Background There are few reports of a biological role for glycosyltransferases in the infiltration of osteoarthritic synovitis. The aim of this research was to investigate the expression and cellular location of β-1,...Background There are few reports of a biological role for glycosyltransferases in the infiltration of osteoarthritic synovitis. The aim of this research was to investigate the expression and cellular location of β-1,4-galactosyltransferase Ⅰ （β-1,4-GaIT-Ⅰ） in a surgically-induced rat model of knee osteoarthritis （OA）, and explore the role of β-1,4-GalT-Ⅰ in the pathogenesis of OA.Methods Male Sprague-Dawley rats were randomly divided into three groups： OA group, sham group and normal group. The model of OA was established in the right knees of rats by anterior cruciate ligament transaction （ACLT） with partial medial meniscectomy. Fibroblast-like synoviocytes （FLSs） obtained from normal rat synovial tissue were cultured.The expression of β-1,4-GalT-Ⅰ mRNA in the synovial tissue, articular cartilage and FLSs treated with tumor necrosis factor-α （TNF-α） were assayed by real-time PCR. Western-blotting and immunohistochemisty were used to observe the expression of β-1,4-GalT-Ⅰ at the protein level. Double immunofluorescent staining was used to define the location of the β-1,4-GalT-Ⅰ with macrophage-like synoviocytes, FLSs, neutrophils, and TNF-α in the OA synovium. The alteration of TNF-α in FLSs which were treated with lipopolysaccharide （LPS） and β-1,4-GalT-Ⅰ-Ab were detected by enzyme-linked immunosorbent assay （ELISA）.Results The mRNA and protein expression of β-1,4-GalT-Ⅰ increased in synovial tissue of the OA group compared with the normal and sham groups at two and four weeks after the surgery, however, no significant difference appeared in the articular cartilage. Immunohistochemistry also indicated that the β-1,4-GalT-Ⅰ expression in OA synovium at four weeks after surgery increased sharply compared with the control group. β-1,4-GalT-Ⅰ co-localized with macrophage-like synoviocytes, FLSa, neutrophils and TNF-α in rat OA synovitis. Moreover, in vitro β-1,4-GalT-Ⅰ mRNA in FLSs was affected in a dose- and time-dependent manner in response to TNF展开更多
Background： Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome lq-l...Background： Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome lq-linked open-angle glaucoma （GLC1A） in a Chinese family with primary open-angle glaucoma （POAG）. Methods： A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Results： Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene （MYOC）. Furthermore, we also identified a novel mutation c.G322A in beta- 1,4-galactosyltransferase 3 （B4GALT3） gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. Conclusion： The mutations c.1456C〈T （p.L486F） in MYOC and c.322G〈A （p.V1081） in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.展开更多
By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyl-tr...By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyl-transferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids) . In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I) , the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-Hl) . Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3 .展开更多
b-1,4-galactosyltransferase (b4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of b4Gal-T genes have been cloned, separated from mouse, chick, bovine...b-1,4-galactosyltransferase (b4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of b4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse b4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopro-pyl-b-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse b4Gal-T. The transcriptional product of b4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.展开更多
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30300099 and No. 30770488）, Natural Science Foundation of Jiangsu Province （No. BK2003035 and No. BK2006547）, College and University Natural Scientific Research Program of Jiangsu Province （No. 03KJB180109）, ＂Ke-Jiao-Xing-Wei-Zhong-Dian-Yi-Xue-Ren-Cai＂ Financial Assistance of Jiangsu Province （No. RC2007087）.
文摘Background There are few reports of a biological role for glycosyltransferases in the infiltration of osteoarthritic synovitis. The aim of this research was to investigate the expression and cellular location of β-1,4-galactosyltransferase Ⅰ （β-1,4-GaIT-Ⅰ） in a surgically-induced rat model of knee osteoarthritis （OA）, and explore the role of β-1,4-GalT-Ⅰ in the pathogenesis of OA.Methods Male Sprague-Dawley rats were randomly divided into three groups： OA group, sham group and normal group. The model of OA was established in the right knees of rats by anterior cruciate ligament transaction （ACLT） with partial medial meniscectomy. Fibroblast-like synoviocytes （FLSs） obtained from normal rat synovial tissue were cultured.The expression of β-1,4-GalT-Ⅰ mRNA in the synovial tissue, articular cartilage and FLSs treated with tumor necrosis factor-α （TNF-α） were assayed by real-time PCR. Western-blotting and immunohistochemisty were used to observe the expression of β-1,4-GalT-Ⅰ at the protein level. Double immunofluorescent staining was used to define the location of the β-1,4-GalT-Ⅰ with macrophage-like synoviocytes, FLSs, neutrophils, and TNF-α in the OA synovium. The alteration of TNF-α in FLSs which were treated with lipopolysaccharide （LPS） and β-1,4-GalT-Ⅰ-Ab were detected by enzyme-linked immunosorbent assay （ELISA）.Results The mRNA and protein expression of β-1,4-GalT-Ⅰ increased in synovial tissue of the OA group compared with the normal and sham groups at two and four weeks after the surgery, however, no significant difference appeared in the articular cartilage. Immunohistochemistry also indicated that the β-1,4-GalT-Ⅰ expression in OA synovium at four weeks after surgery increased sharply compared with the control group. β-1,4-GalT-Ⅰ co-localized with macrophage-like synoviocytes, FLSa, neutrophils and TNF-α in rat OA synovitis. Moreover, in vitro β-1,4-GalT-Ⅰ mRNA in FLSs was affected in a dose- and time-dependent manner in response to TNF
文摘Background： Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome lq-linked open-angle glaucoma （GLC1A） in a Chinese family with primary open-angle glaucoma （POAG）. Methods： A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Results： Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene （MYOC）. Furthermore, we also identified a novel mutation c.G322A in beta- 1,4-galactosyltransferase 3 （B4GALT3） gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. Conclusion： The mutations c.1456C〈T （p.L486F） in MYOC and c.322G〈A （p.V1081） in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.
基金Project supported by the 863 High-technology Programthe National Outstanding Young Scientist Foundationthe National Natural Science Foundation of China (Grant No. 39680019)
文摘By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyl-transferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids) . In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I) , the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-Hl) . Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3 .
文摘b-1,4-galactosyltransferase (b4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of b4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse b4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopro-pyl-b-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse b4Gal-T. The transcriptional product of b4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.
