Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica最新文献

Both pituitary adenylate cyclase activating polypeptide PACAP and growth hormone-releasing hormone GHRH belong to the vasoactive intestinal polypeptide- glucagon-secretin family. They are encoded on the same gene in vertebrates (birds, amphibian, fish). Although the gene encoding PACAP and GHRH has been cloned in other fish species, characterization of this gene in the commercially important grouper (Epinephelus coioides) has not been previously reported. In this study, the GHRH/PACAP cDNA was cloned from grouper hypothalamic tissue. Two cDNA variants of the GHRH/PACAP precursor gene were identified as a result of alternative splicing, a long form encoding both PACAP and GHRH and a short form encoding only PACAP. Both the long and the short forms of the GHRH/PACAP precursor cDNA were identified in grouper 22 issues as well as expression in embryos and larvae by semi-quantitative RT-PCR detection. PACAP / GHRH precursor mRNA was expressed at a high level in the central nervous system than in several peripheral tissues. The data presented here provide the report of PACAP/GHRH mRNA expression in eye and gill tissues in fish. PACAP/GHRH mRNA was expressed in grouper embryo and all larval stages examined and was expressed at a high level starting on neurula stage onwards. The result suggests that PACAP/GHRH had an important role in the development of embryos and larvae, especially in neurula appearance stage.

A novel kinin-releasing and fibrin(ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the A alpha-chain of human fibrinogen with lower activity towards B beta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Many efforts have been paid to the separation of an integrated NA(D)PH dehydrogenase (NDH) complex. Several hydrophilic subcomplexes of NDH have been purified from the cyanobacterium Synechocystis PCC6803. However, no hydrophobic NDH subcomplex has ever been separated from cyanobacteria yet. In this paper, two NDH subcomplexes were separated from n-dodecyl beta-D-maltoside(DM)-treated whole cell extracts of Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Both subcomplexes contained the hydrophobic subunit NdhA, suggesting that they were hydrophobic NDH subcomplexes. Of the two subcomplexes, only one subcomplex contained NdhH. These subcomplexes showed NADPH-nitroblue tetrazolium (NBT) oxidoreductase activity and could specifically oxidize NADPH when several quinone analogues were used as electron acceptors, such as ferricyanide, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichlorophenol indophenol (DCPIP), duroquinone, ubiquinone-0 (UQ-0), etc.

A synthetic spider dragline gene s600 was cloned into fusion protein expression vector pGEX-KG and expressed in Escherichia coli. Protein S600 was purified and rabbit antiserum was prepared. Amino acid composition analysis confirmed the right expression of S600. Western blot analysis revealed that anti-S600 antiserum could react with natural spider silk, so the synthetic dragline protein, designed by the authors, shares similar immunological characteristics with the natural spider silk. An ELISA system was also established for the quantitative detection of synthetic dragline protein expression in silk gland (or in the cocoon) of transgenic silkworm.

Gene networks is the collection of gene-gene regulatory relations at the expression level. In this study, a combined approach of the linear transcriptional modeling, identification of promoter elements and gene co-expression clustering is developed to decipher yeast gene networks from expression time series. The cell must reorganize the genomic expression to programs required for growth and survival in each environment. The expression of many genes is regulated by environmental stress. The products of many genes that induced in the environmental stress are involved in metabolism of carbohydrates, structural repairs and even sporulation. Interestingly, it is identified that transcription factors Mcm1 and Dal82 matched their binding sites TT[bond]CC[triple bond]T[double bond]GGAAA and TGAAAAWTTT in cell cycle progression and environmental stress response, respectively. These conclusions agree with the known observations. The results indicate that the approach may be useful for modeling gene networks from microarray data.

The possibility of using fluorescent proteins as probes to study the twin-arginine translocation (Tat) system was assessed in Escherichia coli. When fused to the twin-arginine signal peptide of trimethylamine N-oxide reductase, the DsRed2 red fluorescent protein from the Discosoma sp. was successfully synthesized and folded in E. coli cells. However, RR-DsRed2 aggregated inside the cells. Therefore, although DsRed2 has been engineered from DsRed for faster maturation and lower non-specific aggregation, it is still not compatible with Tat-dependent translocation. In contrast, the jellyfish green fluorescent protein (GFP) was efficiently exported into periplasm even when the RR motif was changed to KR or RK. These results show that GFP can be used as an efficient reporter protein to study Tat system, but DsRed2 is not suitable for such purpose because of its aggregation property. In addition, when the protein concentration was similar, the fluorescence intensity of KR-GFP and RK-GFP decreased compared with RR-GFP, which would suggest that the twin-arginine signal peptide is not only essential for mediating protein translocation, but also important for the folding of down-stream protein.

The cDNA encoding Kringle 1-5 domains of human plasminogen (designated as K1-5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-5 was transformed into Pichia pastoris GS115 and the recombinant yeast was induced by methanol to express the recombinant protein. The expressed protein was purified by lysine affinity chromatography. The recombinant K1-5 inhibited the growth of bovine aortic endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner, with a half maximal concentration of 14 mg/L. And rhK1-5 inhibited 47% of the BAEC migration stimulated by bFGF at the concentration of 50 mg/L. rhK1-5 also affected the cell cycle of BAEC and caused G(0)-G(1) arrest at the concentration of 14 mg/L.

Signal transducer and activator of transcription 3 (STAT3) plays a central role in mediating signals related to cell growth, differentiation, survival and movement. In this report, the results of immunocytochemistry and Western blot analysis showed that the active form of Stat3 protein existed in COS7 cells. The endogenous Stat3 protein could also induce the expression of m67-sequence-directed reporter genes. The transient transfection of Stat3 cDNA into COS7 cells increased Stat3 protein level and the expression of the reporter gene. The overexpression of Stat3 protein in COS7 cells caused prominent cell morphological changes. These cells had a much larger cell body, extended long processes with branches, lamellipodia and filopodia. These results suggest that Stat3 protein may play important roles in cell adhesion, migration and cytoskeleton reorganization.

TyrR is a global regulation protein encoded by tyrR gene which controls several transcriptional units involving biosynthesis and transportation of aromatic amino acids in Escherichia coli. In this work, the tyrR gene was knocked out by a double-cross homologous recombination. The tyrR- mutant was verified by structural identification by PCR and sequencing, and functional demonstration using lacZ reporter gene. In tyrR- mutant, the activities of two key enzymes in the phenylalanine biosynthesis pathway, whose expression was controlled by TyrR, DAHPS and AT, had been shown to elevate by a 1.08-fold and 2.70-fold compared with the parent strain, respectively. The yield of phenylalanine biosynthesis in the mutant was 1.59 times higher than that of wild type strain. The repression on the transcription of aroP, encoding an aromatic amino acid permease, was eliminated, resulting in a 70.2% increase of the aromatic amino acid transportation in tyrR- mutant strain.

High throughput scoring algorithms that are used to find the match of a tandem mass spectrum to a predicted mass spectrum of a peptide within a database have been applied in shotgun proteomics. However, these algorithms could produce a significant number of incorrect peptide identifications. Here a novel approach was developed to scoring tandem mass spectra against a peptide database, in which fragment ion probabilities, number of enzymatic termini of candidate peptides, matching quality and match pattern between experimental and theoretical spectrum were considered. Benchmarking the novel scorer on a large set of experimental MS/MS spectra, it is demonstrated that PepSearch performs significantly better than the widely used software SEQUEST. The PepSearch software is available at http://compbio.sibsnet.org/projects/pepsearch.