The action of Taiwan cobra venom on methionine enkephalin: a useful assay for oligopeptidase content

The pentapeptide methionine enkephalin is readily hydrolysed by the oligopeptidase activity contained in Taiwan cobra (Naja naja atra) venom. It is a significantly better substrate than the peptides previously used to identify the presence of this enzyme, but it retains many of the sequence characteristics shared by these other peptides. Analysis of the manner of hydrolysis by means of high-performance liquid chromatography and electrospray mass spectrometry revealed the simultaneous actions of at least two types of oligopeptidase on the neuropeptide, producing two routes of initial breakdown. By one route, an endopeptidase cleaved the Gly-Phe bond of enkephalin first to release Tyr-Gly-Gly and Phe-Met. By the other route, an aminopeptidase was able to release Tyr and Gly-Gly-Phe-Met by cleaving the Tyr-Gly bond first. From amongst the various peptide fragments produced, Tyr-Gly-Gly was subject to immediate aminopeptidase action to release Tyr and Gly-Gly. The free Tyr produced in these reactions was in turn quickly transformed by the L-amino acid oxidase in the venom. The kinetic qualities of the enkephalin hydrolysis, and the conversions of the fragments Tyr-Gly-Gly and Tyr, were measured. Methionine enkephalin has potential as a routine assay for venom oligopeptidases, either in testing the venoms from other species or in attempts to purify these enzymes. Moreover, the ease of hydrolysis of this bioactive peptide, coupled with the revelation of the other enzymic steps involving the fragments generated, may provide important clues as to the possible role of the oligopeptidases (and L-amino acid oxidase) in the venom.     

Mapping a conserved conformational epitope from the M protein of group A streptococci

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.     

Purification of cobra venom factor from phospholipase A contaminant

It has been demonstrated that cobra venom factor prepared by the usual combination of ion exchange chromatography and sephadex gel filtration is contaminated by substantial amounts of a 'heavy' phospholipase A. The two activities may be separated by isoelectric focusing. Cobra venom factor focuses at pH between 5-75 and 6-75 whereas the phospholipase is all found at pH below 7-75. In certain test systems, particularly in vitro, and particularly where albumin concentrations are low, the contaminating phospholipase may produce effects that have been attributed to complement activation.     

Enkephalin-processing oligopeptidases in cobra venom: inhibition by thiorphan and bestatin reveals co-operative actions

The peptidase inhibitors thiorphan and bestatin were tested for their ability to inhibit the actions of the oligopeptidases contained in the venom of the Taiwan cobra (Naja naja atra). With methionine enkephalin (TyrGlyGlyPheMet) as substrate, thiorphan was an effective inhibitor of cleavage of the GlyPhe peptide bond while bestatin inhibited cleavage of the TyrGly peptide bond. Thiorphan and bestatin also inhibited subsequent cleavage of the fragments GlyGlyPheMet and TyrGlyGly respectively. These inhibitors reveal an interplay between the venom oligopeptidases in which the enzymes provide additional substrates for each other following their initial competitive attack on the neuropeptide. A possible explanation is that the system is intended to ensure a steady release of Tyr, GlyGly and PheMet over time. Significantly, Tyr is the favoured substrate of the L-amino acid oxidase present in the venom, which rapidly transforms this aromatic amino acid into phenolic derivatives. The efficacies of these inhibitors also suggest that there are similarities between the venom oligopeptidases and the peptidases associated with the processing of enkephalin in its normal contexts.     

Purification and some properties of two phospholipases and a toxin from the venom of desert cobra (Walterinnesia aegyptia)

An acidic and a basic phospholipase A (PLA) and a toxin have been purified from the venom of W. aegyptia using a TSK G 3000 SW gel filtration column and a Mono Q ion-exchange column. Both phospholipases and the toxin were found to be in a homogeneous state on SDS-PAGE and their purity was verified by isoelectric focusing. The acidic PLA showed higher enzymatic activity and concentration in venom when compared to basic PLA. The acidic and the basic PLA showed 16.5 +/- 0.5 Kd and 14.5 +/- 0.5 Kd molecular weights respectively, while the toxin showed 12.0 +/- 0.5 Kd molecular weights on SDS-PAGE. The isoelectric points for the acidic and basic PLA were at pH 4.5 and 7.8 respectively. The toxin was focused in the basic region at pH 10. Both phospholipases constitute about 20% of the venom protein and were nontoxic. However, only basic PLA when mixed with the toxin reduced the time required by the toxin alone to kill the mice, while the toxin was highly lethal to mice and its LD50 was 0.4 micrograms/g of body weight of mouse. The toxin showed on phospholipase activity.     

Nerve growth factor from the venom of the Chinese cobra Naja naja atra: purification and description of non-neuronal activities

Nerve growth factor (NGF) was separated from crude Naja naja atra venom by using weak cation-exchange chromatography, followed by reversed-phase liquid chromatography. The yield of the purification was 0.2-0.5% (w/w). The mol. wt was determined to be 13,600 and the protein still induced the typical fibre outgrowth of cultured PC-12 cells in a concentration range of 5-10 ng/ml. Beside this neuronal effect we demonstrated non-neuronal effects of cobra venom NGF, such as induction of plasma extravasation and histamine release from whole blood cells. With human leucocyte preparations, including enriched basophils, there was an increase in C5a-induced histamine release, whereas NGF alone was inactive. Cobra NGF was one-tenth as potent as human recombinant NGF, with a half-maximal stimulation occurring at 10 ng/ml. Cobra NGF and human recombinant NGF showed a modulatory effect on histamine release comparable to the haematopoietic growth factor IL-3. Thus, the non-neuronal effects of cobra NGF may account for immunomodulatory activities during inflammatory events.     

Kinetics of the inhibition of acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom by local anesthetics: procaine and tetracaine

The N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene, which is responsible for autosomal recessive mucopolysaccharidosis IVA (MPSIVA), has been assigned to the long arm of chromosome 16, subregion 24.3, an area where the adenine phosophoribosyltransferase (APRT) gene and renal dipeptidase (DPEP I) gene are also localized. Molecular genetic studies on a severely affected patient with MPSIVA (Morquio disease), without karyotypic abnormality, revealed a partial submicroscopic deletion of 16q24.3 and a single point mutation on the other allele, with no functional GALNS activity. The patient, his mother, and siblings were hemizygous for GALNS and APRT loci, evidenced by informative RFLP and gene dosage analyses combined with a fluorescence in situ hybridization, utilizing a partial genomic clone of GALNS, but heterozygosity was retained at the DPEP I locus and proximal D16S7. Haplotyping of the family members revealed recombinational events between DPEP I locus and three other polymorphic loci on the paternal chromosome, localizing GALNS gene on the proximal side to DPEP I gene. As estimated from the genetic distance between two flanking markers of proximal D16S7 and distal DPEP I locus, size of the deletion was less than 3Mb. Mother of the boy and two older siblings were asymptomatic, despite this interstitial deletion of the Giemsa-light G band. The remaining paternal allele had no gene rearrangement but GALNS activity was not encoded as Arginine at 386 was replaced with Cysteine (R386C), suggesting this alteration accounts for the severe phenotype. Allelic loss of APRT is frequently observed in cancer tissues, thereby suggesting that the tumor suppressor gene locates near the APRT locus. No family member has evidence of any malignant disease. This study is apparently the first documentation of interstitial deletion of 16q24.3, involving GALNS and APRT genes.     

Characterization of a lectin-like protein isolated from Lachesis muta snake venom

A lectin-like protein was isolated from L. muta venom by gel filtration on BIO Gel P-100 followed by column Chromatography on DEAE-sephades A-50. The protein eluted at 0.4 M Nacl in 0.01 Tris pH 7.3 and exhibited agglutinin activity toward 0+ human erythrocytes. The protein is a dimer with Mr 28 kDa. Amino acid analysis revealed high content of tryptophan and acid recidues and low content of cysteine and methionine residues. No neutral carbohydrates and sialic acid were detected. Circular dichroic spectrum shows 78% of B structure and 1% of alpha structure. In vitro experiments with erythrocytes from rat, rabbit and dog revealed strong agglutination while red blood cells from mice, sheep and goat were not agglutinated. In vivo experiments using anesthetized rats, a sharp and prolonged fall in the blood pressure was observed at protein dose of 1.5 mg/kg. Double dose of protein caused the death of the animal.     

Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein

Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 +/- 0.7 (mean +/- SE, n = 6) and 103 +/- 10 microM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 amino acid residues and was 68% identical to the sequence of the A chain of IX/X-bp. The 15 kDa chain (B chain) consisted of 123 amino acid residues and was 87% identical to IX/X-bp. Three-dimensional model construction from the known fold of IX/X-bp showed that amino acid residues different from those of IX/X-bp are mostly on the molecular surface. Some of these are concentrated on a part of the concave surface which is considered to be the coagulation factor-binding site, presumably acting as a discriminator for ligand binding. These results indicated that X-bp isolated from D. acutus venom was a GD-binding protein, and the C-terminal region of GD peptide was critical for folding of the peptide.     

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

It has been well documented that drug-associated cues are important for the development and expression of drug tolerance. The Pavlovian conditioning analysis of tolerance emphasizes the importance of a drug-associated cues to tolerance by equating a drug administration with a learning trial. According to this analysis, tolerance should be subject to external inhibition, the disruption of a conditional response by a novel stimulus. We previously reported that tolerance to the ataxic effect of ethanol was attenuated by a novel strobe/noise presentation (31). In this article we report evidence of a compensatory CR in rats tolerant to the ataxic effect of ethanol as tested on the tilting plane. Both the compensatory CR and tolerance were disrupted by the presentation of a novel strobe/noise stimulus providing converging evidence that the attenuation of tolerance by a novel stimulus results from external inhibition of Pavlovian conditioning. The disruption of ethanol tolerance and the conditional response mediating tolerance was also apparent when the novel omission of the strobe/noise stimulus was used as the external inhibitor in rats made tolerant to ethanol with the stimulus on. Finally, we have shown that the disruptive effect of a novel stimulus on ethanol tolerance is decreased when there is a 10-day delay between the final tolerance development session and testing, demonstrating that the interval between training and testing is important when assessing associative tolerance.     

Amino acid sequence of a lectin-like protein from Lachesis muta stenophyrs venom

The primary structure of the lectin-like protein from Lachesis muta stenophyrs venom was deduced from analysis of the N-terminus and the sequence of peptides obtained after digestion with trypsin, Arg-C enzyme, Staphylococcus aureus V8 protease and endoproteinase Asp-N. Peptides generated by cleavage of the lectin with cyanogen bromide and o-iodosobenzoic acid were also sequenced. Comparison of the complete 135 amino acid residues sequence with those of the lectin from the venom of Crotalus atrox, with platelet coagglutinin from Bothrops jararaca beta-fragment and with the anticoagulant B protein chain from Trimeresurus flavoviridis venom, revealed 92, 46 and 29% identity, respectively. Significant homology was also found with C-type carbohydrate-recognition domain-like structures from invertebrate and vertebrate lectins. To our knowledge, this is the second known primary structure of a lectin-like protein from snake venom.     

Effect of fasciculin on hydrolysis of neutral and choline esters by butyrylcholinesterase, cobra venom and chicken acetylcholinesterases

Acetylcholinesterases (AChEs) very sensitive to fasciculin inhibition (KiS in picomolar range) have a distinctive group of aromatic amino acids in the peripheral region (Y70, Y121, W279 in Torpedo AChE). Enzymes that lack these amino acids like butyrylcholinesterases (BChEs) or one or two of them like cobra venom, insect and chicken AChEs are 1000 to 1,000,000 times less sensitive. Fasciculin is a non-competitive inhibitor of the hydrolysis of choline and neutral esters by very sensitive AChEs. For the other group of enzymes, differences arise according to the type of substrate. Fasciculin still behaves as a non-competitive inhibitor with choline esters. In contrast, hydrolysis of phenylacetate was unaffected or slightly increased with BChEs and a partial competitive inhibition was observed with cobra venom and chicken enzymes.     

Isolation and characterization of an endogenous inhibitor of phospholipase A2 from Indian cobra (Naja naja naja) venom

PURPOSE: To evaluate the efficacy of stent deployment in the treatment of recurrent stenosis of transplant renal arteries (TRAs). PATIENTS AND METHODS: This retrospective study includes six consecutive patients who underwent a mean of 3.66 previous treatments of TRA stenosis per patient before stent implantation (20 angioplasties and two surgical procedures). The endoprostheses were a Wallstent in four patients and a Palmaz stent in two patients. Clinical, laboratory, and duplex scanning follow-up was performed every 6 months after stent placement in all patients. RESULTS: The procedure was a technical success in all patients. At 6 months, mean systolic blood pressure decreased from 179 to 152 mm Hg (P = .018) and mean diastolic blood pressure decreased from 102 to 90 mm Hg (P = .09). Mean serum creatinine level dropped from 269 to 182 mmol/L (P = .03) and the number of antihypertensive drugs per patient decreased from 2.5 to 1.6. At a mean follow-up of 34 months (range, 7-60 months), all TRAs were patent, with a stenosis less than 50% without clinical consequences in one patient. No secondary procedure was necessary. CONCLUSION: Stent placement seems to be an effective treatment of TRA recurrent stenosis. Midterm follow-up shows satisfactory clinical results and TRA patency rates. This technique might be considered as a valuable therapeutic option for the treatment of TRA recurrent stenosis.     

Intravital microscopic investigation of xenogeneic microcirculation and impact of complement depletion by cobra venom factor

Simple bone cyst (SBC) is an enigma to the radiologist, pathologist and the orthopaedic surgeon. The etiology of this asymptomatic lesion that frequently causes pathological fracture is still unknown. It is probably self limited in nature, seen in children but rare among adults. The biological behavior is unpredictable as is the clinical course in various anatomical sites. This reflects on the high recurrence rate that has been associated with various treatment modalities. The clinical, radiological and biological features are discussed together with comparative review of treatment options from resection, curettage, and bone grafting to steroid injection and the latest experience of the use of percutaneous autologous marrow grafting in SBC.     

Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom

Three acidic phospholipases A2 (NN-I2c-PLA2, NN-I2d-PLA2 and NN-I2c-PLA2) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE Sephadex A-25 columns. They have molecular weights of 13,000-14,500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (approximately 345 nm). All the three phospholipases A2 were enzymatically highly active with specific activities 9-17 micromol min(-1) mg(-1). They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.     

1H NMR conformational study on N-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure-activity relationships

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1-9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, 1H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1-9), I5-Tat(1-9) and L6-Tat(1-9) and to examine the influence of amino acid exchanges on structural features of these peptides. The defined structures revealed differences in the conformations what might be the reason for different interactions of these Tat(1-9) analogues with certain amino acids of the active site of DP IV.     

Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2-deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh1-deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes. Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.     

Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin

Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.