An antimicrobial peptide with antimicrobial activity against Helicobacter pylori

An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had si

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Protective effects of melatonin against low- and high-LET irradiation

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/mu m carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/ cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

IL-2基因的突变及其与肠毒素融合基因的克隆和表达

由金黄色葡萄球菌分泌到胞外的单亚基蛋白中有肠毒素A和B。近年来两种毒素在肿瘤治疗研究方面取得了很大进展。白介素-2作为靶向分子，在抗肿瘤的药物中很有应用前景。本文分别对肠毒素A、肠毒素B和白介素-2进行了克隆和表达，并将白介素-2（125Ala）分别与肠毒素A227AI。肠毒素B进行了融合表达。从筛选到的天然金黄色葡萄球菌STSw的基因组中，通过PCR方法扩增出seb基因，同时突变了该基因两端几个稀有密码子。并将其克隆到7ZTS载体上进行表达，表达量占细胞总蛋白的33.5％。以seam基因为模板，通过重叠PCR将其227位天冬氨酸突变为丙氨酸，以降低其毒性。该突变基因重组到7ZTS载体中，并在JM109（DE3）中表达，表达量占细胞总蛋白的51.5％。通过重叠PCR法，对人的IL-2基因进行定点突变。共突变60个碱基，涉及51个氨基酸，其中第125位的半肤氨酸被突变为丙氨酸。该基因在大肠杆菌中表达量占总蛋白的30％。分别对上述三个工程菌的表达条件进行了探索。先制备出融合基因，再对融合基因进行表达，得到两种融合蛋白。它们是以6个甘氨酸和1个苏氨酸为链，将IL一2（125Ala）与肠毒素A227（Ala）、B分别连接起来，即IL-2（125Ala）-SEA227（Ala）和IL-2（125Ala）-SEB二者在大肠杆菌中表达量分别占总蛋白的10％和12％。以上实验结果为将几种蛋白开发成抗肿瘤靶向药物奠定了坚实基础。

重离子诱导正常组织细胞的突变及损伤效应

对于重离子束辐照诱导的人类肝L02细胞hprt基因突变和DNA损伤效应目前还很少有报导。在重离子治癌和载人空间飞行当中，评价重离子辐照对病灶周围正常组织的辐射危害和空间重离子射线对宇航员的辐射风险越来越重要。本论文通过研究hprt基因突变频率、突变谱以及DNA损伤情况，为正确评价重离子对人体正常组织细胞的辐射风险及危害提供基础数据和依据。利用兰州重离子研究装置（HIRFL）提供的12C6+离子束(到达细胞样品处的能量为83.6MeV/n，对应的LET值约为30keV/m)和医用电子直线加速器提供的X射线（8MV，对应的LET值约为0.2keV/m）对体外培养的L02细胞进行0~6Gy照射后，在含有6-TG的培养基中克隆、6-TG筛选hprt突变细胞株，细胞克隆法测定hprt基因突变频率。各照射剂量分别随意挑取9~10个hprt基因突变株扩大培养，分别提取DNA后用多重PCR法扩增hprt基因的九个外显子，利用琼脂糖凝胶电泳观察其缺失突变谱。利用常规彗星电泳方法检测12C6+离子束（LET为30 keV/μm）辐照后人类肝L02细胞的DNA损伤情况，以CASP软件逐个分析彗星图像，主要检测头部DNA（HDNA％）尾部DNA（TDNA％）、彗星全长（CL）、尾长（TL）、尾矩（TM）和Olive尾矩（OTM）等指标的剂量-效应关系，并用SPSS11.5软件进行统计学分析。最后绘制出并线性拟合TM-剂量曲线。实验结果显示，人类肝细胞L02细胞系hprt基因对12C6+离子束和X射线辐照是敏感的。L02细胞对12C6+离子的存活分数明显低于对X射线的存活分数，即12C6+离子对L02细胞的致死效应强于X射线。两种射线照射后，每106个存活细胞中突变克隆的个数随照射剂量增大而增大。受致死效应影响，受照细胞的突变频率先增大后减小，都在1Gy处达到最大值，与文献报道的其他重离子辐照细胞诱导突变的结果相似。在所分析的突变细胞克隆中，发生缺失突变的概率最大，且大多数为大片段缺失突变。文献报道X射线多诱导微小突变和小片段缺失突，说明高LET的重离子辐射比低LET的X射线所引起的细胞损伤更大。另外，随着照射剂量的增加，完全缺失突变几率呈逐渐增大的趋势。但由于能够分析的细胞克隆数有限，进一步的研究是必要的。彗星电泳实验结果发现辐照以剂量依赖的方式引起L02细胞彗星图像TDNA％、CL、TL、TM和OTM等指标的增大，且TM值与剂量成线性正相关。说明该LET的12C6+离子束对DNA有较强的致损伤效应，且与剂量成正相关。本文实验所用碳离子束的LET(30keV/m)恰处于重离子治癌当中离子束贯穿健康组织到达肿瘤靶区之前入射通道上所具有的LET范围之内。本文的突变频率实验结果显示重离子束诱导正常组织细胞hprt基因突变的频率要高于X射线，也就是重离子束辐射对正常组织的远后效应及辐射风险与危害要大于X射线。突变谱实验结果表明单次大剂量碳离子照射对于hprt基因乃至细胞和组织都会造成很大的损伤。彗星电泳的结果也显示重离子对正常细胞一定的致DNA损伤效应。因此，应在充分考虑重离子束对正常组织辐射风险的前提条件下合理地制订治疗计划，在治疗当中应尽量避免对正常细胞组织的单次大剂量照射，使得重离子治癌成为一种最有效且安全的放射治疗模式。另外，在宇宙空间中，碳离子虽然在空间重离子中所占的比例较小，但从本文结果可以看到其也存在一定的辐射风险。因此，在载人空间飞行当中也应充分考虑其对宇航员的辐射风险及危害，并制定相应的辐射防护计

Rapid molecular weight determination of recombinant hirudin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of recombinant hirudin ( rHV-2) was determined rapidly by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The effects of the three types of matrixes were compared and discussed, alpha-cynao-4-hydroxycinnamic acid was proved to be the best matrix. It showed that MALDI-TOF-MS was superior to the traditional method of molecular weight determination of the biological macromolecules. The mass spectrum data proved that the primary structure of rHV-2 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding and purification.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...

A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory

Neuropsin (kallikrein 8, ELKS) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only express

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Moderate mutation rate in the SARS coronavirus genome and its implications

Background: The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) an

mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample f

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in d

Mitochondrial DNA Haplogroups M7b1 ' 2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G -> A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two