Cloning and characterization of a trypsin-like serine protease gene, a novel regeneration-related gene from Apostichopus japonicus.

Trypsin-like serine protease (TLS) plays an important role in many physiological processes including wound healing, phlogosis reaction, blood clotting, regeneration etc. In this paper, a 1216 bp full-length cDNA sequence of TLS including 39 bp 5' UTR and 355 bp 3'UTR coding for a theoretical 273 amino acids protein was cloned from Apostichopus japonicus by means of the RACE technique for the first time. Bioinformatic analysis revealed that the gene with a 20 residues N-terminal signal peptide and a conserved C-terminal domain belongs to the trypsin-like serine protease superfamily. His78, Asp130 and Ser223 are the principal residues of the catalytic center. In-situ hybridization (ISH) analysis revealed that the TLS gene was widely distributed in different tissues. The expression patterns during different regeneration stages of the TLS gene in the body wall, intestine and respiratory trees were investigated using real-time quantitative PCR. The results show that there was a remarkable and temporary up-regulation of TLS gene expression in the body wall within 1h and subsequent down-regulation of TLS similar to intestine and respiratory trees. With the recovery of tissues, the expression level of the TLS gene was gradually up-regulated and finally reached normal levels. TLS was regulated during different regeneration stages suggesting that TLS is important in the regeneration process of A. japonicus.

Molecular cloning, characterization and expression analysis of a C-type lectin (AJCTL) from the sea cucumber Apostichopus japonicus.

For the sea cucumber Apostichopus japonicus, a C-type lectin (AJCTL) was identified using rapid amplification of cDNA ends (RACE) PCR techniques. The full-length cDNA of AJCTL is composed of 710bp with a 618bp open reading frame (ORF) that encodes a polypeptide of 205 amino acids with a N-terminal signal peptide and a C-terminal C-type lectin domain (CTLD). The calculated molecular mass of the whole protein is 22.5kDa and its predicted isoelectric point is 5.59. AJCTL belongs to the group VII of regulatory proteins and it might function as a Ca(2+)-dependent monosaccharide binding lectin specifically and recognizing mannose-type ligands. In situ hybridization demonstrated that the expression of AJCTL was located in the body wall, longitudinal muscles, intestinum and respiratory tree. This became apparent especially in the cytoplasm of epidermal cells and granular haemocytes. Real-time PCR data suggested that AJCTL was mostly synthesized in the longitudinal muscles and intestinum and less pronounced in the respiratory tree and body wall of adults. After 12h stimulation by Vibrio harveyi, at increasing bacterial concentration gradient, the expression of AJCTL in sea cucumber increased as well. This indicated that CTL is related to an innate immune response.

[Gene polymorphism of the endothelial nitric oxide synthase enzyme and pulmonary hypertension in patient with chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the relationship between the gene polymorphism of the endothelial nitric oxide synthase enzyme (eNOSG894T) with pulmonary hypertension of Chinese patient with chronic obstructive pulmonary disease (COPD). METHODS: Fifty normal volunteers (29 male, 21 female, mean age 67 +/- 3) and 50 patients with COPD complicated by pulmonary hypertension (31 male, 19 female, mean age 69 +/- 10) were included in the study. Lung function, arterial blood gases, and echocardiographic examination were performed. The genotype of eNOSG894T was determined with PCR. RESULTS: The frequencies of GT + FT genotype and T allele were significantly greater in the patients compared to the controls. The levels of NOx and NOx/ET were lower in the GT + T genotype [(48 +/- 8) micromol/L, (0.51 +/- 0.20)] than in the GG genotype [(60 +/- 24) micromol/L, (1.43 +/- 0.64)] of COPD. The level of ET was higher in the GT + TT genotype [(104 +/- 38) microg/L] than in the GG genotype [(50 +/- 26) microg/L] of COPD patients. The mean systolic pulmonary artery pressure was higher in the GT + TT genotype [(57 +/- 15) mm Hg, 1 mm Hg = 0.133 kPa] than in the GG genotype [(47 +/- 10) mm Hg] of COPD patients. In stepwise logistic regression analysis for predicting pulmonary artery pressure, the polymorphism of eNOS gene, the level of Nox and smoking were found to be independent variables. CONCLUSION: The G894T polymorphism of eNOS gene is associated with pulmonary hypertension of COPD in a Chinese population. T allele may be involved in the etiology of pulmonary hypertension with COPD by reducing NO release of the endothelium.