微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.