一株紫罗兰蓝色素产生菌的分离、鉴定及其活性产物的特性分析

[目的]调查秦巴山地区微生物物种的多样性,建立用于活性产物筛选的微生物代谢产物库和菌种资源库,通过数据库平台进行资源共享.[方法]采用不同的分离方法,分离存在于不同生境的微生物类群,通过形态、生理生化及分子生物手段鉴定菌种,发酵并通过甲醇提取代谢产物.[结果]从本地区土壤样本中分离获得一株产水溶性紫罗兰蓝色素的放线菌Str-4331,在高氏培养基上其气生菌丝为白色,产生水溶性紫罗兰色素,镜检观察孢子丝螺旋形,孢子椭圆形至长圆形,通过形态、生理生化特征初步确定为链霉菌.利用ClustalX对其16S rRNA进行序列比对,用Neighbor-Joining (NJ)构建系统发育树,并用Bootstraping法对其评价.实验结果表明,该菌基因序列与砖红链霉菌(Streptomyces lateritius) LMG 19372基因序列有99％的最高相似性.抑菌试验表明,活性产物具有较强的抗革兰氏阳性细菌活性.色素在水溶液中颜色随着pH的变化而变化.质谱分析显示3个主要化合物,分子量分别为557.68、588.43、485.18 kD,其中分子量为557.68 kD化合物与榴菌素B分子量一致.[结论]根据菌株的16S rRNA序列分析结果,结合菌株的培养特性和生理生化特性,将菌株Str-4331归属为砖红链霉菌(S.lateritius).该菌种首次在本地区分离到,具有进一步研究开发的价值.     

A Novel Integrated Method Tor Large-scale Detection,Identification, and Quantification of Widely Targeted Metabolites： Application in the Study of Rice Metabolomics

Liquid chromatography-mass spectrometry （LC-MS）-based metabolomics has been facilitated by the con- struction of MSz spectral tag （MS2T） library from the total scan ESI MS/MS data, and the development of widely targeted metabolomics method using MS/MS data gathered from authentic standards. In this report, a novel strategy called step- wise multiple ion monitoring-enhanced product ions （stepwise MIM-EPI） was developed to construct the MS2T library, in which stepwise MIM was used as survey scans to trigger the acquisition of EPI. A total number of 698 （almost） non- redundant metabolites with MS2 spectra were obtained, of which 135 metabolites were identified/annotated. Integrating the data gathered from our MS2T library and other available multiple reaction monitoring （MRM） information, a widely targeted metabolomics method was developed to quantify 277 metabolites, including some phytohormones. Evaluation of the dehydration responses and natural variations of these metabolites in rice leaf not only suggested the coordinated regulation of abscisic acid （ABA） with metabolites such as serotonin derivative（s）, polyamine conjugates under drought stress, but also revealed some C-glycosylated flavones as the potential markers for the discrimination of indica and japonica rice subspecies. The new MS2T library construction and widely targeted metabolomics strategy could be used as a tool for rice functional genomics.     

The neuroprotective effect of acute moderate alcohol consumption on caspase-3 mediated neuroapoptosis in traumatic brain injury: The role of lysosomal cathepsin L and nitric oxide

Our aim in this study was to investigate the effect of moderate acute alcohol administration on cysteine protease mediated neuronal apoptosis and nitric oxide production in the traumatic brain injury. A total of 29 adult Sprague–Dawley male rats weighing 250–300 g were used. The rats were allocated into four groups. The first group was the control (sham-operated) group in which only a craniotomy was performed, the others were alcohol, trauma and trauma + alcohol groups. Caspase-3 enzyme activity in the trauma group increased significantly in comparison with the control group. The alcohol given group showed a decreased caspase-3 enzyme activity compared to the trauma group. The level of caspase-3 enzyme activity in the alcohol + trauma group decreased in comparison to the trauma group. SF/FEL ratio of cathepsin-L enzyme activity in the trauma group was significantly higher than in the control group. Our results indicate that moderate alcohol consumption may have protective effects on apoptotic cell death after traumatic brain injury. Protective effects of moderate ethanol consumption might be related to inhibition of lysosomal protease release and nitric oxide production.     

In vivo phage display — A discovery tool in molecular biomedicine

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration.     

Identification of fragments targeting an alternative pocket on HIV-1 gp41 by NMR screening and similarity searching

The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein–protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ～500 μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor.     

黄萎病菌诱导下野生茄‘托鲁巴姆’SSH文库的构建与分析

由大丽轮枝菌(Verticillium dahliae Kleb.)引起的茄子黄萎病是一种严重的土传病害,但在茄子栽培品种中缺少高抗种质,野生茄‘托鲁巴姆’高抗黄萎病。为探讨野生茄‘托鲁巴姆’抗黄萎病的分子机制,该试验以‘托鲁巴姆’为试材,利用抑制性差减杂交技术,分别以黄萎病菌侵染与未侵染的根系为检测方(tester)和驱动方(driver),构建了1个黄萎病菌诱导下的根器官正向差减cDNA文库。结果表明:(1)随机挑取170个阳性克隆,经测序,共获得109个非冗余EST,其中61个EST与其他植物的抗逆相关基因同源,如几丁质酶、细胞色素P450、过氧化物酶、蛋白酶抑制子等。(2)借助NCBI中的EST序列,经过电子拼接结合RT-PCR验证,克隆了1个通用胁迫蛋白基因———StUSP1。(3)表达分析结果显示,‘托鲁巴姆’受黄萎病菌侵染后,StUSP1在根中上调表达。     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

Recently, high-throughput screening (HTS) has become the mainstream technique for drug discovery. Compounds that are synthesized by combinatorial chemistry might be more suitable than natural products to apply to HTS, because the purification procedure is a drawback of using natural products. Nevertheless, natural products remain an extremely important source of drugs. To overcome the demerits of natural products, we are constructing the RIKEN Natural Products Depository (NPDepo) that is focused primarily on microbial metabolites. In this review, I describe (i) engineering pathways for biosynthetic gene clusters of microbial metabolites, (ii) construction of fraction libraries of microbial metabolites, and (iii) the development of a new screening system using a chemical array and a protein library produced by GLORIA.