Gene cloning, heterologous expression and activity identification of latroeggtoxin-Ⅵ / 生物工程学报

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.

Recent progress in protein chemistry and proteomics of Latrodectus tredecimguttatus toxins / 生物工程学报

Latrodectus tredecimguttatus (commonly known as black widow spiders) have toxins not only in their venom glands, but also in other parts of their body, in their eggs and even in the newborn spiderlings. The study on the toxins in venom and materials outside the venom glands of the spiders to elucidate their differences and similarities, evolutional relationship and biological functions is of important theoretical and applicable significance. The development of modern protein chemistry and proteomics techniques has provided efficient means for the study of protein and peptide toxins of L. tredecimguttatus. By using such techniques, the molecular base and action mechanism of the toxins can be revealed at the levels of both single purified proteins and omics. Up to now, although protein chemistry and proteomics study on L. tredecimguttatus toxins have achieved a certain progress, the relevant work particularly that on the toxins in the materials outside the venom glands has to be further deepened.

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.

Aqueous Polymer Two-phase Partition for The Proteomic Analysis of Plasma Membrane From Rat Dorsal Root Ganglion Neurons / 生物化学与生物物理进展

Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. To comprehensively identify proteins of plasma membrane (PM) from small amount of dorsal root ganglion (DRG) neurons, a proteomics strategy that utilizes aqueous polymer two-phase partition in combination with differential velocity centrifugation was adopted to enrich the PM, followed by SDS-PAGE, CapLC-MS/MS and bioinformatics analysis. Western blot analysis showed that the concentration of PM in purified plasma membrane(PPM) was 2.3 times higher than that in crude plasma membrane(CPM), 15 times higher than that in whole tissue lysate (WTL). By searching against the rat IPI protein sequence database, a total of 729 non-redundant proteins were identified from the PM preparation, of which 547 had a gene ontology (GO) annotation indicating a cellular component, and 159 (21.8%) were unambiguously identified as PM proteins. A data set of plasma membrane proteins of DRG as well as a tool to study PM proteins were provided in a small amounts of sample.

Effects of Arg20 mutation on sodium channels activity of JZTX-V / 生物工程学报

Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.

The effect of continuous cervical epidural for postoperative analgesia on endocrine and respiratory function in patients after replantation of amputated fingers / 中华麻醉学杂志

0 05) Conclusions Cervical epidural for postoperative analgesia can effectively inhibit the stress response in patient after the replantation of amputated fingers but dose not affect respiratory function Fentanyl provides best analgesia with less adverse effects and is suitable for postoperative analgesia in these patients

Effect of the different intravenous anesthetics on the levels of four neuropeptides in patients with hypertension during induction / 中华麻醉学杂志

95mmHg) were randomly divided into 3 equal groups of 20 each : thiopental group, etomidate group and propofol group. All patients were prmedicated with intramuscular phenobarbital 0. 1g and atropine 0.5mg. Anesthesia was induced with thiopental 4mg?kg-1 or etomidate 0.3mg?kg-1 or propofol 2mg?kg-1, midazolam 0.1mg?kg-1, fentanyl 5?g?kg-1 and vecuronium 0.1mg?kg-1 and maintained with isoflurane inhalation and intermittent boluses of fentanyl and vecuronium. Blood samples were taken before anesthesia(To), 2min after induction(T1) and 2 and 10 min after tracheal intubation (T2, T3) for determination of plasma levels of endothelin(ET), calcitonin gene-related peptide(CGRP), neuropeptide Y (NPY) and neurotensin (NT). Results The three groups were compared with regard to age, sex and weight. BP significantly decreased after induction with thiopental or propofol but increased significantly after intubation in thiopental group and etomidate group. ET and NPY decreased but CGRP and NT increased significantly after induction in thiopental and propofol group. The plasma levels of all four neuropeptides increased significantly after tracheal intubation in thiopental and etomidata group but remained unchanged in propofol group. Conclusions Propofol is the intravenous anesthetic of choice for induction of anesthesia in patients with hypertension.