation of Type 1 and Type 2 for cobras (old world snakes) and rattlesnakes (new world snakes), respectively.2 During that same period, studies on the porcine pancreatic digestive enzyme that hydrolyzes phospholipids led to the determination that this mammalian enzyme (and also the human pancreatic enzyme) had the same disulfide bonding pattern as cobras and hence the designation as IB with the cobra enzyme as IA.






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Figure 1


The specific reaction catalyzed by phospholipase A2 at the sn-2 position of the glycerol backbone is shown. X, any of a number of polar headgroups; R1, fatty acids, or alkyl, or alkenyl groups and R2, fatty acids or acyl moieties.

/pdf/phospholipase-a2-enzymes-physical-structure-biological-usiwyw5zej.pdf

Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.

The phospholipase A2 superfamily and its group numbering system

An ultrasensitive electrochemical enzyme immunoassay (EEIA) for the detection of proteins on an 8x8 array is described. The assay is based on wired enzyme technology. Briefly, capture antibody was covalently immobilized on a self-assembled 11-mercaptoundecanoic acid (MUA) monolayer coated gold electrode. After incubating with a target protein (antigen), the gold electrode was treated sequentially in biotinylated detection antibody solution and in avidin-horseradish peroxidase conjugate (A-HRP) solution. A cationic redox polymer (electrochemical mediator) overcoating was applied to the gold electrode through layer-by-layer electrostatic self-assembly. The formation of a bilayer brought the HRP in electrical contact with the underlying electrode, making the bilayer an electrocatalyst for the reduction of hydrogen peroxide where the redox polymer acts as an artificial mediator. Consequently, the concentration of protein could be quantified amperometrically. This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the electrochemical detection. The applicability of the system in protein detection was demonstrated with a snake toxin, beta-bungarotoxin, a neurotoxin from the venom of the snake Bungarus multicinctues. Under optimized experimental conditions, the assay allowed the detection of beta-bungarotoxin in the range of 20 pg/mL to 1.5 ng/mL with a detection limit of 10 pg/mL (20 fg). A higher detection limit of 25 pg/mL was obtained in serum.

An ultrasensitive protein array based on electrochemical enzyme immunoassay.

Background: The Botulinum neurotoxins (BoNTs) are a major threat in military settings. The mouse bioassay is the “gold standard” method, however, it has practical limitations. Purpose: To standardize a rapid, sensitive and fieldable optical immunoassay (OIA) for the detection of BoNTs A, B, E and F in food matrices. Materials and methods: The toxicity of BoNTs A, B, E and F was assessed by using a modified LD 50 assay. OIA was used to detect BoNTs in a variety of food matrices representing liquids, solids, and semisolid food. For specificity studies, the assay was used to test varying concentrations of epsilon toxin (ETX) from Clostridium perfringens, toxin A (TcdA) from C.difficile and staphylococcus enterotoxin B (SEB) from S.aureus in spiked assay buffer. To evaluate the OIA performance, five concentrations (serially five-fold diluted, viz. 20, 4, 0.8, 0.16 and 0.032 in ng/mL) of BoNT/A were made in orange juice and tested by both OIA and mouse bioassay. Results: The mouse intraperitoneal (i.p.) LD 50 values (in ng/kg) of BoNTs A, B, E and F were determined to be 1.6, 1.8, 1.3, and 2.4 respectively. OIA could readily detect 20ng/mL of BoNTs A, B, E and F in all the food samples tested. OIA demonstrate limits of detection lesser in range to the gold standard mouse bioassay. Conclusion: The OIA was found to

Fieldable Assay For Botulinum Neurotoxins

By using micro-scale polystyrene beads as a platform, a rapid and sensitive method for the detection of snake venom was established. In the method, anti-venom antibody or BSA was covalently fixed onto the microsbead to form the capture-bead or control bead. In first step of the experiment, the venom binds to the capture-bead to form the complex through the antibody-antigen interaction. The Qdot conjugated second antibody was then added. The second antibody targeted the Qdot to the capture-bead/antigen complex and form Qdot-second antibody-antigen-capture-bead complex. This complex can be directly observed under UV-microscope. The system was applied to the testing of Naja kaouthia venom and the detection limit of this method was 5–10 ng/ml.

https://www.tandfonline.com/doi/pdf/10.1080/17458080701713953

Immuno-fluorescence detection of snake venom by using single bead as the assay platform

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA

L-amino acid oxidases (LAAOs), which are largely present in snake venoms, are a group of flavoenzymes, known to catalyze oxidative deamination of L-amino acid substrates, resulting in the formation of corresponding α-keto acids, hydrogen peroxide and ammonia. LAAOs have attracted interest in biomedical and pharmacological research because of their antimicrobial, anti-HIV, anticoagulant, inhibition of platelet aggregation and apoptotic-inducing activities. The aim of this study is to evaluate the anticancer effects of LAAO from Crotalas adamanteus snake venom in human NUGC-3 gastric cancer cells in vitro. Cell viability and cytotoxicity were determined by Alamar Blue and Lactate Dehydrogenase assays. Cell cycle phases were analyzed by flow cytometry. Apoptosis was identified by flow cytometric analysis with Annexin-V and PI staining. The morphological changes were observed by fluorescence microscopy, transmission electron microscopy and scanning electron microscopy. The ROS level was quantified by DCFDA staining as well as malondialdehyde formation. The results showed that LAAO reduced cell viability and induced cytotoxicity in a dose-dependent manner. There was associated cell cycle arrest at the G2/M phase. Morphological changes induced by LAAO treatment included membrane blebbing, cell shrinkage and chromatin condensation. Apoptosis induced by LAAO in NUGC-3 gastric cancer cells as evidenced by Annexin-V and PI positive staining, was found to be mediated via a caspase-independent pathway without activating caspase-3 and Poly ADP-ribose polymerase. Moreover, a concomitant increase of intracellular oxidative stress and the translocation of the apoptosis inducing factor (AIF) from mitochondria to the nucleus were observed. It would appear that LAAO derived from the Crotalas adamanteus venom, induced a caspase-independent apoptotic pathway which was incited by ROS and AIF translocation. This study provides new biological insights into the application of LAAO from a snake venom in cancer therapeutics.

Citation Format: Jian Ding, Boon-Huat Bay, P Gopalakrishnakone. L-amino acid oxidase from Crotalas adamanteus venom induces caspase-independent apoptosis in human NUGC-3 gastric cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2014-5425

Ponnampalam Gopalakrishnakone

Papers

An ultrasensitive protein array based on electrochemical enzyme immunoassay.

Fieldable Assay For Botulinum Neurotoxins

Immuno-fluorescence detection of snake venom by using single bead as the assay platform

Abstract 5425: L-amino acid oxidase from Crotalas adamanteus venom induces caspase-independent apoptosis in human NUGC-3 gastric cancer cells

A computer based colour-photo database system for dangerous animals and plants: academic and public information networks