Comparative study of three short-chain neurotoxins from the venom of Naja kaouthia (Yunnan, China)

Three short-chain neurotoxins named NT-I, NT-II, and NT-III were purified from the venom of Naja kaouthia, a snake distributed throughout the south of Yunnan province, China, by a series of chromatographic steps, including an FPLC Resource S column. Their molecular weights, determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, were 6952.19 Da, 6854.92 Da, and 6828.80 Da, respectively. NT-I consisted of 62 amino acid residues, and the other two consisted of 61 amino acid residues, including 8 cysteines. After hydrolysis by endoproteinase Glu-C, their primary sequences were determined. A test of their activities demonstrated that they effectively inhibited muscle contractions induced by electric stimulation. Furthermore, the extent of inhibition caused by NT-II and NT-III was less than that of NT-I. The IC(50)s were 0.04 mug/ml, 0.20 mug/ml, and 0.23 mug/ml for NT-I, NT-II, and NT-III, respectively. Compared with NT-II and NT-III, the higher activity of NT-I may be a result of the amino acid residue substitution Ile36 to Arg36.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) lb is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCgamma2. Mucetininduced phosphorylation of the Fcgamma chain of platelet was greatly promoted by inhibition of alpha(llb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream Of alpha(llb)beta(3) activation are involved in dephosphorylation of Fcgamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(llb)beta(3). Inhibition Of alpha(llb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation Of alpha(llb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxaneA(2) are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Polymorphisms of the estrogen receptor alpha (ESR1) gene and the risk of Alzheimer's disease in a southern Chinese community

Background: Alzheimer's disease (AD) is a neurodegenerative disease with a higher prevalence in women. Expression of estrogen receptor 1 (ESR1) gene has been identified throughout the brain. Owing to the putative neuroprotective effects of estrogen, estro

Polymerase chain reaction based C4AQ0 and C4BQ0 genotyping: association with systemic lupus erythematosus in southwest Han Chinese

Objective: To investigate the association of complement C4 null genes (C4QO, including C4AQO and C4BQO) and C2 gene with systemic lupus erythematosus (SLE) in southwest Han Chinese; 136 patients with SLE and 174 matched controls were genotyped. Methods: C4 null genes were determined by a polymerase chain reaction (PCR) procedure with sequence specific primers (PCR-SSP). The 2 bp insertion in exon 29, which was previously identified in non-Chinese populations and caused defective C4A genes, was directly typed by sequencing the whole exon 29 using exon specific primers. The exon 6 of complement C2 was also sequenced in both the patients and controls. Results: The frequency of homozygous C4AQO allele was 12.5% (17/136) in patients with SLE compared with 1.1% (2/174) in controls (p<0.001, odds ratio (OR)=12.286, 95% confidence interval (95% CI) 2.786 to 54.170). There was no significant difference for homozygous C4BQO allele between patients with SLE and controls (p=0.699). Patients with the C4AQO gene had an increased risk of acquiring renal disorder, serositis, and anti-dsDNA antibodies compared with those without C4AQO (for renal disorder, p=0.018, OR=8.951, 95% Cl 1.132 to 70.804; for serositis, p=0.011, OR 4.891, 95% CI 1.574 to 15.198; for anti-dsDNA, p=0.004, OR 7.630, 95%Cl 1.636 to 35.584). None of the patients or controls had the 2 bp insertion in exon 29 of the C4 gene. The type I C2 deficiency was not detected in the 3 10 samples. Conclusion: It is suggested that deficiency of C4A (not due to a 2 bp insertion in exon 29), but not C4B or C2, may be a risk factor for acquiring SLE in south west Han Chinese; this results in increased risk of renal disorder, serositis, and anti-dsDNA antibodies in patients with SLE. Racial differences seem to be relevant in susceptibility to SLE.

Natural selection on EPAS1 (HIF2 alpha) associated with low hemoglobin concentration in Tibetan highlanders

By impairing both function and survival, the severe reduction in oxygen availability associated with high-altitude environments is likely to act as an agent of natural selection. We used genomic and candidate gene approaches to search for evidence of such genetic selection. First, a genome-wide allelic differentiation scan (GWADS) comparing indigenous highlanders of the Tibetan Plateau (3,200 3,500 m) with closely related lowland Han revealed a genome-wide significant divergence across eight SNPs located near EPAS1. This gene encodes the transcription factor HIF2 alpha, which stimulates production of red blood cells and thus increases the concentration of hemoglobin in blood. Second, in a separate cohort of Tibetans residing at 4,200 m, we identified 31 EPAS1 SNPs in high linkage disequilibrium that correlated significantly with hemoglobin concentration. The sex-adjusted hemoglobin concentration was, on average, 0.8 g/dL lower in the major allele homozygotes compared with the heterozygotes. These findings were replicated in a third cohort of Tibetans residing at 4,300 m. The alleles associating with lower hemoglobin concentrations were correlated with the signal from the GWADS study and were observed at greatly elevated frequencies in the Tibetan cohorts compared with the Han. High hemoglobin concentrations are a cardinal feature of chronic mountain sickness offering one plausible mechanism for selection. Alternatively, as EPAS1 is pleiotropic in its effects, selection may have operated on some other aspect of the phenotype. Whichever of these explanations is correct, the evidence for genetic selection at the EPAS1 locus from the GWADS study is supported by the replicated studies associating function with the allelic variants.

Alpha-momorcharin inhibits HIV-1 replication in acutely but not chronically infected T-lymphocytes

AIM: To identify the anti-human immunodeficiency virus type 1 (HIV-1) activities of alpha-momorcharin ( alpha-MMC) from Momordica charantia in acutely and chronically infected lymphocytes. METHODS: The anti-HIV activities of alpha-MMC were examined by 1) the inhibition of syncytia formation induced by HIV-1 III B; 2) reduction of p24 core antigen expression level and decrease in numbers of HIV antigen positive cells in acutely and chronically infected cultures. The cytotoxic effects of alpha-MMC was tested by trypan blue dye exclusion or colorimetric MTT assay. RESULTS: alpha-MMC was found to obviously inhibit HIV-1 III B-inducing C8166 syncytia formation and markedly reduced both expression of p24 core antigen and the numbers of HIV antigen positive cells in acutely but not chronically HTV-1-infected culture. The median effective concentration (EC50) in these assays were 0.016, 0.07, and 0.32 mg.L-1, respectively. CONCLUSION: alpha-MMC is a unique component of momorcharin with anti-HIV activity, and markedly inhibited HIV-1 replication in acutely but not chronically HIV-1-infected T-lymphocytes.

奶牛胚胎移植中GnRH、PGF_(2)#alpha#同期发情处理效果的比较

193头健康、经产的黑白花奶牛分为3个组,用3种不同的方法进行同期发情处理和胚胎移植。第1组:注射1次Lutalyse;第2组:注射2次Lutalyse;第3组:注射Fertagyl-Lutalyse-Fertagyl。结果表明:第1组和第2组之间的同期发情胚胎移植率无显著差异(P>0.05),分别为26.5%(21/79)和27.0%(18/68);第3组的同期发情胚胎移植率为60.9%(28/46),显著高于第1组和第2组(P<0.05)。

Opiate withdrawal induces dynamic-expressions of AMPA receptors and its regulatory molecule CaMKII alpha in hippocampal synapses

Adaptive changes in brain areas following drug withdrawal are believed to contribute to drug seeking and relapse. Cocaine withdrawal alters the expression of GluR1 and GluR2/3 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) rec

灵长类大脑前额叶NE Alpha-2Ⅰ亚型受体参与记忆过程的证据

灵长类大脑前额皮质与感觉联合皮质和皮质下结构有广泛的纤维联系, 可广 泛接受来自体内外的感觉信息。前额皮质与注意和短时记忆过程都有关, 可能 主沟区与短时空间记忆的关系更为密切, 北区的去甲肾上腺素能系统参与短时记 忆过程, alpha-2Ⅰ去甲肾上腺素亚型受体与增强记忆有关。参23

POSTSYNAPTIC ALPHA-2 RECEPTOR STIMULATION IMPROVES MEMORY IN AGED MONKEYS - INDIRECT EFFECTS OF YOHIMBINE VERSUS DIRECT EFFECTS OF CLONIDINE

Very low doses (0.00001 mg/kg) of the alpha-2 adrenergic antagonist, yohimbine, improved working memory performance in a subset of aged monkeys. Improvement appeared to result from increased norepinephrine (NE) release onto postsynaptic alpha-2 adrenoceptors, as the response was blocked by the ''postsynaptic'' alpha-2 antagonist, SKF104078. Cognitive-enhancing effects of low dose yohimbine treatment may depend on aged animals retaining an intact, endogenous NE system. In contrast to yohimbine, the alpha-2 agonist, clonidine, has improved working memory in air aged animals examined. In the present study, clonidine's beneficial effects were also blocked by the postsynaptic antagonists SKF104078 and SKF104856, suggesting that clonidine acts by directly stimulating postsynaptic alpha-2 adrenoceptors. Beneficial doses of clonidine (0.01 mg/kg) and yohimbine (0.00001 mg/kg) were combined to see if they would produce additive effects on memory enhancement. This strategy was successful in young monkeys with intact NE systems but was not effective in the aged monkeys. These findings demonstrate that drugs that indirectly stimulate postsynaptic alpha-2 receptors by increasing NE release are not as reliable in aged monkeys as directly acting agonists that can replace NE at postsynaptic alpha-2 receptors.

RESERPINE IMPAIRS SPATIAL WORKING-MEMORY PERFORMANCE IN MONKEYS - REVERSAL BY THE ALPHA-2-ADRENERGIC AGONIST CLONIDINE

Repeated daily treatment with the catecholamine-depleting agent, reserpine, dramatically reduced performance on the delayed response task, a test of spatial working memory that depends upon the integrity of the prefrontal cortex. Delayed response performance fell from an average of 27.2/30 trials correct before reserpine treatment to an average of 20.4/30 trials correct after repeated reserpine administration. Injection of the alpha2-adrenergic agonist, clonidine (0.0001-0.05 mg/kg), to chronic reserpine-treated monkeys significantly restored performance on the delayed response task; performance after an optimal dose averaged 27.8/30 trials correct. Clonidine's beneficial effects on delayed response performance were longlasting; monkeys remained improved for more than 24 h after a single clonidine injection. The finding that clonidine is efficacious in reserpinized animals supports the hypothesis that alpha2-adrenergic agonists improve cognitive function through actions at postsynaptic, alpha2-adrenergic receptors on non-adrenergic cells. In contrast to the delayed response task, reserpine had little effect on performance of a visual discrimination task, a reference memory task which does not depend on the prefrontal cortex. These results emphasize the importance of postsynaptic alpha2-adrenergic mechanisms in the regulation of working memory,

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection. (C) 2009 Elsevier B.V. All rights reserved.

Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2. Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus). Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. (C) 2008 Elsevier B.V. All rights reserved.