Insights into the binding mode and functional components of the analgesic-antitumour peptide from Buthus martensii Karsch to human voltage-gated sodium channel 1.7 based on dynamic simulation analysis.

Voltage-gated sodium (Nav) channels are transmembrane proteins composed of four homologous domains (DI-DIV) that play important roles in membrane excitability in neurons and muscles. Analgesic-antitumour peptide (AGAP) is a neurotoxin from the scorpion Buthus martensii Karsch, and has been shown to exert analgesic effect by binding on site 4 of human Nav1.7 (hNav1.7). Mechanistic details about this binding, however, remain unclear. To address this issue, we compared the binding modes of AGAP/AGAPW38G/AGAPW38F and the hNav1.7 voltage-sensing domain on DII (VSD2hNav1.7) using homology modeling, molecular docking, molecular dynamics simulation and steered molecular dynamics. Results revealed the key role of tryptophan at position 38 on the binding of AGAP to VSD2hNav1.7. Pivotal roles are played also by residues on the ß-turn and negatively charged residues at the C-terminal. We further show that electrostatic interaction is the main contributor to the binding free energy of the complex. Agreement between our computational simulation findings and prior experimental data supports the accuracy of the described mechanism. Accordingly, these results can provide valuable information for designing potent toxin analgesics targeting hNav1.7 with high affinity.Communicated by Ramaswamy H. Sarma.

Association of IL-28B, TBX21 gene polymorphisms and predictors of virological response for chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. The outcomes of both spontaneous HCV clearance and response to therapy depend on both viral and host factors. To investigate the influence of polymorphisms of IL-28B rs12979860 and TBX21 rs17250932, rs4794067 as well as viral factors (HCV genotype, F protein) on the outcome of HCV infection, we genotyped 565 patients with chronic HCV infection, 191 patients spontaneously resolved from HCV infection, 359 healthy controls and 383 treatment-naïve CHC patients with pegylated interferon-α and ribavirin (PEG IFN-α/RBV). Results showed that TBX21 rs4794067 variant genotypes significantly correlated with increased risk of HCV chronic infection (dominant model: OR = 5.690, 95% CI = 2.024-16.000) and susceptibility (dominant model: OR = 5.658, 95% CI = 2.514-12.735). We also found that the rs12979860, rs2227982 and rs36084323 polymorphisms showed no significant associations with susceptibility or spontaneous clearance of HCV in the anti-F antibody subgroup; however, the anti-F antibody positive subgroup might show an increased risk of N-SVR (all P < 0.001). Our results demonstrate that variant factors in both the host and pathogen are commonly important for HCV clearance. In addition rs4794067 and F protein status may be strong predictive markers in the Chinese population.

Antinociceptive Effects of AGAP, a Recombinant Neurotoxic Polypeptide: Possible Involvement of the Tetrodotoxin-Resistant Sodium Channels in Small Dorsal Root Ganglia Neurons.

Antitumor-analgesic peptide (AGAP) is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG) neurons. The results showed that AGAP (3-1000 nM) inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R) sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide.

PD-1/PD-L1 signal pathway participates in HCV F protein-induced T cell dysfunction in chronic HCV infection.

Programmed cell death-1/programmed cell death-1 ligand 1 (PD-1/PD-L1) inhibitory signal pathway has been verified to be involved in the establishment of persistent viral infections. Blockade of PD-1/PD-L1 engagement to reinvigorate T cell activity is supposed to be a potential therapeutic scheme. Studies have verified the participation of PD-1/PD-L1 in hepatitis C virus (HCV) core protein-regulated immune response. To determine the roles of PD-1/PD-L1 signal pathway in HCV F protein-induced immunoreaction in chronic HCV infection, variations in T cells were examined. The results showed that PD-1 expression on CD8(+) and CD4(+) T cells was increased with HCV F stimulation in both chronic HCV patients and healthy controls, and could be reduced partly by PD-1/PD-L1 blocking. Additionally, by PD-1/PD-L1 blocking, HCV F-induced inhibition of T cell proliferation and promotion of cellular apoptosis were partly or even totally recovered. Furthermore, levels of both Th1 and Th2 cytokines were elevated in the presence of anti-PD-L1 antibody. All these results indicated that PD-1/PD-L1 signal pathway also participates in HCV F protein-induced immunoregulation. PD-1/PD-L1 blocking plays important roles in the restoration of effective functionality of the impaired T cells in chronic HCV patients.

HCV F protein amplifies the predictions of IL-28B and CTLA-4 polymorphisms about the susceptibility and outcomes of HCV infection in Southeast China.

Cytotoxic T lymphocyte associated antigen-4(CTLA-4) is an inhibitory receptor with great value in the progression of hepatitis C virus (HCV) infection related diseases. To determine the potential associations of IL-28B rs12979860 and CTLA-4 rs231775, rs3087243 and rs5742909 polymorphisms with the generation of HCV F protein, susceptibility and outcomes of HCV infection, a total of 375 healthy controls, 219 HCV spontaneous recovered patients and 600 chronic HCV patients from Southeast China were recruited and genotyped in this study. And the relative mRNA levels of CTLA-4 in T cells were detected. Logistic regression analysis showed that rs231775 A allele was associated with significantly higher rate of spontaneous viral clearance in anti-HCV F antibody negative patients (adjusted OR=0.512, P=0.008), but allele A was related to higher mRNA level of CTLA-4 with the generation of HCV F protein. And rs5742909 T allele added up to the risk of HCV infection chronicity significantly in patients with the presence of HCV F protein (adjusted OR=2.698, P=0.003). Also, the rs5742909 CC genotype, along with the presence of HCV F protein, indicated a significantly higher CTLA-4 level than that in anti-HCV F antibody negative patients. The AG+AA genotype of rs3087243 significantly increased the susceptibility to HCV infection in subjects over 56 years old (adjusted OR=1.595, P=0.011). Genotype-genotype interaction between IL-28B rs12979860 and CTLA-4 rs3087243 was found to be significantly associated with increased susceptibility to HCV infection (adjusted OR=1.509, P=0.005). Haplotype analysis in CTLA-4 also showed significant association with the generation of HCV F protein. All these results indicated the importance of IL-28B and CTLA-4 polymorphisms and their associations with HCV F protein in the risk and chronicity of HCV infection in Chinese Han population in Southeast China.

Site-directed mutagenesis of BmK AGP-SYPU1: the role of two conserved Tyr (Tyr5 and Tyr42) in analgesic activity.

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.

Purification, characterization, and bioactivity of a new analgesic-antitumor peptide from Chinese scorpion Buthus martensii Karsch.

Scorpion venoms are complex mixtures of dozens or even hundreds of distinct proteins, many of which have diverse bioactivities. In this study, after bioassay-driven chromatographic purification, a new dual-function peptide with analgesic and antitumor activities was isolated and designated BmK AGAP-SYPU2. The first 12 amino acid residues were sequenced with Edman degradation. The cDNA was cloned by using rapid amplification of cDNA ends from the cDNA pool from scorpion glands. The amino acid sequence of BmK AGAP-SYPU2 was then deduced, and is consistent with the molecular mass measured with MALDI-TOF-MS. A preliminary pharmacological analysis revealed the following: in the dose-effect curve plotted with the mouse-twisting test, BmK AGAP-SYPU2 showed analgesic activity with an ED50 value of 1.42 mg/kg; in the time-effect curves plotted with a hot-plate procedure, BmK AGAP-SYPU2 had similar effects to those of the painkiller morphine, except for its longer duration. BmK AGAP-SYPU2 also showed antitumor activity against Ehrlich ascites tumor and S-180 fibrosarcoma models in vivo. Sequence alignment and homology modeling showed that BmK AGAP-SYPU2 is highly conserved relative to other scorpion α-toxins. However, a few different amino acids endow it with unique molecular properties, which may be responsible for its specific bioactivities. BmK AGAP-SYPU2, a new scorpion neurotoxin with dual functions, is a potential candidate drug amenable to exploitation and modification.

The role of glycine residues at the C-terminal peptide segment in antinociceptive activity: a molecular dynamics simulation.

Elucidating structural determinants in the functional regions of toxins can provide useful knowledge for designing novel analgesic peptides. Glycine residues at the C-terminal region of the neurotoxin BmK AGP-SYPU2 from the scorpion Buthus martensii Karsch (BmK) have been shown to be crucial to its analgesic activity. However, there has been no research on the structure-function relationship between the C-terminal segment of this toxin and its analgesic activity. To address this issue, we performed three MD simulations: one on the native structure and the other two on mutants of that structure. Results of these calculations suggest that the existence of glycine residues at the C-terminal segment stabilizes the protruding topology of the NC domain, which is considered an important determinant of the analgesic activity of BmK AGP-SYPU2.

Purification, characterization and functional expression of a new peptide with an analgesic effect from Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1).

In this study, a new peptide named BmK AGP-SYPU1 with an analgesic effect was purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) through a four-step chromatographic process. The mouse twisting test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU1 was further qualified by RP-HPLC and HPCE. The molecular mass determined by the MALDI-4800-TOF/TOF MS for BmK AGP-SYPU1 was 7544 Da. Its primary structure of the N-terminal was obtained using Edman degradation. The gene sequence of BmK AGP-SYPU1 was cloned from the cDNA pool and genomic of scorpion glands, respectively, and then expressed in Escherichia coli. The sequence determination showed that BmK AGP-SYPU1 was composed of 66 amino acid residues with a new primary structure. The metal chelating affinity column and cation exchange chromatography were used to purify the recombinant BmK AGP-SYPU1. Consequently, the native and recombinant BmK AGP-SYPU1 showed similar analgesic effects on mice as assayed using a mouse twisting model. These results suggested that BmK AGP-SYPU1 is a new analgesic component found in the Chinese scorpion Buthus martensi Karsch.

Site-directed mutagenesis of the toxin from the Chinese scorpion Buthus martensii Karsch (BmKAS): insight into sites related to analgesic activity.

This study utilized the E. coli expression system to investigate the role of amino acid residues in toxin from the Chinese scorpion--Buthus martensii Karsch (BmKAS). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, ten mutants of BmKAS were obtained by PCR. Using site-directed mutagenesis, all of these residues were substituted with different amino acids. This study represents a thorough mapping and elucidation of the epitopes that form the molecular basis of the toxin's analgesic activity. Our results showed large mutant-dependent differences that emphasize the important roles of the studied residues.

Effect of ANEPIII, a novel recombinant neurotoxic polypeptide, on sodium channels in primary cultured rat hippocampal and cortical neurons.

Previous studies have shown that the recombinant neurotoxic polypeptide BmK ANEP (ANEPIII) displayed good anti-neuroexcitation activity as demonstrated by pharmacological tests of the blockade of chemical-induced convulsive seizures. In order to search for further anticonvulsant mechanism of action of ANEPIII, the effects of ANEPIII on sodium channels were assessed using the whole-cell patch clamp recordings in primary cultures of rat hippocampal and cortical neurons. ANEPIII decreased the sodium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in hippocampal and cortical neurons revealed an IC(50) value of 124.6 nM and 192.7 nM, respectively. Furthermore, ANEPIII 1000 nM significantly shifted the activation curves of sodium current in hippocampal and cortical neurons to more positive potentials and the recovery from inactivation of sodium current was significantly slower. Voltage-dependent inactivation curves of sodium channels in hippocampal and cortical neurons did not change in the presence of 1000 nM ANEPIII. Thus, our results demonstrated that ANEPIII in submicromolar concentrations was a voltage-dependent, reversible blocker of sodium current in hippocampal and cortical neurons. It is concluded that these phenomena may explain, at least in part, the anti-neuroexciting properties of this peptide.

Induction of G(2)/M phase arrest and apoptosis by oridonin in human laryngeal carcinoma cells.

Oridonin (1), an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects. In this study, the mechanism involved in 1-induced growth inhibition, including apoptosis and G(2)/M phase arrest, in human laryngeal carcinoma HEp-2 cells deficient in functional p53, was investigated for the first time. Compound 1 triggered the mitochondrial apoptotic pathway, as indicated by increased Bax/Bcl-2 ratios, reduction of mitochondrial membrane potential (DeltaPsi(m)), and substantial increase in apoptosis-inducing factor (AIF) and cytochrome c. Inhibition of caspase-9 in HEp-2 cells did not protect the cells from 1-induced apoptosis, and cleaved caspase-9 was not detected, indicating that apoptosis occurred via a caspase-9-independent pathway. The results also suggested that G(2)/M phase arrest and apoptosis mediated by 1 occurred via a p53-independent but in a p21/WAF1-dependent manner in HEp-2 cells. In addition, the generation of reactive oxygen species (ROS) was found to be a critical mediator in growth inhibition induced by 1. Taken together, the results indicate that oridonin (1) is a potentially effective agent for the treatment of laryngeal squamous cell carcinoma.

Inhibition of EGFR signaling augments oridonin-induced apoptosis in human laryngeal cancer cells via enhancing oxidative stress coincident with activation of both the intrinsic and extrinsic apoptotic pathways.

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to have anti-tumor effects, while the epidermal growth factor receptor (EGFR) signal pathway has been reported to play a vital role in the biological progression of several tumors and to be a target for therapeutic intervention. In this work, we show that inhibition of EGFR with tyrphostin AG1478 enhances oridonin-induced cell death in human laryngeal cancer cells HEp-2, a cell line characterized by EGFR gene amplification. The enhanced apoptotic effect correlates with high expression and activation of Bax, FADD, caspase-8 as well as caspase-3 and decreased protein levels of Bcl(2) and SIRT1, suggesting that both the extrinsic and intrinsic apoptosis pathways are involved in the apoptotic processes. However, treatment with oridonin and AG1478 greatly enhances nuclear translocation of apoptosis inducing factor (AIF) without caspase-9 activation, indicating that the apoptosis occurs via a caspase-9-independent mitochondrial pathway. Here, it is the active form of caspase-8 but not caspase-9 that activates downstream effector caspase-3, resulting in the cleavage of critical cellular proteins and apoptosis. Furthermore, the combined use of AG1478 and oridonin augments the production of reactive oxygen species (ROS). Incubation of cells with N-Acetylcysteine (NAC) attenuates the apoptosis and the mitochondrial membrane potential (Deltapsim) disruption induced by the combination of oridonin and AG1478, which indicates that ROS plays a pivotal role in cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should be a potentially very effective anti-neoplastic therapy for laryngeal cancer.

Structure and function relationship of toxin from Chinese scorpion Buthus martensii Karsch (BmKAGAP): gaining insight into related sites of analgesic activity.

In this study, an effective Escherichia coli expression system was used to study the role of residues in the antitumor-analgesic peptide from Chinese scorpion Buthus martensii Karsch (BmKAGAP). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, nine mutants of BmKAGAP were obtained by PCR. Using site-directed mutagenesis, all of these residues were individually substituted by one amino acid. These were then subjected to a circular dichroism analysis, and an analgesic activity assay in mice. This study represents a thorough mapping and elucidation of the epitopes that underlie the molecular basis of the analgesic activity. The three-dimensional structure of BmKAGAP was established by homology modeling. Our results revealed large mutant-dependent differences that indicated important roles for the studied residues. With our ongoing efforts for establishing the structure and analgesic activity relationship of BmKAGAP, we have succeeded in pinpointing which residues are important for the analgesic activity.

Location of the analgesic domain in Scorpion toxin BmK AGAP by mutagenesis of disulfide bridges.

An increasing number of analgesic peptides have been found in the tail toxicyst, but there has been little research into their analgesic domains. Where are the analgesic domains in a conservative betaalphabetabeta topology conformation of the analgesic peptides? We have carried out research to address this question. On account of the importance of disulfide bonds in the study of protein structure, the conformational stability, catalytic activity and folding, and site-directed mutagenesis in disulfide bridges have been used to look for the analgesic domain in a mature antitumor-analgesic peptide from the venom of the Chinese scorpion Buthus martensii Karsch (BmK AGAP). The mouse-twisting assay was used to examine the analgesic activity of 12 mutants, in which two mutants (C22S, C46S) and (C16S, C36S), exhibited lower relative activity. Following the conformational analysis, one domain, called the "core domain", was found to be the key to the analgesic activity.

Dinucleotides docking to scorpion polypeptide toxins: a molecular modeling method for protein functional site recognition.

To understand the principles underlying protein folding, many molecular modeling methods are being developed for predicting functional positions. In this work, fully flexible dinucleotides d(pApA), d(pApC), d(pApG), d(pApT), d(pCpA), d(pCpC), d(pCpG), d(pCpT), d(pGpA), d(pGpC), d(pGpG), d(pGpT), d(pTpA), d(pTpC), d(pTpG), and d(pTpT) were first docked onto the surface of scorpion polypeptide toxins (LqhIT2, PDB ID:2I61) and homology modeled ANEPIII. Automated docking was able to identify sites on scorpion polypeptide toxins where favorable nucleotide interactions can occur, and those sites were in agreement with the mutation data of this protein published recently [I. Karbat, R. Kahn, L. Cohen, N. Ilan, N. Gilles, G. Corzo, O. Froy, M. Gur, G. Albrecht, S.H. Heinemann, D. Gordon, M. Gurevitz, The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail, Febs J 274 (2007) 1918-1931]. Simulation results suggested that dinucleotides docking is a suitable molecular modeling method that could be developed for protein functional site recognition.

Cloning, expression, and pharmacological activity of BmK AS, an active peptide from scorpion Buthus martensii Karsch.

BmK AS is a beta long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.

[Analysis of chromosome aberrations in the cell derived from primary cell culture of laryngeal carcinoma and the Hep-2 cell line].

OBJECTIVE: To search for characteristic chromosome changes in primary laryngeal squamous cell carcinoma (LSCC) and Hep-2 cell line and to realize the relationship between the cytogenetic abnormality and the pathogenetic mechanism in LSCC. METHODS: The fresh resulted samples of LSCC were analyzed with an improved primary cell culture for chromosome preparation and G-banding technique. Hep-2 cell line was analyzed by high resolution banding technique. Molecular cytogenetics analysis was made by chromosome 6 painting probe. RESULTS: Four primary LSCC succeeded in primary cell culture and obtained metaphases, one was tetraploid, the other three were triploid. The chromosome mode of Hep-2 cell line was from 68 to 75 and fifteen marker chromosomes were found. The most structural abnormalities of chromosome in primary LSCC and HEP-2 cell line were unbalance translocation, terminal deletion and isochromosome. The complicate aberration in chromosome 6 was common in LSCC and Hep-2. CONCLUSION: 6q-, I(5p), 17p-, 5q- are considered as characteristic chomosome changs in LSCC. Fluorescence in situ hybridization (FISH) may enhance the ability of detecting complicated chromosome rearrangements and marker chromosomes, which could provide more value data to verify the chromosome characteristic aberration in LSCC.

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons.

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.

Identification of the thymidylate synthase within the genome of white spot syndrome virus.

Thymidylate synthase (TS) (EC 2.1.1.45) is essential for the de novo synthesis of dTMP in prokaryotic and eukaryotic organisms. Within the white spot syndrome virus (WSSV) genome, an open reading frame (WSV067) that encodes a 289 amino acid polypeptide showed significant homology to all known TSs from species including mammals, plants, fungi, protozoa, bacteria and DNA viruses. In this study, WSV067 was expressed in Escherichia coli, and the purified recombinant protein showed TS activity in dUMP-folate-binding assays using ultraviolet difference spectroscopy. RT-PCR and Western blot analyses showed that WSV067 was a genuine and early gene. Phylogenetic analysis revealed that WSSV-TS was more closely related to the TSs of eukaryotes than to those from prokaryotes.