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.
Animals ; Arthropod Proteins ; chemistry ; Black Widow Spider ; chemistry ; Proteomics ; Venoms ; chemistry

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.
Chromatography, High Pressure Liquid ; Mass Spectrometry ; Membrane Proteins ; analysis ; metabolism ; Membranes, Artificial ; Oxidation-Reduction ; Polyvinyls ; chemistry ; Proteome ; analysis ; Proteomics

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

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.

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.
Amino Acid Substitution ; Animals ; Arginine ; genetics ; Ganglia, Spinal ; drug effects ; Mutagenesis, Site-Directed ; Mutant Proteins ; pharmacology ; Neurons ; drug effects ; Patch-Clamp Techniques ; Peptides ; chemistry ; genetics ; pharmacology ; Rats ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; drug effects ; Spider Venoms ; chemistry ; genetics ; isolation & purification ; pharmacology ; Spiders ; Tetrodotoxin ; pharmacology

The neurotoxin peptide, hainantoxin-Ⅵ (HNTX- Ⅵ), has been isolated from the venom of Chinese tarantula Ornithoconus hainana by a combination of ion exchange chromatography and reverse phase HPLC. The toxin was found to contain 34 amino acid residues with 6 conserved cysteine residues. The effects of HNTX-VI on voltage-gated sodium channels were studied via whole-cell patch clamp techniques. Although several inhibitors of mammalian neuronal sodium channel activation (hainantoxin Ⅰ-Ⅴ) had been characterized from the same venom, the present study indicated that HNTX-Ⅵ had the ability to slow the inactivation kinetics of the sodium channels in Cockroach Periplaneta Americana dorsal unpaired median (DUM) neurons in a similar manner to δ-atractoxins. After HNTX-Ⅵ treatment, steady-state sodium channel inactivation became incomplete, leading to a non-inactivating component at potentials more positive than - 55 mV. The novel function of the tarantula toxin HNTX-Ⅵ not only supplies a useful tool for exploring the gating mechanisms of sodium channels but also provides theoretical foundations for exploiting novel and safe insecticides.

A subcellular proteomic method was applied to investigate the protein expression profiles of nasopharyngeal carcinoma (NPC) cell lines,CNE1 and CNE2,at various differentiation levels.Plasma membrane (PM) proteins were obtained by Percoll density grade centrifugation and subjected to twodimensional electrophoresis (2DE) followed by PDQuest software analysis.Nine proteins expressed with more than two folds difference were identified by MALDI-TOF-TOF,of which functions involved in cell differentiation,signal transduction,and metabolism.Half of these proteins,such as galectin-1 and annexin Ⅱ,were analyzed with real-time quantitative PCR or Westem blotting.We have tested a proteomic method to study differentiated NPCs at different levels and found several proteins that might be related to their biological characteristics.

Nasopharyngeal carcinoma (NPC) poses serious health problems in Southern China and yet the molecular mechanism of the carcinogenesis remains unclear. We used modern proteomic technologies to compare the protein expression profiles between the NPC cell lines (HNE1 and CNE1 ) and an immortalized nasopharyngeal epithelial cell line NP69 to identify cancer related proteins. Cell lysates were separated by two-dimensional gel electrophoresis (2 DE ) and analyzed by PDQuest software. The differentially expressed proteins were identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). We discovered 15 up-regulated proteins and 18 down-regulated proteins in both HNE1 and CNE1 cell lines compared with NP69. These proteins are correlative with various functions, such as cell proliferation, apoptosis, cancer metastasis, metabolism, cytoskeleton and signal transduction. Western blotting analyses were further carried out to verify the differential expression of individual proteins. Several identified proteins in our research might be used as potential molecular markers to understand the molecular mechanism of NPC development and metastasis, and might be used as candidate targets for NPC treatments.

0.05). The mainly side-effects were myelosuppression, nausea, and vomiting. Conclusions:Docetaxel/cisplatin and gemcitabine/cisplatin regimens were well toleranced in advanced NSCLC patients with long term survival.

The antinociceptive effect of epidural administration of huwentoxin-I was elucidated in a tonic visceral pain rat model produced by acute colon inflammation. The nociceptive behaviors were induced by perendoscopically injecting dilute formalin (50 μl) into the depth of the colonic wall in rats. Both ω-conotoxinMVIIA and morphine hydrochloride were given epidurally as positive control while saline as negative control.Similar to ω-conotoxin-MVIIA and hydrochloride morphine, the epidural administration of HWTX-Ⅰ significantly reduced the nociceptive responses in a dose-dependent manner in tonic visceral pain rat model ( P ＜ 0.05). The suppression effects of both huwentoxin- Ⅰ and ω-conotoxin-MVIIA at 20 μg/kg were kept steady compared with the saline group and reached their maximum effects at the doses of 50 ～ 75 μg/kg within 1 hour when the nociception had been observed. It was also found that at the same doses, huwentoxin- Ⅰ was less effective in antinociception than ω-conotoxin-MVIIA. However, ω-conotoxin-MVIIA, but not huwentoxinⅠ , caused an obvious motor dysfunction at these doses. The action of morphine hydrochloride was initiated faster, but lasted for a shorter time than that of huwentoxin- Ⅰ and ω-conotoxin-MVIIA. Thus, huwentoxinⅠ , a potent blocker of neuronal N-type voltage-sensitive calcium channels, induced a remarkable dosedependent restrain effect similar to ω-conotoxin-MVIIA and morphine on the tonic visceral pain produced by colonic wall injection of formalin in conscious rats.