Investigation of the catalytic mechanism of the hotdog-fold enzyme superfamily Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.     

A noncanonical cysteine protease USP1 is activated through active site modulation by USP1-associated factor 1

Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.     

Hemiboea sinovietnamica sp. nov. (Gesneriaceae) from a limestone area along the boundary of Sino‐Vietnam

Hemiboea sinovietnamica W. B. Xu & X. Y. Zhuang, a new species of Gesneriaceae from a limestone area along the boundary of Sino‐Vietnam, is described and illustrated. The new species is similar to H. longgangensis Z. Y. Li with its yellowish corolla, but differs in having leaf blades glabrous on both sides, involucre trigonous, 2–3 cm in diameter, glabrous outside, glabrous cymes, a white calyx that is glabrous outside, and glabrous pistil and capsule.     

Modification of chitosan membrane with poly(vinyl alcohol) and biocompatibility evaluation

This work aimed to overcome chitosan (CS) membrane' drawbacks: mainly stiffness and hydrophobic surface by adding poly(vinyl alcohol) (PVA) and evaluate their biocompatibility. The chemical structure, crystalline and thermal properties were studied by FT-IR, XRD and DSC. The mechanical properties and wettability of CS/PVA membranes were studied by tensile test and static contact angle measurement. In vitro biocompatibility was also evaluated by MTS cytotoxicity assay and SEM examination. The results suggest that adding PVA into CS membrane could greatly improve CS membrane's flexibility and wettability. All the membranes prepared were biocompatible and have potential applications in GTR technology.     

Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

ABSTRACT: BACKGROUND: The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. RESULTS: In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively). CONCLUSIONS: The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.     

Selection of a DNA barcode for Nectriaceae from fungal whole-genomes

A DNA barcode is a short segment of sequence that is able to distinguish species. A barcode must ideally contain enough variation to distinguish every individual species and be easily obtained. Fungi of Nectriaceae are economically important and show high species diversity. To establish a standard DNA barcode for this group of fungi, the genomes of Neurospora crassa and 30 other filamentous fungi were compared. The expect value was treated as a criterion to recognize homologous sequences. Four candidate markers, Hsp90, AAC, CDC48, and EF3, were tested for their feasibility as barcodes in the identification of 34 well-established species belonging to 13 genera of Nectriaceae. Two hundred and fifteen sequences were analyzed. Intra- and inter-specific variations and the success rate of PCR amplification and sequencing were considered as important criteria for estimation of the candidate markers. Ultimately, the partial EF3 gene met the requirements for a good DNA barcode: No overlap was found between the intra- and inter-specific pairwise distances. The smallest inter-specific distance of EF3 gene was 3.19%, while the largest intra-specific distance was 1.79%. In addition, there was a high success rate in PCR and sequencing for this gene (96.3%). CDC48 showed sufficiently high sequence variation among species, but the PCR and sequencing success rate was 84% using a single pair of primers. Although the Hsp90 and AAC genes had higher PCR and sequencing success rates (96.3% and 97.5%, respectively), overlapping occurred between the intra- and inter-specific variations, which could lead to misidentification. Therefore, we propose the EF3 gene as a possible DNA barcode for the nectriaceous fungi.     

Transmissibility of caprine scrapie in ovine transgenic mice

ABSTRACT: BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.     

携带人二氢叶酸还原酶基因慢病毒表达载体构建及鉴定

目的:构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法:采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindIII及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。结果:成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达。结论:成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。     

乙型肝炎实验动物模型的研究进展

乙型肝炎是一种影响人类健康的严重疾病,尤其在亚洲.近年来,随着安全有效的预防性疫苗普遍使用,乙肝的流行已得到很好的控制.但仍有很多慢性乙型肝炎病毒感染者,其中部分为慢性乙肝患者.近年来,不同乙肝动物模型的建立和应用在乙肝病毒的研究中起到了重要作用.对近年来乙肝动物模型的研究进行了综述.