[Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I].

OBJECTIVE: To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I. METHODS: Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system. RESULTS: A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls. CONCLUSION: The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

Increased expression of hLRH-1 in human gastric cancer and its implication in tumorigenesis.

Altered signaling pathways or deregulated transcription factors represent an important category of molecular events leading to aberrant gene regulation in gastric cancer, among which the role of WNT/beta-catenin pathway remains unclear. LRH-1 is a critical transcription factor in controlling cell proliferation via crosstalk with the beta-catenin signaling pathway. In order to gain a knowledge of the expression of hLRH-1v1 and hLRH-1 in gastric cancer, a Q-PCR analysis was carried out. Our results showed that in about 50 and 47.6% of 42 tested patients with gastric cancer, the mRNA expression of hLRH-1v1 and hLRH-1 was significantly upregulated, as compared with self-paired normal control, respectively. Besides, overexpression of hLRH-1 was shown to promote the proliferation of gastric adenocarcinoma cell SGC-7901 via induction of cyclin E1. Taken together, our present study demonstrated for the first time the increased expression of hLRH-1v1 and hLRH-1 in human gastric cancer, an alteration which may implicate in tumorigenesis.

[Prokaryotic expression and characterization of N-terminal truncated glycoprotein gp85 of Epstein-Barr virus].

AIM: To construct a prokaryotic recombinant vector of Epstein-Barr virus (EBV) membrane protein gp85, to express the protein in E.coli and characterize the antigenicity of this non-glycosylated protein. METHODS: The BXLF2 gene coding 5'-terminal truncated of EBV gp85 was amplified from the EBV strain B95-8 cell line with specific primers. After identification by the restriction digestion with Hind III and Xho I, the PCR product was inserted into the prokaryotic expression plasmid pGEX-5T and confirmed by sequencing. The constructed prokaryotic expression vector pGEX5T-85N was transformed into the competent E.coli BL21. The expressed recombinant protein gp85N was purified by affinity chromatography, characterized by Western blot, and used immunize BALB/c mice. The titer of antiserum from the immunized mice was detected by ELISA. RESULTS: Sequencing analysis revealed that the obtained truncated BXLF2 gene was identical to that published in GenBank and successfully cloned into pGEX-5T. SDS-PAGE showed that the expressed recombinant protein was partially soluble with a relative molecular mass of 45,000. ELISA results indicated that the expressed gp85N was recognized by that the anti-gp85 mAb and contiserum with high titer was obtained from the immunized mice. CONCLUSION: The obtained recombinant gp85N with an excellent antigenicity should provide preliminary data for characterization of the antibody produced by the immunized mice.

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.

[Expression of matrix metalloproteinases-9 and tissue inhibitors of matrix metalloproteinases-1 in connective tissue of vaginal wall of women with stress urinary incontinence].

OBJECTIVE: To study semi-quantitatively mRNA expression of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), in vaginal wall connective tissue in women with stress urinary incontinence (SUI) compared to continent controls, and to explore the relationship between MMP-9, TIMP-1 and SUI. METHODS: Vaginal wall tissues were obtained from 24 women with SUI who were followed-up (12 cases are > 60 years old and 12 cases < or = 60 years old). Seven patients undergoing total hysterectomy for carcinoma in situ of cervix without urinary incontinence served as control group. RNA was extracted and quantified. Semi-quantitative competitive reverse transcription was carried out with oligo-nucleotide primers to quantify MMP-9 and TIMP-1 mRNA expression. RESULTS: We used GeneSnap to analyze the data. MMP-9 in three groups (> 60, < or = 60 years and control) was 0.56 +/- 0.20, 0.56 +/- 0.19, 0.37 +/- 0.18, significantly decreased (P < 0.05). There was no difference between > 60 and < or = 60 year age groups (P > 0.05). TIMP-1 in three groups was 0.23 +/- 0.11, 0.31 +/- 0.12, 0.41 +/- 0.13, significantly increased (P < 0.05). There was a great difference between > 60 and < or = 60 year age groups in TIMP expression (P > 0.05). The ratio of MMP-9/TIMP-1 in > 60, < or = 60 year age groups and control group was 2.49 +/- 1.82, 1.82 +/- 1.58, 0.90 +/- 1.38, significantly decreased (P < 0.05). CONCLUSION: Stress urinary incontinent women demonstrate a significant increase in MMP-9 mRNA expression and significant decrease in TIMP-1 mRNA expression. In SUI patients, proportion of MMP-9 and TIMP-1 was overbalanced. Both these findings are consistent with increased collagen breakdown and may play an important role in the onset and development of SUI.

[Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris].

AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.