• Korean Journal of Clinical Laboratory Science
• /
• v.36 no.2
• /
• pp.163-172
• /
• 2004

Mast cells (MCs) have been implicated in the pathogenesis of tissue fibrosis. However, the role of MC in the development of liver fibrosis has not been fully elucidated. Stem cell factor (SCF) is known to recruit MCs to the liver following injury as it induces mast cell proliferation, survival and differentiation from resident tissue precursors. This study examines the interaction between activated hepatic stellate cells (HSCs) and MCs in rat fibrotic liver, and SCF production by HSCs during culture in vitro. Rats were studied 4, 7, 14 and 21 days after bile duct ligation (BDL). Fibrogenesis was assessed by a measurement of collagen stained with sirius red F3B. Activated HSCs and MCs were identified by ${\alpha}$-smooth muscle actin (${\alpha}-SMA$) immunohistochemical and alcian blue staining and measured by a computerized image analysis system. SCF production was determined in rat HSC cultures using Western blotting. Mild fibrotic changes were noted in BDL rat livers as early as 4 days after induction of cholestasis. Significant expansion and organization of fibrous tissue has occurred in day 14 BDL rats which progressed to bridging fibrosis by day 21. In BDL rats, both a large number of activated HSCs and MCs were detected in portal tracts and fibrous septa. Both area of activated HSCs infiltration and density of MCs were significantly higher in all BDL group compared with Shams. In BDL rats, both areas of activated HSCs infiltration and density of MCs were no significant difference between day 4 and 7 and were significantly higher in day 14. However, the areas of activated HSCs infiltration were significantly lesser in day 21 and the densities of MCs were significantly higher in day 21 compared with day14 BDL. In BDL rats, both areas of activated HSCs infiltration and density of MCs were highly correlated with areas of fibrosis. Western blotting showed that SCF protein was consistently produced in activated HSCs by culture on plastic and freshly isolated HSCs expressed relatively little 30kD SCF compared to late primary culture activated HSCs (day 14) and passaged HSCs. These results suggest that HSCs activated in vitro produce SCF, and may play an important role in recruiting mast cells to the liver during injury and fibrosis.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.6
• /
• pp.1070-1075
• /
• 2008

IscA was proposed to be involved in the iron-sulfur cluster assembly encoded by the iscSUA operon, but the role of IscA in the iron-sulfur cluster assembly still remains controversial. In our previous study, the IscA from A. ferrooxidans was successfully expressed in Escherichia coli, and purified to be a [$Fe_4S_4$] -cluster-containing protein. Cys35, Cys99, and Cys101 were important residues in ligating with the [$Fe_4S_4$] cluster. In this study, Asp97 was found to be another ligand for the iron-sulfur cluster binding according to site-directed mutagenesis results. Molecular modeling for the IscA also showed that Asp97 was a strong ligand with the [$Fe_4S_4$] cluster, which was in good agreement with the experimental results. Thus, the [$Fe_4S_4$] cluster in IscA from A. ferrooxidans was ligated by three cysteine residues and one aspartic acid.

• Microbiology and Biotechnology Letters
• /
• v.30 no.4
• /
• pp.312-319
• /
• 2002

The Sporosarcina psychrophilia pyrB gene, which encodes aspartate transcarbamylase (ATcase), was cloned on Sau3AI restriction endonuclease fragment inserted into pUC19 plasmid vector, S. psychrophilia pyrB gene was expressed in Escherichia coli pyrB mutant for the complementation test. The sequence of 2,606 nucleotides including putative pyrB gene was determined. The region contained one full open reading frame (ORf) and two partial ORFs. The deduced amino acid sequence of the second ORF showed 59% identity with that of Bacillus caldolyticus ATCase. The first and third partial ORFs were closely related to the uracil permease (pyrP) and dihydroorotase (pyrC), respectively. Besides, potential terminator, antiterminator, and anti-antiterminator structures were found in the intergenic region between pyrP and pyrB. These results suggested that S. psychrophilia pyrimidine nucleotide biosynthesis genes are clustered as well as other Bacillus sp. Over-expressed product of pyrB encoding ATCase was purified and analyzed by the SDS-PAGE. The purified PyrB protein turned out to be molecular mass of 27 kDa and showed ATCase activity.