Haemolytic effect of the scorpion Heterometrus fulvipes venom

The haemolytic effect of the venom from scorpion Heterometrus fulvipes was studied using sheep erythrocytes. While there was no haemolytic effect seen directly by the erythrocytes, erythrocytes sensitized with homologous haemolysin were lysed by the venom. This factor in Heterometrus fulvipes venom having a 'complement like' haemolytic effect was found to be thermolabile and dialysable and sensitive to the action of 2-mercaptoethanol. It was identified as a low molecular weight peptide containing disulphide groups.     

Pharmacological and immunological evaluation of scorpion (Heterometrus bengalensis) venom antiserum

An antiserum was prepared for the first time against the venom of a common scorpion, H. bengalensis, by hyperimmunization of rabbit. This antiserum showed positive precipitin bands in immunogeldiffusion and immunoelectrophoresis. The serum showed a high titre value tested by indirect haemagglutination test. The antiserum developed in rabbit protected mice against the lethal action of the venom. Smooth muscle contractile response of venom on guinea pig ileum, and rat uterus was antagonized by the antiserum. This antiserum effectively antagonized the venom induced neuromuscular paralysis tested on rat phrenic nerve diaphragm and chick biventer cervices. Antiserum also protected the venom-induced cardiac arrest tested on isolated guineapig heart and auricle preparations.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Proteomic analysis of Tityus discrepans scorpion venom and amino acid sequence of novel toxins

The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.     

Comparative detoxification and protection of scorpion (Heterometrus bengalensis) venom by toxoid antiserum

Comparative detoxification of scorpion venom by using different chemical agents was investigated. Detoxification by formalin showed the best optimum detoxifying agent. The formalin treatment resulted in 2.3% protein loss with 6-fold detoxification. This formal toxoid was immunogenic in rabbit giving high neutralizing antibodies as revealed from indirect haemagglutination test. Toxoid antiserum protected mice against the lethal action of venom. It also effectively antagonized the smooth muscle contractile response of venom, and venom-induced neuromuscular paralysis. This toxoid antiserum also protected the venom-induced cardiac arrest. The possibility of using this formal toxoid for antisera production and immunization for therapeutic use needs to be explored.     

Proteomic analysis from the mineralized radular teeth of the giant Pacific chiton, Cryptochiton stelleri (Mollusca)

The biomineralized radular teeth of chitons are known to consist of iron-based magnetic crystals, associated with the maximum hardness and stiffness of any biomineral. Based on our transmission electron microscopy analysis of partially mineralized teeth, we suggest that the organic matrix within the teeth controls the iron oxide nucleation. Thus, we used Nano-LC-MS to perform a proteomic analysis of the organic matrix in radular teeth of the chiton Cryptochiton stelleri in order to identify the proteins involved in the biomineralization process. Since the genome sequence of C. stelleri is not available, cross-species similarity searching and de novo peptide sequencing were used to screen the proteins. Our results indicate that several proteins were dominant in the mineralized part of the radular teeth, amongst which, myoglobin and a highly acidic peptide were identified as possibly involved in the biomineralization process.     

Purification and properties of phospholipase A2 from the venom of scorpion, (Heterometrus fulvipes)

Phospholipase A2 was purified about 78 fold from the venom of scorpion Heterometrus fulvipes. The molecular weight of the enzyme was found to be 16,000 and it was optimally active at pH 7.4 and at 50 degrees C. The Km value of the enzyme was 1.8 x 10(-3) M. Calcium, Magnesium and Zinc ions stimulated whereas Mercury ion and EDTA inhibited the enzyme activity. This enzyme exhibited fluorescence emission maximum between 310-320 nm.     

Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome*

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Highlights

• •Reported proteasomal spliced HLA peptides do not fit the consensus binding motifs.
• •Their MS/MS spectrum matches suggest that many of them are ambiguous.
• •Our workflow is based on de novo sequencing, alignment, and multiple search tools.
• •The upper bound proportion of cis-spliced peptides is 2–6% and likely much smaller.

     

Detection and distribution of scorpion (Heterometrus bengalensis) venom in rabbit tissues by enzyme-linked immunosorbent assay (ELISA).

ELISA was carried out to detect distribution of scorpion venom in experimental animal tissues. The venom content of different tissues was in the order, liver greater than kidney greater than spleen greater than lung greater than heart greater than diaphragm greater than brain. Tissue distribution of venom antigen in the envenomental subject by ELISA will provide a better approach for serotherapy.     

De novo assembly of the Pseudomonas syringae pv. syringae B728a genome using Illumina/Solexa short sequence reads

Illumina's Genome Analyzer generates ultra-short sequence reads, typically 36 nucleotides in length, and is primarily intended for resequencing. We tested the potential of this technology for de novo sequence assembly on the 6 Mbp genome of Pseudomonas syringae pv. syringae B728a with several freely available assembly software packages. Using an unpaired data set, velvet assembled >96% of the genome into contigs with an N50 length of 8289 nucleotides and an error rate of 0.33%. edena generated smaller contigs (N50 was 4192 nucleotides) and comparable error rates. ssake and vcake yielded shorter contigs with very high error rates. Assembly of paired-end sequence data carrying 400 bp inserts produced longer contigs (N50 up to 15 628 nucleotides), but with increased error rates (0.5%). Contig length and error rate were very sensitive to the choice of parameter values. Noncoding RNA genes were poorly resolved in de novo assemblies, while >90% of the protein-coding genes were assembled with 100% accuracy over their full length. This study demonstrates that, in practice, de novo assembly of 36-nucleotide reads can generate reasonably accurate assemblies from about 40 × deep sequence data sets. These draft assemblies are useful for exploring an organism's proteomic potential, at a very economic low cost.     

Isolation, purification and immunological evaluation of toxin Hb from scorpion Heterometrus bengalensis (C.L. Koch) venom

Toxin-Hb, a lethal toxic antigenic protein, isolated from the venom of H. bengalensis by CM-cellulose ion-exchange chromatography was a heat labile basic protein with a molecular weight of 10 kDa. It produced irreversible blockade on the isolated rat phrenic nerve diaphragm and chick biventer cervicis. LD50 of toxin Hb was 0.48 mg/kg (iv) in mice. Antiserum was raised in mice by hyperimmunization against toxin Hb. Antitoxin Hb antiserum was immunologically potent as revealed by immunogel-diffusion and immunoelectrophoresis. Five fold protection against the lethal action of toxin Hb was achieved by the antiserum. It also effectively antagonised toxin Hb induced neuromuscular blockade on isolated rat phrenic nerve diaphragm and chick biventer cervicis preparations.     

Neurotoxins from the venom of the Central Asian scorpion Buthus eupeus

Three main polypeptide neurotoxins M9, M10, M14, possessing paralytic activity in mice, have been isolated from the venom of the Central Asian scorpion Buthus eupeus. The amino acid composition of these toxins was determined. Toxins M9 and M14 were digested with trypsin, chymotrypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The complete amino acid sequences of the toxins M9 and M14 were established.     

Cloning, sequence analysis and homology modeling of a novel phospholipase A2 from Heterometrus fulvipes (Indian black scorpion).

We report the cloning and sequencing of group III phospholipaseA(2) from Heterometrus fulvipes (HfPLA(2)), Indian black scorpion. The cDNA sequence codes for the mature portion of the group PLA(2) of 103 amino acids. The sequence has 85% identity with Mesobuthus tamulus (Indian red scorpion) PLA(2) and a 40% identity with bee venom PLA(2) and human group III PLA(2). Most of the essential features of group III PLA(2) like Ca(2+) binding loop and catalytic residues are conserved. Homology modeling was done with the known structure of group III bee venom PLA(2). All the secondary structural motifs and the disulfide bridges are as predicted. The variation like the replacement of aspartic acid residue with glutamic acid in the well known histidine-aspartic acid dyad is a rare feature. This is the first structural model report of an Indian black scorpion PLA(2).     

A comprehensive analysis of LACK (Leishmania homologue of receptors for activated C kinase) in the context of Visceral Leishmaniasis

The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.     

OVNIp: An open source application facilitating the interpretation,the validation and the edition of proteomics data generated by MS analyses and de novo sequencing

Several academic software are available to help the validation and reporting of proteomics data generated by MS analyses. However, to our knowledge, none of them have been conceived to meet the particular needs generated by the study of organisms whose genomes are not sequenced. In that context, we have developed OVNIp, an open‐source application which facilitates the whole process of proteomics results interpretation. One of its unique attributes is its capacity to compile multiple results (from several search engines and/or several databank searches) with a resolution of conflicting interpretations. Moreover, OVNIp enables automated exploitation of de novo sequences generated from unassigned MS/MS spectra leading to higher sequence coverage and enhancing confidence in the identified proteins. The exploitation of these additional spectra might also identify novel proteins through a MS‐BLAST search, which can be easily ran from the OVNIp interface. Beyond this primary scope, OVNIp can also benefit to users who look for a simple standalone application to both visualize and confirm MS/MS result interpretations through a simple graphical interface and generate reports according to user‐defined forms which may integrate the prerequisites for publication. Sources, documentation and a stable release for Windows are available at http://wwwappli.nantes.inra.fr:8180/OVNIp .