The role of calcium-sensing receptor on ischemia/reperfusion-induced rat cardiomyocyte apoptosis / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the role of calcium-sensing receptor (CaR) on ischemia/reperfusion-induced rat cardiomyocyte apoptosis.</p><p><b>METHODS</b>The isolated rat hearts were subjected to 40 min ischemia followed by 2h of reperfusion with or without CaR agonist GdCl3 at the beginning of reperfusion. Control hearts (without ischemia) and ischemic hearts (40 min ischemia without reperfusion) served as controls. The protein expressions of CaR, Bcl-2 and cyt C were detected by Western blot. Cardiomyocyte apoptosis was detected by TUNEL. Mitochondrial potential (Deltaphim) was detected by laser confocal microscopy.</p><p><b>RESULTS</b>Compared to controls groups, the expressions of CaR and apoptotic cells were significantly increased, Deltaphim and expressions of mitochondria cyt C and Bcl-2 were significantly reduced in ischemia/reperfusion hearts with or without GdCl3.</p><p><b>CONCLUSION</b>CaR was involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion via mitochondrial pathway.</p>

Knockout of the ptsG gene in Escherichia coli and cultural characterization of the mutants / 生物工程学报

Metabolic engineering provide powerful tools for the systematic manipulation of cellular metabolic activities. The ptsG gene for glucose-specific transporter Enzyme II CBGlc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette with short shared sequences on both ends generated by PCR was electroporated into Escherichia coli DH5alpha and JM109. Recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombinase functions. Therefore, the ptsG gene was disrupted to construct the mutants called DH5alphaP and JM109P. There was no difference between the mutants and parent strains in LB media.However, in LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass, together with exploiting more glucose. The maximal cell density obtained with DH5alphaP was approximately 3 times more than that of DH5alpha, then the result of JM109P increased fourfold. The products of recombinant protein TNF respectively accounted for 24.3% of total cellular protein in DH5alphaP with A600 8.28 and 20.8% of total cellular protein in JM109P with A600 7.62. The specific volume expression amount of TNF was greater in the ptsG mutant than in its parent strain. These results demonstrate that the ptsG-mutant strains will be available for high cell-density culture.

Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli / 生物工程学报

Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.

Construction of gene library of Arthrobacter BT801 and isolation & expression of hydantoinase gene / 生物工程学报

Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.

The construction of shuttle vectors of Brevibacillus brevis-Escherichia coli / 生物工程学报

The 5' region of the cell wall protein(CWP) gene containing multiple tandem promoters and the signal peptide-coding sequence was isolated by PCR from Br. brevis 50, and used to construct the shuttle vector pBKE50, which included the replication origin of pUB110 and the erythromycin-resistance gene of pGK12. The alpha-amylase gene of Bacillus subtilis 168 was ligated to pBKE50, producing plasmid pBKE50/alpha-amy. After the resulting plasmid was introduced into Br. brevis 50, soluble and biologically active alpha-amylase was secreted directly into the culture medium. The expression level of alpha-amylase in the recombinant Br. brevis 50 was twice higher than that of the donor strain.

EXPRESSION OF L-N-CARBAMOYLASE GENE IN PICHIA PASTORIS / 微生物学通报

N-carbamoylase is a part of hydantoinase operon which can transform N-carbamoylamino acid to corresponding ammo acids. The L-N-carbamoylase of Arthrobacter BT801, codied by the HyuC gene, is the rate-limiting and the only stereoselective enzyme. HyuC DNA fragment was amplified by PCR from the plasmid of pUC18-169. The target fragment was introduced into pPIC3. 5K plasmid to construct the pPIC3. 5K-hyuC expressing vector which was then transduced into Pichia pastoris GS115 cells after being linearized by BglⅡ digestion. Multi-copies insertion transformants were screened on G418 plates. The recombinant protein was proved to have biological activity of hydrolyzing N-carbamoylphenylalanine into phenylalanine through enzyme activity determination.