黑尾胡蜂毒液抗菌肽的分离纯化及功能研究

由于病原微生物对众多传统抗生素药物的耐药性问题日益突显,因此寻找新型的抗菌药物显得尤为紧迫,多肽类制剂因其具有广谱的抗菌活性,已成为克服病原微生物耐药性的潜在候选药物.从黑尾胡蜂毒液中分离并鉴定了一种具有抗菌活性的多肽,命名为VT-1.VT-1对临床致病菌均表现出广谱的抗菌活性.此外,VT-1对人红细胞显示出微弱的溶血活性.     

10种蔬菜内生细菌的分离筛选

以抑制植物主要病害病原菌和内生防病相关酶活性为评价标准,从蔬菜作物体内分离筛选植物内生细菌资源。从苦瓜、丝瓜、空心菜、大豆等10种蔬菜体内分离到101株细菌,发现内生细菌在不同种类的蔬菜中出现的频率不同,其中丝瓜叶上的内生细菌数量最多,洋葱体内分离到的内生细菌数量最少。运用平板对峙法从中筛选出了对黄瓜枯萎病菌、番茄青枯病菌、水稻细条病菌、荔枝霜疫霉病菌等有较强拮抗作用的菌株,获得对以上4种病菌均有拮抗作用的内生细菌有41株,占总株数的40．6％。对101株细菌进行纤维素酶、蛋白酶和几丁质酶活性测定表明,同时具有3种酶活性的菌株有32株,占总菌数的31．7％。从中选出13株拮抗效果较好的内生细菌。经初步鉴定均属于芽孢杆菌属（Bacillus sp）。进一步对筛选获得的同时对以上4种病原菌均有拮抗作用,且具有纤维素酶、蛋白酶和几丁质酶活性的13号、14号、59号和82号菌株进行16SrDNA序列分析,表明4个菌株均为枯草芽孢杆菌（B．subtilis）或其近缘种。     

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.     

水溶性C60-脱氢枞胺衍生物的合成及生物活性

以C60-脱氢枞胺吡咯烷衍生物和C60-脱氢枞胺氮烯加成物为原料,通过两种不同的水性化途径分别合成了两种水溶性的C60-脱氢枞胺富勒醇衍生物和两种水溶性的C60-脱氢枞胺甘氨酸衍生物。产物经TEM、FT-IR、ESI-MS、1H NMR和HPLC等分析方法检测确认为目标产物,且为加成度不同的混合物。对合成出的4种水溶性C60-脱氢枞胺衍生物进行了生物活性测试。结果表明,4种水溶性C60-脱氢枞胺衍生物对rTaq DNA聚合酶和HIV-1反转录酶均表现出一定的抑制作用,对卵巢癌细胞株几乎不表现出抑制活性。     

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin-the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.     

High resolution X-ray analyses of renin inhibitor-aspartic proteinase complexes

Inhibitors of the conversion of angiotensinogen to the vasoconstrictor angiotensin II have considerable value as antihypertensive agents. For example, captopril and enalapril are clinically useful as inhibitors of angiotensin-converting enzyme. This has encouraged intense activity in the development of inhibitors of kidney renin, which is a very specific aspartic proteinase catalysing the first and rate limiting step in the conversion of angiotensinogen to angiotensin II. The most effective inhibitors such as H-142 and L-363,564 have used non-hydrolysable analogues of the proposed transition state, and partial sequences of angiotensinogen (Table 1). H-142 is effective in lowering blood pressure in humans but has no significant effect on other aspartic proteinases such as pepsin in the human body (Table 1). At present there are no crystal structures available for human or mouse renins although three-dimensional models demonstrate close structural similarity to other spartic proteinases. We have therefore determined by X-ray analysis the three-dimensional structures of H-142 and L-363,564 complexed with the aspartic proteinase endothiapepsin, which binds these inhibitors with affinities not greatly different from those measured against human renin (Table 1). The structures of these complexes and of that between endothiapepsin and the general aspartic proteinase inhibitor, H-256 (Table 1) define the common hydrogen bonding schemes that allow subtle differences in side-chain orientations and in the positions of the transition state analogues with respect to the active-site aspartates.     

中华大蟾蜍尾退化过程中尾蛋白酶活性的变化

采用活性电泳方法对中华蟾蜍尾退化过程中尾蛋白酶活性的变化进行了研究,结果显示：（1）酸性条件（pH3．5）下,从35期到38期,检测到的蛋白酶带数目较少,活性较弱;碱性条件（pH9．5）下,除25kD的蛋白酶带之外,检测到的其它酶带与酸性条件下相似;（2）中性条件（p H7.0)下,从35期到38期,检测到的蛋白酶带逐渐增多,活性也明显增强,尤其是在尾退化的高峰期（37期、38期）,110kD、92kD、74kD的酶带活性达到峰值;另外在尾退化的高峰期还检测到了7条新酶带,其中55kD的酶带活性也达到峰值。以上结果显示,蛋白酶在中华大蟾蜍蝌蚪尾退化过程中起重要作用。     

运动训练对小鼠心肌蛋白酶活性的影响

采用活性电泳方法，研究了运动训练对小鼠心肌蛋白酶活性的影响。结果表明：（1）随着运动强度的增加，不同运动训练组小鼠心肌的中性和碱性蛋白酶活性有不同程度的升高。酸性条件下，未检测到蛋白酶活性；（2）运动心心肌碱性蛋白酶活性的影响比对中性蛋白酶活性的影响更为显。     

X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes

Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.     

3株拮抗内生放线菌的鉴定及其抑菌活性初步研究

对分离自江西东乡野生稻根部的3株内生放线菌进行鉴定和抑菌活性研究,根据菌株的形态与培养特征、生理生化特性及16S rRNA基因序列系统发育分析,将菌株FRo1、FRo2和FRo3分别鉴定为委内瑞拉链霉菌、娄彻氏链霉菌和肉质链霉菌.采用管碟法和菌丝生长速率法分析菌株拮抗病原细菌与病原真菌的活性.结果表明:菌株FRo1发酵液对所有测试细菌均有抑制作用,显示较广谱抑细菌活性; 菌株FRo2显示出较强抑制金黄色葡萄球菌和7种病原真菌的活性,其中对金黄色葡萄球菌抑菌直径为22.33 mm,对小麦赤霉、水稻纹枯病菌、车前草核盘菌及胶胞炭疽菌的抑制率分别为70;、45;、58;和65;; 而菌株FRo3发酵液对金黄色葡萄球菌、枯草芽孢杆菌及伤寒杆菌表现出不同程度的抑制,除车前草核盘菌外对所测试的病原真菌均有抑制活性,但抑制率均低于50;,这些菌株抑菌效果良好,显示出较好的生防潜力.     

渗透压对细菌的影响

在等渗溶液中,微生物正常生长繁殖;在高渗溶液中,细胞失水收缩,而水分为微生物生理生化反应所必需,失水会抑制其生长繁殖.根据不同盐质量分数下细菌的繁殖情况,可以判断渗透压对细菌的影响.用含不同质量分数NaCl的牛肉膏蛋白胨培养基培养细菌来观察渗透压对细菌的影响,选用的菌种为金黄色葡萄球菌,大肠杆菌,枯草芽孢杆菌.大肠杆菌耐高渗透压的能力较差,在质量分数为3%以下的NaCl溶液中能正常生长,在5%的NaCl溶液中受到抑制;枯草芽孢杆菌和金黄色葡萄球菌有较强的耐盐能力,枯草芽孢杆菌在5%的NaCl溶液中能正常生长,在10%的NaCl溶液中受到抑制,而金黄色葡萄球菌在10%的NaCl溶液中仍能正常生长,在高于10%的NaCl溶液中生长受到抑制.     

Monoclonal antibodies to lymphocytic choriomeningitis virus react with pathogenic arenaviruses

Certain arenaviruses have become widely recognized as important human pathogens, the most notable among these being Lassa virus, the causative agent of Lassa fever. Two other members of the group, Junin and Machupo virus, are the aetiological agents of Argentine and Bolivian haemorrhagic fevers, respectively. All these agents share both morphological features and to varying degrees serological cross-reactivity with other non-pathogenic arenaviruses. Despite the evident clinical importance of these viruses, work to define their physiochemical structure and to develop rapid and precise diagnostic techniques has been slow. Consequently, the definitive relationships among West African Lassa strains, strains of a related agent from Mozambique and of an Old World arenavirus, lymphocytic choriomeningitis (LCM), have not been established. This problem is of more than simple taxonomic importance in view of the fact that a Mozambique virus strain produced subclinical infection in experimental monkeys which were then resistant to challenge with monkey and human virulent Lassa virus from Sierra Leone. We have explored the use of monoclonal hybridoma antibodies generated against relatively less hazardous arenaviruses to define antigens cross-reactive with the important human pathogens of the group. Here we describe the use of monoclonal antibodies directed against LCM, virus to define antigenic specificities shared among LCM, Lassa and Mozambique viruses.     

鲎素抗菌肽分子结构稳定性及生物活性的研究

鲎素是一种高效的、广谱的抗菌肽,已经成为研究热点.为了实现鲎素在生产实践中的应用,研究鲎素在体外分子结构、生物活性的稳定性.本文采用不同温度、pH值、体外蛋白酶(胃蛋白酶、胰蛋白酶、弹性蛋白酶、羧肽酶B )分别处理鲎素.通过检测鲎素最小抑菌浓度(MIC)的变化,来衡量鲎素生物活性稳定性;采用HPLC法检测鲎素残留量,通过HPLC图谱变化来评定鲎素分子结构稳定性情况.结果表明:在t < 120℃、pH < 10.0作用情况下,鲎素对大肠杆菌K88的MIC为1.25～2.5 mg/L,高效液相色谱检测鲎素残留量在65.5～70.2μg之间;鲎素对胃蛋白酶的作用表现出很高的稳定性,对胰蛋白酶、羧肽酶、弹性蛋白酶的作用稍微敏感.在胃蛋白酶浓度小于 320 U/mL,胰蛋白酶浓度小于40 U/mL,羧肽酶浓度小于20 U/mL,弹性蛋白酶浓度小于2.5 U/mL作用下鲎素仍具有活性.通过实验得出结论:鲎素抗菌肽具有较强的高温耐受性,在酸性条件下具有稳定性,同时鲎素具有一定的耐受蛋白酶降解的能力.     

辣椒碱抗病原菌活性及其在番茄酱防腐中的应用

从红辣椒中提取的辣椒碱对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌这3种常见的病原细菌及黑曲霉、啤酒酵母等7种病原真菌进行拮抗试验。结果表明,辣椒碱具有广谱的抗菌范围和较强的拮抗病原菌的活性,且抗菌活性有明显的量效关系,其中辣椒碱对供试细菌的拮抗作用远大于对供试真菌的作用。将辣椒碱添加到番茄酱中进行防腐试验的结果表明,辣椒碱可有效的抑制微生物的生长,从而延长番茄酱的贮藏期。     

鱼皮胶原蛋白的纯化及酶解性质的研究

分别对鲨鱼、鲢鱼、草鱼、罗非鱼鱼皮的胶原蛋白进行提取、纯化并进行SDS PAGE电泳分析,对它们的酶解性质进行研究.结果表明,提取的胶原蛋白有较高纯度,是典型的I型胶原蛋白.4种鱼皮胶原蛋白均能被胰蛋白酶和蛋白酶K水解,两种酶酶切位点有差异.不同鱼种鱼皮胶原蛋白的一级结构不同.本实验的酶解条件是适宜的.     

微生物复合菌剂的生物安全评价

微生物复合菌剂在石油污染土壤生物修复中的应用潜力在不断扩大,有着广阔的发展前景,但同时对人体健康和生态环境的安全问题也存在较大的不确定性和潜在风险.文章中采用八株降油菌制成混合菌剂,通过动物试验和陆生植物生长试验,对微生物复合菌剂进行生物安全评价.结果表明,试验用微生物复合菌剂对动物皮肤无刺激性,对植物生长无毒害作用,具有相对安全性.     

魔芋内生细菌抗软腐病菌株筛选

通过组织分离法从魔芋块茎中分离得到内生细菌,用牛津杯法皿内测试内生细菌对魔芋软腐病菌的拮抗作用,以筛选拮抗软腐病菌能力强的菌株。结果显示从魔芋块茎中分离得到35株内生细菌,35株内生细菌对魔芋软腐病菌均有不同程度的拮抗活性,不同菌株间拮抗活性存在显著差异。初筛中,对软腐病菌相对抑制率(RIR)达到50%以上的内生细菌有17株,100%的有11株。复筛中RIR为100%的内生细菌有8株,其中7株胞外产物的RIR超过了150%。     

1株产细菌素乳杆菌的鉴定及其所产抑菌物质的特性

从分离自内蒙古传统乳制品的67株乳杆菌中筛选得到1株产细菌素的乳杆菌KLDS 1.0355。通过API 50生化反应实验和16S rDNA序列同源性分析,鉴定该菌株为短乳杆菌。应用琼脂扩散法研究了KLDS 1.0355无细胞发酵上清液的部分特性,KLDS 1.0355的无细胞发酵上清液对热（121℃,30min）和pH值（pH 2.0～10.0）稳定,可被多种蛋白酶如胃蛋白酶、胰蛋白酶、木瓜蛋白酶、α-糜蛋白酶和蛋白酶K失活,不能被过氧化氢酶失活,说明该抑菌物质为1种蛋白质。进一步证明了该抑菌物质的作用方式是杀菌,且抑菌谱广,可抑制多种革兰氏阳性菌和革兰氏阴性菌。