[Identification and Molecular Biology of Variant D Blood Group of RHD*95A Genotype].

OBJECTIVE: To explore the molecular biology of D variant blood group with RHD*95A genotype and the genetic mechanism of its generation. METHODS: A total of 6 samples from 3 generations of a family were analyzed. RHD blood group was identified by saline test tube and microcolumn gel card method. 10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and analyzed by direct sequencing. Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein. RESULTS: The proband was identified as D variant by serological identification, RHD gene sequencing directly detected a c. 95 c > A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr (T) into aspartic acid Asn (N), the rest of the exon sequences were normal compared with the normal RHD*01 gene. In the family, the proband's father, grandmather and uncle were all carried the same RHD*95A allele. Protein modeling results suggested that the hydrogen chain connected to the 32nd amino acid residue was changed after p.T32N mutation, which affected the structural stability of RHD protein. CONCLUSION: The first genetic lineage of the RHD*95A gene was identified in a Chinese population. The c.95C>A mutation in RHD gene was found in the family, which resulted in reduced expression of RHD antigen and showed D variant, the mutation could be stably inheritable. Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.

[Nitric oxide regulates c-fos expression in osteoblastic cells induced by wall-shear].

OBJECTIVE: To observe regulation of nitric oxide on c-fos expression in osteoblastic cells in response to changes in wall-shear stress in vitro. METHODS: Isolated and purified osteoblastic cells from the calvaria of newborn SD rats were cultured and passaged. The third generation cells, pre-treated with 10% FBS DMEM, 0.3 mmol/L L-NMMA DMEM and 0.1 mmol/L SNP DMEM separately, were subjected to wall-shear stress of 1.2 Pa. Gene expression of the c-fos and NOS activity were studied before (0 min) and 10 min, 15 min, 30 min, 60 min after treated with wall-shear stress. RESULT: The expression of c-fos mRNA was increased transiently after application of 1.2 Pa wall-shear stress in osteoblastic cells and peaked at 15 min. The expression of c-fos mRNA was decreased after pre-application with L-NMMA and increased after use of SNP. CONCLUSION: Changes in the osteoblastic cells mechanical environment may cause a dramatic induction of NO and c-fos expression.

Nitric oxide induces oral squamous cell carcinoma cells apoptosis with p53 accumulation.

Nitric oxide has been reported to have cytotoxic effects in several tumor cells. The objective of this study was to investigate the effects of exogenous nitric oxide on apopotosis in oral squamous cell carcinoma cells and to reveal its possible mechanism. Tca8113 cells were cultured with various concentrations of nitric oxide that were released from sodium nitroprusside (SNP). Nitrite/nitrate levels in the culture supernatant were determined using a commercial available nitric oxide kit. Cellular proliferation was determined by MTT assay. Apoptosis was detected by flow cytometry. Expression of inducible nitric oxide synthase (iNOS) was determined by immunocytochemistry. p53 expression was assessed by Western blot. SNP can release nitric oxide into the culture medium in a dose-dependent manner. Nitric oxide remarkably inhibits proliferation in a dose and time-dependent manners and lead to apoptosis of the Tca8113 cell. The p53 expression was elevated accompanying by the increased apoptotic cells. No difference of iNOS was found whether or not the cells were treated with SNP. Exogenous nitric oxide had an inhibitory effect on Tca8113 cells proliferation in a dose and time-dependent manners and possibly via p53 dependent apoptosis pathway. Exogenous nitric oxide had no significant effect on cellular iNOS protein.