Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

The effect of rebamipide on gastric xanthine oxidase activity and type conversion in ethanol-treated rats

Rebamipide, a novel antipeptic ulcer drug, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic acid, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intraperitoneal administration of rebamipide at 60 mg/kg body weight reduced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of rebamipide on alcohol-induced xanthine oxidase-type conversion and enzyme activity. It was also observed that the activity of xanthine oxidase was significantly inhibited by administration of rebamipide at 60 mg/kg body weight, leading to a significant reduction of lipid peroxide content in alcohol-treated rats. The results suggest that alcohol-induced gastric mucosal lesions might be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and the protective effect of rebamipide on gastric mucosal lesions would result from its ability to protect against oxidative stress on gastric mucosal lesions of alcohol-treated rats.