Biomedical subjects

K Xing

Publications and source records attributed to K Xing.

6 recordsLinked to original sources

PubMed2003

Cladistic analysis of iridoviruses based on protein and DNA sequences.

Cladograms of iridoviruses were inferred from bootstrap analysis of molecular data sets comprising all published protein and DNA sequences of the major capsid protein, ATPase and DNA polymerase genes of members of the Iridoviridae family Iridovirus. All data sets yielded cladograms supporting the separation of the Iridovirus, Ranavirus and Lymphocystivirus genera, and the cladogram based on data derived from major capsid proteins further divided both the Iridovirus and Ranavirus genera into two groups. Tests of alternative hypotheses of topological constraints were also performed to further investigate relationships between infectious spleen and kidney necrosis virus (ISKNV), an unclassified fish iridovirus for which the complete genome sequence data is available, and other iridoviruses. Cladograms inferred and results of Shimodaira-Hasegawa tests indicated that ISKNV is more closely related to the Ranavirus genus than it is to the other genera of the family.

Amino Acid Sequence↗

PubMed2001

Human lens thioltransferase: cloning, purification, and function.

PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

Amino Acid Sequence↗

PubMed2001

Regulation of thioltransferase expression in human lens epithelial cells.

PURPOSE: To study how the expression of thioltransferase (TTase), a critical thiol repair and dethiolating enzyme, is regulated in human lens epithelial cells under oxidative stress. Also to examine whether depleting the primary cellular antioxidant glutathione (GSH) in these cells has any influence on TTase expression under the same conditions. METHODS: Human lens epithelial cells (B3) were grown to confluence (1.6 million) and gradually weaned from serum in the medium before exposing to 0.1 mM H2O2 for 2 hours. Cells were removed at the time intervals of 0, 5, 10, 15, 30, 60, and 120 minutes for protein measurements of GSH and TTase activity and for reverse transcription-polymerase chain reaction (RT-PCR) or Northern hybridization analysis to quantify TTase mRNA. The effect of GSH depletion on TTase mRNA expression was examined by treating the cells with buthionine S,R-sulfoximine (BSO); 1-chloro, 2,4-dinitrobenzene (CDNB); or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Lens epithelial cells, depleted of cellular GSH by treatment with BCNU, were subjected to oxidative stress to examine the effect on TTase activity and mRNA level. RESULTS: A transient increase was detected in TTase mRNA after 5 minutes of H2O2 treatment. The upregulation reached a maximum of 80% above the normal level by 10 minutes and gradually decreased as the oxidant was detoxified by the cells. Manipulation of cellular GSH level by treatment with BSO, CDNB, and BCNU resulted in a minimum change in TTase expression. It is noteworthy that when cells depleted of GSH were subjected to oxidative stress, TTase expression was also found to be strongly upregulated. CONCLUSIONS: These observations suggest that the upregulation of TTase expression in the lens epithelial cells could be an adaptive response of the cells to combat oxidative stress to restore the vital functions of the lens proteins and enzymes. Such regulation is independent of cellular GSH concentration.

Buthionine Sulfoximine↗

PubMed2001

[High level secretion expression of porcine somatoropin gene in Pichia pastoris and N-glycosylation analysis of products].

The porcine somatoropin gene was inserted into the Pichia pastoris expression vector of pPICZ alpha A which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid of pPICZ alpha A-pST was linearnized by Sac I and transformed into X-33 by electroporation. The multi-copy insert transformants were selected and cultivated in flasks. SDS-PAGE and Western blot analysis showed that PST gene products were observed in the supernants with a little larger molecular weight size than the natural PST's, however, the molecular weight size of the PST gene products in the soluble cellular proteins were identical to the natural PST's. Retransformation of the linearnized pPICZ alpha-pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one. The expressed rPST accumulated up to about 956 mg/L. The N-glycosylation analysis showed rPST had no N-glycosylation.

Animals↗

PubMed2000

Modulation of lens glycolytic pathway by thioltransferase.

The observation that the level of S-thiolated proteins (protein-thiol mixed disulfides) was transiently increased in the lens epithelial cells correlation with the transient inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD), a key glycolytic enzyme, when the cells were treated with a bolus of hydrogen peroxide, prompted our speculation that G-3PD may have been transiently thiolated at the SH sensitive active center. In the meantime, thioltransferase (TTase), a thiol regulating enzyme, whose activity remained constant under the same condition, may be regulating G-3PD and other sulfhydryl-sensitive glycolytic enzymes through thiol-disulfide exchange reactions ( Lou et al., 1998 ). To prove this hypothesis, several purified glycolytic enzymes from a commercial source, including hexokinase (HK), G-3PD, pyruvate kinase (PK) and fructose 1,6-bisphosphatase (FBPase), an enzyme in gluconeogenesis, were made into protein-thiol mixed disulfide and used for this study. Glycolytic enzymes in cultured rabbit lens epithelial cells pre-exposed to H(2)O(2)(0.5 m M for 15 min) were also studied for this purpose. Recombinant human lens thioltransferase (RHLT), which was isolated and purified previously in this laboratory, reactivated these pure glycolytic enzymes inactivated by forming protein-S-S-gluthathione (PSSG), protein-S-S-cysteine (PSSC) or, protein-S-S-cysteamine after thiolating with oxidized glutathione, cystine or cystamine respectively. RHLT also reactivated these enzymes in the cell extract of cultured rabbit lens epithelial cells after being briefly exposed to 0.5 m M H(2)O(2). The S-thiolation and dethiolation of FBPase however, showed an opposite effect to that of glycolytic enzymes. These results suggest that TTase may participate in the repair process of glycolytic enzymes during oxidative stress and restore their activities in situ.

Adenosine Triphosphate↗

PubMed1999

Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads.

The immobilization of trypsin on porous glycidyl methacrylate (GMA-GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple "contrast matching" method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media. Copyright 1999 Academic Press.

Journal Article↗