BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

12种中草药的乙醇提取液对印鼠客蚤的毒效研究

目的:探讨中草药灭蚤的可行性。方法:用乙醇提出12种中草药的可溶成分,以瓶膜法测定其对印鼠客蚤的毒力。结果:在0.5g/ml(生药)的浓度下,10种中草药对印鼠客蚤有速效击倒作用,其中鸡血藤、巴豆仁、除虫菊花的10min击倒率大于50％;5种中草药的KD_(50)小于15min,比2％DDVP为优;巴豆仁接触30min恢复24h死亡率为70％。结论:除虫菊花、黎芦、鸡血藤、巴豆仁、贯众用于杀蚤有一定价值。     

通调督脉化瘀排毒法治疗尿毒症晚期患者92例临床研究

目的观察应用通调督脉化瘀排毒法治疗尿毒症晚期的临床疗效。方法将92例尿毒症晚期患者随机分为两组,治疗组48例,除一般治疗外,加用海洋生命平衡超导治疗仪,将中药干粉剂作用于督脉、足太阳膀胱经的五脏六腑之俞穴,每日3次,每次1小时;对照组44例,给予一般治疗。结果治疗组总有效率及生化指标与对照组比较均有显著改善(P<0.05)。结论应用调督通脉化瘀排毒法,能提高尿毒症晚期患者的临床疗效。     

艰难梭茵毒素基因3''''末端重复区域基因片段的PCR克隆

目的探寻一种简单、快速、灵敏的检测艰难梭菌的方法.方法以艰难梭菌毒素A和毒素B基因3'末端高度保守重复区分别设计一对引物,进行PCR反应,同时在同一体系相同反应条件下使用相同引物分别以大肠杆菌和乳杆菌DNA为模板进行扩增,比较二者结果.结果从艰难梭菌成功克隆了毒素A基因3'端重复区域960 bp的基因片段及毒素B基因3'端重复区域1851 bp的基因片段,不同来源的菌株出现了相同的2条电泳带,并通过测序鉴定;而对照的大肠杆菌和乳杆菌株无特异电泳带出现.结论从艰难梭菌毒素A、毒素B基因3'末端重复区域克隆的基因片段具有保守性,可以作为基因探针在临床上进行艰难梭菌检测,该方法简便、灵敏,可直接用粪便标本进行检测.此外,这些基因编码的多肽具有较高的疏水性并存在着膜受体结合区域,可以此为基础进行基因工程蛋白质疫苗的研究.     

健康人携带的白喉棒状杆菌生物学性状分析

目的：探讨健康人携带的白喉棒状杆菌的生物学特性及其与标准的产毒菌株的生物学性状的差异。方法：采用鸡蛋斜面培养基，亚碲酸钾血琼脂平板和肉汤培养基观察其生长特性，测定多种系列化反应性，毒力和药物敏感性。结果：健康人携带的白喉杆菌的培养特性典型均无毒力；发酵麦芽糖和发酵蔗糖的菌株较多，有较多的耐麦迪霉素，苯唑青霉素和洁霉素的菌株。结论：健康人所携带的白喉杆菌的生物学特性不同于标准的有毒力的菌株。     

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesinencoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap⁺ and/or sfa⁺ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing a-haemolysin (Hly⁺) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesinencoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.La PCR a permis de détecter les opérons pap, sfa et afa chez 243 souches de Escherichia coli isolées d'infections de l'arbre urinaire. On observe que respectivement 54, 53 et 2% des souches sont du génotype pap, sfa et afa. Les souches pap⁺ et/ou sfa⁺ sont plus fréquentes dans les pyelonephritis aiguës (94%) que dans les cystites (67%) (P < 0,001) et dans les bactériuries asymptomatiques (57%) (P < 0,001). Les opérons pap et/ou sfa sont décelés chez 90% des souches MRHA⁺ (hémagglutination mannoserésistante) et 37% des souches MRHA⁻ (P < 0,001). La présence des opérons pap et sfa est spécialement significative chez les souches appartenant au type III de MRHA (100%) sans adhésines P, et au type IVa (97 %) exprimant la liaison Gai-Gal typique des adhésines P. Les opérons pap et sfa sont tous deux étroitement associés aux souches toxigéniques produisant l'α-hémolysine (Hly⁺) et le facteur cytotoxique nécrotique de type 1. Une corrélation apparaît entre les opérons pap et sfa et le sérogroupe: 93 % des souches appartenant aux groupes O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 et O83 possèdent ces opérons, et seulement 37 % des souches appartenant à d'autres sérogroupes (P < 0,001) les possèdent. Ces résultats confirment l'utilité de notre typage MRHA pour l'identification (présomptive) des souches pathogènes de E. coli porteuses de divers facteurs de virulence. Ainsi, 85 % des souches possédant à la fois les opérons pap et sfa codant les adhésines sont du type III ou IVa (de MRHA) en relation avec la virulence des souches responsables d'infections urinaires et de bactériémies.     

产气荚膜梭菌毒素研究进展

产气荚膜梭菌通过产生大量的毒素导致人类和动物患气性坏疽、肠炎和肠毒素血症。目前,已知产气荚膜梭菌可产生20多种毒素和水解酶。不同的毒素类型与特定的疾病类型相关。毒素分型已由毒素基因的分子检测替代了传统的血清分型方法。因此本文围绕产气荚膜梭菌毒素种类、基本特征、致病机制以及与疾病的关系进行系统回顾总结和展望,为后续的毒素分型等快速检测技术的建立、免疫抗原筛选、抗体制备以及相关致病机制研究提供基础。     

Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin.

Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.     

A new membrane-attack complex/perforin (MACPF) domain lethal toxin from the nematocyst venom of the Okinawan sea anemone Actineria villosa.

The Okinawan sea anemone Actineria villosa causes severe cases of stinging. We isolated the 60 kDa A. villosa toxin (AvTX-60A) as the major toxin from the isolated nematocysts of this species. AvTX-60A showed fatal toxicity to mice with intraperitoneal injection at a minimum lethal dose of less than 250 microg/kg. The N-terminal amino acid sequence was determined and the corresponding cDNA encoding AvTX-60A was sequenced. The deduced amino acid sequence of AvTX-60A showed high similarity with PsTX-60A, which had been isolated as one of the major toxins from the venomous sea anemone Phyllodiscus semoni. These sea anemone toxins are new members of the family of proteins containing membrane-attack complex/perforin (MACPF) domains, best known in pore forming proteins such as perforin. These are the first examples of MACPF domain proteins as toxins for prey acquisition or repelling predators in nature.     

Rapid and efficient identification of cysteine-rich peptides by random screening of a venom gland cDNA library from the hexathelid spider Macrothele gigas.

We identified novel 10 multi-cysteine peptides, namely Magi 7-16, from the spider Macrothele gigas by simple random cDNA screening of the venom gland. Mass analysis of the crude venom detected the mass numbers of the cross-linked forms of all peptides, confirming their presence in the venom. Magi 11, a C-terminus amidated peptide, was chemically synthesized and was indistinguishable from the native peptide proving the feasibility of the method for peptide identification. Moreover, toxicological assays showed diverse lethal or paralytic activities of these peptide toxins on mice and/or insects.