Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

General Information

A novel cDNA clone encoding a COR413-like gene was isolated by suppression subtraction hybridization and cDNA library screening from sea-island cotton (Gossypium barbadense). This gene (designated as GbCOR413, Accession number: AY761065) has a total length of 893 bp with an open reading frame of 600 bp, encoding a predicated polypeptide of 200 amino acids with a molecular weight of 22.74 kDa and a predicated pI of 9.2. Bioinformatics analyses revealed that this gene belonged to a novel stress-regulated multi-spanning transmembrane protein family without signal peptide. By means of semi-quantities RT-PCR analysis, the expression of GbCOR413 under short-term cold treatment at 4°C, water submergence and abscic acid treatment was investigated. Our studies suggested that the cloned gene was a new member of COR gene family which was slowly responsive to cold stress in cotton. Jin Wang and Kai-Jing Zuo are co-first authors of this paper.     

呼吸道不同部位的微生物分布特点及药敏分析

目的指导临床合理选用抗生素,预防减少呼吸道感染的发生及深入.方法取痰、喉室的分泌物及喉癌术后气管内的分泌物进行细菌培养和药敏试验.结果呼吸科呼吸道感染的病原菌中以白色念球菌居首位,其次革兰阴性杆菌检出率高,对亚胺培南的耐药性最低.耳鼻喉科声带息肉患者喉室的病原菌中以草绿链球菌居首位,对阿莫西林-棒酸,头孢1、2代敏感.耳鼻喉科喉癌术后气管切开患者下呼吸道感染的病原菌中,既有革兰阳性菌（以肺炎链球菌为主）又有革兰阴性杆菌（以铜绿假单胞菌、醋酸钙不动杆菌为主）,革兰阴性杆菌较革兰阳性菌多,均对头孢他啶有较高的敏感率.结论由于细菌的分布特点不一,耐药性不一样,最好依据细菌学选择敏感抗生素,当病原菌不明时,需要经验用药时则根据其筛查出的细菌排序的流行趋势选择抗生素.     

大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

The Relationship Between the Nariations of Fruit-Bearing Organic Characters in Natural Aneurolepidium chinense Population and the Climatic Factors in the Songnen Plain of China

Based on eight years continuous observation and measurement on the ways and intensity of utilization of three fixed plots of the meadow of natural Aneurolepidium chinense in the Songnen Plain of China at definite stage of annual growth season the quantitative analysis on the relationship between the variations of fruit-bearing organic characters of A. chinense population and the climatic factors was investigated. Change of climate was one of the comprehensive factors which led to the variations of fruit-bearing organic characters of the population in different plots, but variations on the characters of the population among the plots and on the degree of correlation or functional relationships between the character and the climatic factor in different stages were only caused by the ways and intensity utilization. The characters of the spike organ were more affected by climatic factors in the stage of growth and development of the winterness plants of the population. There were universal ecological effects of time lap in the climatic factors.     

Changing dendritic field size of mouse retinal ganglion cells in early postnatal development

During early postnatal development, dendrites of retinal ganglion cells (RGCs) extend and branch in the inner plexiform layer to establish the adult level of stratification, pattern of branching, and coverage. Many studies have described the branching patterns, transient features, and regulatory factors of stratification of the RGCs. The rate of RGC dendritic field (DF) expansion relative to the growing retina has not been systematically investigated. In this study, we used two methods to examine the relative expansion of RGC DFs. First, we measured the size of RGC DFs and the diameters of the eyeballs at several postnatal stages. We compared the measurements with the RGC DF sizes calculated from difference of the eyeball sizes based on a linear expansion assumption. Second, we used the number of cholinergic amacrine cells (SACs) circumscribed by the DFs of RGCs at corresponding time points as an internal ruler to assess the size of DFs. We found most RGCs exhibit a phase of faster expansion relative to the retina between postnatal day 8 (P8) and P13, followed by a phase of retraction between P13 and adulthood. The morphological α cells showed the faster growing phase but not the retraction phase, whereas the morphological ON–OFF direction selective ganglion cells expanded in the same pace as the growing retina. These findings indicate different RGCs show different modes of growth, whereas most subtypes exhibit a fast expansion followed by a retraction phase to reach the adult size. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 397–407, 2010     

SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA

We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids’ side chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV and capsid assembly inhibitory activities. The structure–activity relationship (SAR) study showed that compounds bearing proper aromatic side chains had potential antiviral activities. The molecular modeling experiments revealed the molecular mechanism that they could bind to CA in the same manner as CAP-1 and occupy two more grooves.