Endothelial nitric oxide synthase gene polymorphisms associated with susceptibility to high altitude pulmonary edema in Chinese railway construction workers at Qinghai-Tibet over 4 500 meters above sea level / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level.</p><p><b>METHODS</b>A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing.</p><p><b>RESULTS</b>The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both Pü0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both Pü0.001).</p><p><b>CONCLUSIONS</b>Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.</p>

Molecular cloning, tissue distribution and expression in engineered cells of human orphan receptor GPR81 / 生物工程学报

The gpr81 was amplified by polymerase chain reaction (PCR) using human fetus kidney cDNA and whole blood genome DNA as template, respectively. The expression profile of gpr81 in human fetus was analyzed by RT-PCR and the result indicated GPR81 mRNA was most abundant in fetus liver and heart. In addition, the deduced amino acid of GPR81 was compared with other related molecules by Clustal w/x software, and a molecular phylogenetic tree was constructed with Treeview software. It was showed that GPR81 had the highest homology with nicotinic acid receptor in amino acids. After sequence identification, gpr81 was inserted into the plasmid pcDNA3. 1 (-)/his-mycA and then transfected into Chinese hamster ovary cell (CHO-K1). With the selection of G418, an engineered cell line which could stably express gpr81 was obtained by the indication of RT-PCR and Western-blot detection. The establishment of the cell line will serve as means for further study of GPR81.

Subcellular localization and tissues expression profile of hGPCRc: an orphan G protein-coupled receptor / 生物工程学报

As a member of orphan G protein-coupled receptors (oGPCRs), hGPCRc was cloned from human colon tissue and analyzed by bioinformatic softwares. It was showed that the corresponding amino acids of hGPCRc formed seven-transmembrane domains as the key characteristic of GPCRs. Then, the recombinant GFP-hGPCRc was constructed by fussing hGPCRc into pEGFP-N1 carrying green fluorescent protein (GFP) gene, and CHO-K1 cells were subsequently transfected with the GFP-hGPCRc or pEGFP-N1. The green fluorescence protein expression in the two different transfected cells was observed under the laser scanning confocal microscopy (LSCM). It was showed that green fluorescence protein was distributed in the whole bodies of the cells transfected with pEGFP-N1, but mainly distributed on the plasma membrane and cytoplasm membrane transfected with GFP-hGPCRc. Thus, the localization on the membrane of hGPCRc was accorded with the predication by bioinformatic analysis. The expression analysis of hGPCRc by RT-PCR indicated that hGPCRc was abundantly expressed in heart, kidney, cerebel and colon etc., but absent in liver, cerebra, small intestine and muscle etc. The expressing profile of hGPCRc could provide some useful clues to understanding its effects on embryonic development and physiological functions.