Effect of dimerized melittin on gastric cancer cells and antibacterial activity.

Melittin is the peptide toxin found in bee venom and is effective against cancer cells. To enhance its activity, a branched dimeric form of melittin was designed. The monomeric form of the peptide was more cytotoxic against gastric cancer cells at low concentrations (1-5 µM) than the dimer form, while the cytotoxic effect was comparable at higher concentrations (10 µM). Confocal microscopy showed that both the monomer and dimer forms of melittin with fluorescent label at the C terminus penetrated the cytoplasm and localized at the cell nucleus and disrupted the cell membrane. The results indicated that both peptides localized in the nucleus and no significant difference in penetration was observed between monomer and dimer of melittin. Although the C and N termini are important for melittin activity, using C terminus for dimerization of the peptide resulted in similar activity for the monomer and dimer against bacteria and gastric cancer cells.

L-amino acid oxidase from snake venom and its anticancer potential.

L-amino acid oxidase (LAAO) is a protein toxin commonly found in snake venom. It has many applications, ranging from biotechnology to potential anticancer therapeutics. LAAO converts L-amino acid into α-keto acid and release ammonia and hydrogen peroxide as by-products. Induction of oxidative stress in cancer cells is one of the cancer treatment strategies as controlled and targeted release of hydrogen peroxide can theoretically induce sufficient oxidative stress to kill cancer cells. Furthermore, L-amino acid oxidase has been shown to selectively bind to cell membranes of specific phospholipid composition and deliver the hydrogen peroxide to localized regions on the cell surface. In this mini review, we discuss the relevance of L-amino acid oxidase, in terms of its structure and enzyme activity, its potential as a cytotoxic agent and exploitation of its cytotoxic nature as an anticancer therapeutic.

Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom.

Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer, is a K+ channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/ß scaffold that is cross-linked by four disulfide bridges (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7, and Cys4-Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4-Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1-Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3-Cys4 and Cys7-Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1-Cys5 and Cys2-Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.

Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

Wound healing activity and mechanisms of action of an antibacterial protein from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus).

Basic phospholipase A2 was identified from the venom of the eastern diamondback rattlesnake. The Crotalus adamanteus toxin-II (CaTx-II) induced bactericidal effects (7.8 µg/ml) on Staphylococcus aureus, while on Burkholderia pseudomallei (KHW), and Enterobacter aerogenes were killed at 15.6 µg/ml. CaTx-II caused pore formation and membrane damaging effects on the bacterial cell wall. CaTx-II was not cytotoxic on lung (MRC-5), skin fibroblast (HEPK) cells and in mice. CaTx-II-treated mice showed significant wound closure and complete healing by 16 days as compared to untreated controls (**P<0.01). Histological examination revealed enhanced collagen synthesis and neovascularization after treatment with CaTx-II versus 2% Fusidic Acid ointment (FAO) treated controls. Measurement of tissue cytokines revealed that interleukin-1 beta (IL-1ß) expression in CaTx-II treated mice was significantly suppressed versus untreated controls. In contrast, cytokines involved in wound healing and cell migration i.e., monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor-basic (FGF-b), chemokine (KC), granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly enhanced in CaTx-II treated mice, but not in the controls. CaTx-II also modulated nuclear factor-kappa B (NF-κB) activation during skin wound healing. The CaTx-II protein highlights distinct snake proteins as a potential source of novel antimicrobial agents with significant therapeutic application for bacterial skin infections.

Cross-Reactivity against Naja sumatrana (Black Spitting Cobra) Envenoming from the Haffkine Antivenom in a Mouse Model.

Naja sumatrana is the dominant cobra species in Malaysia, Singapore, Borneo, and Sumatra, and it does not have specific antivenom. The Haffkine antivenom has been advocated instead. This study aims to determine the efficacy of this antivenom against Naja sumatrana envenoming using a mouse model. Methods. Male Swiss albino mice were used. Intravenous LD50 was first determined separately for Naja naja and Naja sumatrana venom. ED50 was determined by preincubating antivenom with each venom at 2.5 LD50 before administering the mixture into the tail vein. Validation was carried out using a challenge test. Each mouse received 111 µg of Naja sumatrana venom intramuscularly followed by intraperitoneal administration of dilute Haffkine antivenom. Survival was recorded 24 hours after envenoming. Results. The LD50 of Naja naja venom was 78.13 µg, standard error (SE) 13.3 µg. The ED50 of the Haffkine antivenom against Naja naja venom was 45.9 mg, SE 7.5 mg. The LD50 and ED50 of Naja sumatrana venom were 55.5 µg, SE 12.0 µg; and 73.9 mg, SE 12.0 mg, respectively. The intra-peritoneal ED50 against 111 µg intramuscular Naja sumatrana venom was 136.95 mg, SE 36.74 mg. Conclusion. The Haffkine polyvalent antivenom exhibited cross-neutralisation against Naja sumatrana venom when used at a higher dose.

Synthesis and characterization of amino acid deletion analogs of κ-hefutoxin 1, a scorpion toxin on potassium channels.

Nine analogs of scorpion toxin peptide κ-hefutoxin 1 were synthesized by stepwise deletion of its amino acid residues. Disulfide bond pairings of the synthetic analogs were confirmed by enzymatic digestion followed by MALDI-TOF-MS measurements. Functional characterization shows that analogs in which N-terminal residues were deleted retained biological activity, whereas deletion of middle part residues resulted in loss of activity. Furthermore, κ-hefutoxin 1 and analogs were subjected to a screening on voltage-gated potassium channels in order to determine their subtype selectivity. It is shown that κ-hefutoxin 1 is suitable as template for peptidomimetics in order to design small peptide-based therapeutic compounds.

Therapeutic application of natural inhibitors against snake venom phospholipase A(2).

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.

Therapeutic potential of peptides with neutralizing ability towards the venom and toxin (CaTx-I) of crotalus adamanteus.

The CaTx-I (PLA2) toxin of Crotalus adamanteus venom is responsible for most of the symptoms observed during envenomation. Synthetic peptides were designed and screened for venom (0.8 µg/ml) and CaTx-I (0.1 µM) inhibition at varying doses of the peptide (10000- 0.0001 µM) using a Cayman chemical human secretory phospholipase A2 (sPLA2, Type II) assay kit. Further, in vitro neutralization studies were evaluated by a fixed dose of peptide (1 µM) against venom (0.8 µg/ml) and toxin (0.1 µM). Among the linear peptides (PIP-18, cyclic C and PIP59-67) that showed potent neutralizing effects against the venom/toxin of C. adamanteus. PIP-18 [IC50, 1.23 µM] and cyclic C [IC50, 1.27 µM] peptides possessed the strongest inhibitory effect against CaTx-I. A fixed dose of CaTx-I (75 µg/kg) was administered intraperitoneally (i.p.) into mice followed by an i.p. injection of peptides PIP-18 and cyclic C at (6 µg/mouse), venom (150 µg/kg) and toxin CaTx-I alone served as references. Mice treated with PIP-18 and cyclic C showed a very strong neutralizing effect and markedly reduced mortality compared to the control after 24 h. The CA venom and CaTx-I injected mice showed severe toxicity after 24 h. Peptides PIP-18 and cyclic C were non-hemolytic at 100 µM. They produced a significant decrease in lipid peroxidase (LPx) and enhancement of superoxide dismutase (SOD), catalase (CAT) and Glutathione-s-transferase (GST) levels indicating their antioxidant property against venom-induced changes in mice. This study confirmed the potent snake venom neutralizing properties of peptides.

Evaluation of antibacterial activity of proteins and peptides using a specific animal model for wound healing.

Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and extracellular matrix (ECM). In this study, phospholipase A(2) (PLA(2)) purified from crotalid snake venom was found to express in vitro bactericidal activity against a group of clinical human pathogens. Based on the sequence homology of PLA(2), a series of peptides were derived from the C-terminal region of crotalid PLA(2). These short synthetic peptides were found to reproduce the bactericidal activity of its parent molecule. In vitro assays for bactericidal and cytolytic activities of these peptides showed very high microbicidal potency against Gram-negative and Gram-positive (Staphylococcus aureus) bacteria. Variants of the peptides showed reduced toxicity toward normal human cells, while retaining high bactericidal potency. Here we describe the protocol for evaluating the wound healing process by antibacterial peptides. We evaluated the biological roles of the candidate peptides in skin wound healing, using a specific BALB/c mice model. Peptide-treated animals showed accelerated healing of full-thickness skin wounds, with increased reepithelialization, collagen synthesis, and angiogenesis observed during the healing process. Healing wounds in protein/peptide-treated mice had higher densities of neutrophils, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression were also upregulated. These results demonstrate that the protein/peptide derived from snake venoms promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of collagen and growth factors.

Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch.

In this communication, the full protein sequence of a novel venom hyaluronidase BmHYA1 was reported. It is the first full hyaluronidase amino acid sequence from scorpion venom. It was deduced from nucleotide sequence by 3' Rapid Amplification of cDNA Ends (RACE) PCR cloning, followed by alignment with the N-terminal amino acid sequence, which was obtained by Edman degradation. BmHYA1 has 385 amino acid residues containing five potential N-glycosylation sites. The phylogenetic analysis indicates early divergence and independent evolution of BmHYA1 from other hyaluronidases.

Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

Therapeutic Potential of Plants as Anti-microbials for Drug Discovery.

The uses of traditional medicinal plants for primary health care have steadily increased worldwide in recent years. Scientists are in search of new phytochemicals that could be developed as useful anti-microbials for treatment of infectious diseases. Currently, out of 80% of pharmaceuticals derived from plants, very few are now being used as anti-microbials. Plants are rich in a wide variety of secondary metabolites that have found anti-microbial properties. This review highlights the current status of traditional medicine, its contribution to modern medicine, recent trends in the evaluation of anti-microbials with a special emphasis upon some tribal medicine, in vitro and in vivo experimental design for screening, and therapeutic efficacy in safety and human clinical trails for commercial outlet. Many of these commercially available compounds are crude preparations administered without performing human clinical trials. Recent methods are useful to standardize the extraction for scientific investigation of new phytochemicals and anti-microbials of traditionally used plants. It is concluded that once the local ethnomedical preparations of traditional sources are scientifically evaluated before dispensing they should replace existing drugs commonly used for the therapeutic treatment of infection. This method should be put into practice for future investigations in the field of ethnopharmacology, phytochemistry, ethnobotany and other biological fields for drug discovery.

Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1beta-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice.

INTRODUCTION: Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis. METHODS: Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1beta stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)alpha, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively. RESULTS: PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1beta-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1beta-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice. CONCLUSIONS: Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.

ETS2 regulating neurodegenerative signaling pathway of human neuronal (SH-SY5Y) cells exposed to single and repeated low-dose sarin (GB).

The mechanistic understanding of low-level sarin-induced neurotoxicity after single or repeated doses has yet to be explored at a cellular level. Using the microarray (Affymetrix-GeneChips) transcription profiling approach, the present study examined gene expression in human SH-SY5Y cells exposed to single (3 and 24 h) or repeated (2 x 24 h) doses of sarin (5 microg/mL) to delineate the possible mechanism. Two hundred twenty-four genes whose expression was significantly (P < 0.01) altered by at least 3-fold were selected by GeneSpringGX analysis. The comparative gene expression data confirmed the transcriptional changes to be related to dose and exposure time of sarin. The effect of a single noncytotoxic sarin dose on gene transcription was variable, whereas repeated doses over 48 h persistently down-regulated genes linked to neurodegenerative mechanisms. Thirty persistently altered genes were validated using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Similar qRT-PCR profiles obtained in sarin-treated SH-SY5Y and HCN-1A cells confirmed the cell-independent alterations in expression levels. Genes (ETS2, APOE, PSEN1, DDC, and CD9) implicated mainly in the regulation of sarin-induced neuropathogenesis were further confirmed by Western blot and double-immunofluorescence assays. The regulome pathway suggests a new feasible mechanism by which sarin increases ETS2 expression and takes control over other genes involved in the neurodegenerative pathway. The overall data delineate an in vitro experimental model suitable for studying the neuropathology of cells and may provide novel insights into therapeutic interventions.

Transcriptome profiling of neuronal model cell PC12 from rat pheochromocytoma.

GeneChip microarray is a cutting-edge technology being used to study the expression patterns of genes with in a particular cell type. In this study, the Affymetrix RAE230A platform was used to profile stably expressed mRNA transcripts from PC12 cells at passage 5 and 15. The whole-cell PC12 transcriptome revealed that a total of 7,531 stable transcripts (P < 0.05), corresponding to 6,785 genes, were found to be consistently expressed between passage 5 and 15. The data analysis revealed 3,080 functional proteins, belonging to 13 families, which indicate that about 65% of the proteins expressed in PC12 cells are uncharacterized. By using our custom-built rat neuronal reference genome database, we mapped endogenously expressed stable neuronal transcripts from PC12 cells comprising about 765 genes responsible for neuronal function and disease. These neuronal transcripts were further analyzed to provide a genetic blueprint that can be used by neurobiologist to unravel the complex cellular and molecular mechanisms underlying biological functions and their associated signalling networks for diseases affecting the nervous system.

A study on the differential protein profiles in liver cells of heat stress rats with and without turpentine treatment.

BACKGROUND: Heat stress (HS) and related illnesses are a major concern in military, sports, and fire brigadiers. HS results in physiologic responses of increased temperature, heart rate and sweating. In heat stroke, inflammatory response plays an important role and it is evidenced that turpentine (T) induced circulating inflammatory cytokines reduced survival rate and duration at 42 degrees C. Here we report the alteration in the protein expression in liver cells upon HS with and without T treatment using two dimensional gel electrophoresis (2-DE), tryptic in-gel digestion and MALDI-TOF-MS/MS approaches. RESULTS: The effects of HS and T treatments alone and a combined treatments (T+HS) was performed in Wistar rat models. Proteomic analysis of liver in the HS and T+HS groups were analyzed compared to liver profiles of resting control and T treated groups. The study revealed a total of 25 and 29 differentially expressed proteins in the HS and T+HS groups respectively compared to resting control group. Fourteen proteins showed altered expression upon T treatment compared to resting control group. Proteins that are involved in metabolic and signal transduction pathways, defense, redox regulation, and cytoskeletal restructuring functions were identified. The altered expression of proteins reflected in 2D gels were corroborated by quantitative real time RT-PCR analysis of 8 protein coding genes representing metabolic and regulatory pathways for their expression and normalized with the house keeping gene beta-actin. CONCLUSION: The present study has identified a number of differentially expressed proteins in the liver cells of rats subjected to T, HS and T+HS treatments. Most of these proteins are implicated in cell metabolism, as well as adaptive response to incurred oxidative stress and tissue damage due to T+HS and HS effects.

Use of the upside-down method to prepare porous polymer films with tunable surface pore sizes.

Porous polyurethane films with well-ordered pores were prepared using polystyrene microspheres as a template, and the surface pore sizes were tuned using a new upside-down (USD) method. After polystyrene microspheres were self-assembled on a substrate and the voids between microspheres were infiltrated with a polyurethane prepolymer solution, the samples were placed upside down to allow the prepolymer solution to flow back before it was solidified. The surface pore sizes of the porous films were tuned by changing the time in which the samples were placed upside down. Using microspheres of the same size, we prepared porous polyurethane films with surface pore sizes ranging from 32 to 87% of the templating microsphere size.

Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi.

A hyaluronidase, named BmHYA1, was purified from the venom of Chinese red scorpion (Buthus martensi), using successive chromatography. The homogeneity of BmHYA1 was confirmed by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of BmHYA1 was 48,696 Da determined by MALDI-TOF MS. The optimal temperature and pH of BmHYA1 were 50 degrees C and pH 4.5, respectively. It could be inhibited by DTT, Cu(2+), Fe(3+) or heparin, but not Mg(2+), Ca(2+), reduced glutathione, l-cysteine or EDTA. The sequence of thirty N-terminal amino acids of BmHYA1 was obtained by Edman degradation, as TSADF KVVWE VPSIM CSKKF KICVT DLLTS; but no similarity was found to other venom hyaluronidases. Further, BmHYA1 can hydrolyze hyaluronan into relatively smaller oligosaccharides and modulate the expression of CD44 variant in the breast cancer cell line MDA-MB-231.