Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine (x-chymotrypsin with a K-i of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (PI) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities = 89.5%) and other snake venom protease inhibitors. (C) 2003 Elsevier Inc. All rights reserved.

Molecular Dynamics Simulation of Methane Hydrate Dissociation Process in the Presence of Thermodynamic Inhibitor

The dissociation of methane hydrate in the presence of ethylene glycol (11.45 mol.L-1) at 277.0 K was studied using canonical ensemble (NVT) molecular dynamics simulations. Results show that hydrate dissociation starts from the surface layer of the solid hydrate and then gradually expands to the internal layer. Thus, the solid structure gradually shrinks until it disappears. A distortion of the hydrate lattice structure occurs first and then the hydrate evolves from a fractured frame to a fractional fragment. Finally, water molecules in the hydrate construction exist in the liquid state. The inner dissociating layer is, additionally, coated by a liquid film formed from outer dissociated water molecules outside. This film inhibits the mass transfer performance of the inner molecules during the hydrate dissociation process.

Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92±0.62 and 6.59±1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44±10.20 nM, as well as suppressing HIV-1- induced cytopathic effect with an EC50 value of 3.04±1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/ AIDS patients, particularly for those infected by T20-resistant variants.

金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究

蛇毒和蜂毒是提供药理学活性分子的丰富来源，它们富含肽和蛋白，包括一 些酶类和毒素。 丝氨酸蛋白酶抑制剂广泛存在于动物、植物和微生物体内，参与许多重要的 生理过程，如血液凝集、纤维蛋白溶解、细胞凋亡、发育以及炎症反应和补体活 化等（van Gent D. et al., 2003）。通过凝胶过滤、离子交换和反向高压液相色谱， 我们从金环蛇毒液中纯化得到一种天然的丝氨酸蛋白酶抑制剂，命名为 bungaruskunin。并且从该蛇的毒腺cDNA 文库中克隆到了它的核苷酸序列。 bungaruskunin 预测的前体由83 个氨基酸组成，包括含有24 个氨基酸的信号肽 和含有59 个氨基酸的成熟肽。它与一种由红腹伊澳蛇（Pseudechis porphyriacus） 的cDNA 预测到的丝氨酸蛋白酶抑制剂blackelin 具有最大相似性，达64％。 Bungaruskunin 是一种Kunitz 型的蛋白酶抑制剂，具有一个保守的Kunitz 结构域， 能够抑制胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶。通过对金环蛇毒腺cDNA 文库 的筛选，我们还得到了另外两条β-bungarotoxin B 链，Bungaruskunin 的整体结 构与β-bungarotoxin B 链相似，特别是它们都具有高度保守的信号肽序列。这些 发现强烈地表明蛇毒Kunitz/BPTI 蛋白酶抑制剂与神经毒性的类似物可能起源于 共同的祖先。 肥大细胞脱粒肽是从膜翅目昆虫的毒液中鉴别出的一个小肽家族，是一种具 有潜在的药物治疗作用的诱导活性分子（Xueqing Xu et al., 2006）。来源于蜂类的 缓激肽样的类似物vespakinin 家族是一种具有调节和激素功能的活性成分，与哺 乳动物和两栖动物的缓激肽类似（Nakajima T., 1984）。本研究对三种胡蜂的 毒液进行了一系列的活性检测，发现黑尾胡蜂的蜂毒对白色念珠菌Candida albicans 和金黄色葡萄球菌 Staphylococcus aureus 有抑制作用。凹纹胡蜂和黑尾 胡蜂的蜂毒具有微弱的磷酯酶A2 活性。通过凝胶过滤和反向高压液相色谱，没 有得到相关的活性组分。通过对三种胡蜂毒腺cDNA 文库的筛选，我们得到了2 条来源于黑尾胡蜂的核苷酸序列，Blast 分析表明，其中一条编码类似肥大细胞 脱粒肽，但未克隆到全长，序列比对结果显示其与来源于大胡蜂（Vespa magnifica） 的Mastoparan-like peptide 12c precursor（GenBank accession A0SPI0）的核苷酸序 列相似性达98％（Xueqing Xu et al., 2006）；另一条编码缓激肽类似物，命名为 Hw-bradykinin，序列比对结果显示其与来源于大胡蜂（Vespa magnifica）的 vespakinin-M precursor（GenBank accessionABG75944）的核苷酸相似率达96% (Zouhong Zhou et al., 2006)。

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

N-acetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro

N-Acetylchitooligosaccharide (N-acetyl-COs) was prepared by N-acetylation of chitooligosaccharide (COs). In vitro study using human umbilical vein endothelial cells (HUVECs) revealed that both N-acetyl-COs and COs inhibited the proliferation of HUVECs by inducing apoptosis. Treatment of HUVECs by N-acetyl-COs resulted in a significant reduction of density of the migration cells and repressed tubulogenesis process. The antiangiogenic effects of the oligosaccharides were further evaluated using in vivo zebrafish angiogenesis model, and the results showed that both oligosaccharides inhibited the growth of subintestinal vessels (SIV) of zebrafish embryos in a dose-dependent manner, as observed by endogenous alkaline phosphatase (EAP) staining assay. In contrast, no cytotoxicity was found when treating the NIH3T3 and several other cancer cells with the oligosaccharides. Our results also confirmed the antiangiogenic activity of N-acetyl-COs was significantly stronger than the parent oligosaccharide, COs. (c) 2007 Published by Elsevier Inc.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达

扇贝养殖是我国重要的海水养殖产业，然而自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。丝氨酸蛋白酶抑制因子及丝氨酸蛋白酶在无脊椎动物的免疫应答中起着核心作用，它们的协同作用直接导致外界病源入侵的信号转导和级联放大，并进一步激活一系列防御体系，如黑化反应、血液凝结和抗菌肽的合成等。因此，克隆扇贝参与免疫防御的丝氨酸蛋白酶抑制剂基因并对其功能进行研究，将有助于进一步研究扇贝的免疫防御机制，丰富和发展无脊椎动物免疫学的内容。 运用大规模EST技术和RACE技术从栉孔扇贝中克隆出一个Kazal型丝氨酸蛋白酶抑制剂基因，定名为CfKZSPI。该基因cDNA序列全长1788bp，其中5' 非编码区（Untranslated Region, UTR）为97 bp，3' UTR161 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，开放阅读框（Open Reading Frame, ORF）含有1530 bp，编码509 个氨基酸残基。对其推测氨基酸序列进行分析，发现其中包括22个氨基酸残基组成的信号肽序列和12个Kazal型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR（quantitative real time PCR）对鳗弧菌浸泡刺激后栉孔扇贝血淋巴中CfKZSPI 的 mRNA表达量进行了检测，发现其mRNA 的表达量在鳗弧菌刺激后3h明显上升，达到空白组的43.6倍；然后在6h时有所下降，为空白组的15.0倍；随着菌刺激时间的增长，CfKZSPI基因的 mRNA 表达量急剧增加，在刺激后8h，12h，24h分别达到空白组的174.1，207.8，675.4倍。统计分析发现3h（P=0.019<0.05）和12h（P=0.020<0.05）时，CfKZSPI基因mRNA表达量与空白组差异均显著。为了研究栉孔扇贝CfKZSPI的蛋白活性，将其第十二个结构域克隆到pET-32a（+）载体中，转化大肠杆菌Rosetta-gami（DE3）表达菌株，获得可溶性表达的蛋白rCfKZSPI-12，对其进行抑制蛋白酶活性的分析，发现其对胰蛋白酶有很强的抑制活性，而对凝血酶没有抑制活性。当rCfKZSPI-12与胰蛋白酶分子比率为1：1时，约90%的蛋白酶活性被抑制。运用狄更斯作图法研究rCfKZSPI-12对胰蛋白酶的抑制能力，结果发现其对胰蛋白酶的抑制常数为173 nmol L-1。 采用同样方法从海湾扇贝cDNA文库中克隆出一个Kunitz型丝氨酸蛋白酶抑制剂基因，定名为Aikunitz。该基因全长632 bp，其中5' UTR 为105 bp，3' UTR 为 245 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，ORF 含有282 bp，编码93 个氨基酸残基。推测的氨基酸序列N末端有一个20个氨基酸残基组成的信号肽序列，成熟蛋白包括一个Kunitz型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR对鳗弧菌和藤黄微球菌感染后海湾扇贝血淋巴中Aikunitz 的mRNA的表达量进行了检测，结果发现其在鳗弧菌刺激后3h到9h持续上升，9h时表达量为PBS对照组的4.49倍（P=0.008<0.05），然后开始下降，在72h时表达量为对照组的0.24倍（P=0.021<0.05）；而在藤黄微球菌刺激后3h到12h其表达量上升，其中6h时为空白组的5.95倍（P=0.0004<0.01）；12h以后迅速下降，其中24h的表达量为对照组的0.38倍（P=0.028<0.05）。将Aikunitz基因编码的成熟蛋白按照重组CfKZSPI-12的方法进行重组表达，并对重组蛋白进行抑制蛋白酶和抑菌活性分析。结果发现其对胰蛋白酶和弹性蛋白酶两种丝氨酸蛋白酶都没有抑制作用。抑菌实验同样发现，重组Aikunitz 对供试的革兰氏阳性菌藤黄微球菌和革兰氏阴性菌鳗弧菌和大肠杆菌都不显示明显抑菌活性。

Total synthesis of bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane as potent PTP1B inhibitor

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator and has been proved to be an effective target for the treatment of type 2 diabetes mellitus. Bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane 7 was first reported as a natural bromophenol with significant inhibition against PTP1B which was isolated from red algae Rhodomela confervoides. Intrigued by its astonishing activity (IC50 = 2.4 mu mol/L), compound 7 was synthesized with the overall yield of 24% and evaluated for its PTP1B inhibitory activity compared with natural compound. (C) 2008 Li Jun Han. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Berberine as a natural source inhibitor for mild steel in 1 M H2SO4

Berberine was abstracted from coptis chinensis and its inhibition efficiency on corrosion of mild steel in 1 M H2SO4 was investigated through weight loss experiment, electrochemical techniques and scanning electronic microscope (SEM) with energy disperse spectrometer (EDS). The weight loss results showed that berbefine is an excellent corrosion inhibitor for mild steel immersed in 1M H2SO4. Potentiodynamic curves suggested that berbefine suppressed both cathodic and anodic processes for its concentrations higher than 1.0 x 10(-4) M and mainly cathodic reaction was suppressed for lower concentrations. The Nyquist diagrams of impedance for mild steel in 1 M H2SO4 containing berbefine with different concentrations showed one capacitive loop, and the polarization resistance increased with the inhibitor concentration rising. A good fit to Flory-Huggins isotherm was obtained between surface coverage degree and inhibitor concentration. The surface morphology and EDS analysis for mild steel specimens in sulfuric acid in the absence and presence of the inhibitor also proved the results obtained by the weight loss and electrochemical experiments. The correlation of inhibition effect and molecular structure of berberine was then discussed by quantum chemistry study. (c) 2005 Elsevier B.V. All rights reserved.

Nicotinic acid as a nontoxic corrosion inhibitor for hot dipped Zn and Zn-Al alloy coatings on steels in diluted hydrochloric acid

The inhibition effect of nicotinic acid for corrosion of hot dipped Zn and Zn-Al alloy coatings in diluted hydrochloric acid was investigated using quantum chemistry analysis, weight loss test, electrochemical measurement, and scanning electronic microscope (SEM) analysis. Quantum chemistry calculation results showed that nicotinic acid possessed planar structure with a number of active centers, and the populations of the Mulliken charge, the highest occupied molecular orbital (HOMO), and the lowest unoccupied molecular orbital (LUMO) were found mainly focused around oxygen and nitrogen atoms, and the cyclic of the benzene as well. The results of weight loss test and electrochemical measurement indicated that inhibition efficiency (IE%) increased with inhibitor concentration, and the highest inhibition efficiency was up to 96.7%. The corrosion inhibition of these coatings was discussed in terms of blocking the electrode reaction by adsorption of the molecules at the active centers on the electrode surface. It was found that the adsorption of nicotinic acid on coating surface followed Langmuir adsorption isotherm with single molecular layer, and nicotinic acid adsorbed on the coating surface probably by chemisorption. Nicotinic acid, therefore, can act as a good nontoxic corrosion inhibitor for hot dipped Zn and Zn-Al alloy coatings in diluted hydrochloric acid solution. (c) 2007 Elsevier Ltd. All rights reserved.

Neural Network and Bioinformatic Methods for Predicting HIV-1 Protease Inhibitor Resistance

This article presents a new method for predicting viral resistance to seven protease inhibitors from the HIV-1 genotype, and for identifying the positions in the protease gene at which the specific nature of the mutation affects resistance. The neural network Analog ARTMAP predicts protease inhibitor resistance from viral genotypes. A feature selection method detects genetic positions that contribute to resistance both alone and through interactions with other positions. This method has identified positions 35, 37, 62, and 77, where traditional feature selection methods have not detected a contribution to resistance. At several positions in the protease gene, mutations confer differing degress of resistance, depending on the specific amino acid to which the sequence has mutated. To find these positions, an Amino Acid Space is introduced to represent genes in a vector space that captures the functional similarity between amino acid pairs. Feature selection identifies several new positions, including 36, 37, and 43, with amino acid-specific contributions to resistance. Analog ARTMAP networks applied to inputs that represent specific amino acids at these positions perform better than networks that use only mutation locations.

Prenatal protease inhibitor use and risk of preterm birth among HIV-infected women initiating antiretroviral drugs during pregnancy.

BACKGROUND: Conflicting results have been reported among studies of protease inhibitor (PI) use during pregnancy and preterm birth. Uncontrolled confounding by indication may explain some of the differences among studies. METHODS: In total, 777 human immunodeficiency virus (HIV)-infected pregnant women in a prospective cohort who were not receiving antiretroviral (ARV) treatment at conception were studied. Births <37 weeks gestation were reviewed, and deliveries due to spontaneous labor and/or rupture of membranes were identified. Risk of preterm birth and low birth weight (<2500 g) were evaluated by using multivariable logistic regression. RESULTS: Of the study population, 558 (72%) received combination ARV with PI during pregnancy, and a total of 130 preterm births were observed. In adjusted analyses, combination ARV with PI was not significantly associated with spontaneous preterm birth, compared to ARV without PI (odds ratio [OR], 1.22; 95% confidence interval [CI], 0.70-2.12). Sensitivity analyses that included women who received ARV prior to pregnancy also did not identify a significant association (OR, 1.34; 95% CI, 0.84-2.16). Low birth weight results were similar. CONCLUSIONS: No evidence of an association between use of combination ARV with PI during pregnancy and preterm birth was found. Our study supports current guidelines that promote consideration of combination ARV for all HIV-infected pregnant women.

In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.

BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.