17ß-Oestradiol Attenuates the Photoreceptor Apoptosis in Mice with Retinitis Pigmentosa by Regulating N-myc Downstream Regulated Gene 2 Expression.

Retinitis pigmentosa (RP) is a heterogeneous group of retinal degenerative diseases in which the final pathological feature is photoreceptor cell apoptosis. Currently, the pathogenesis of RP remains poorly understood and therapeutics are ineffective. 17ß-Oestradiol (ßE2) is universally acknowledged as a neuroprotective factor in neurodegenerative diseases and has manifested neuroprotective effects in a light-induced retinal degeneration model. Recently, we identified N-myc downstream regulated gene 2 (NDRG2) suppression as a molecular marker of mouse retinal photoreceptor-specific cell death. ßE2 has also been reported to regulate NDRG2 in salivary acinar cells. Therefore, in this study, we investigated whether ßE2 plays a protective role in RP and regulates NDRG2 in photoreceptor cells. To this end, we generated RP models and observed that ßE2 not only reduced the apoptosis of photoreceptor cells, but also restored the level of NDRG2 expression in RP models. Then, we showed that siNDRG2 inhibits the anti-apoptotic effect of ßE2 on photoreceptor cells in a cellular RP model. Subsequently, we used a classic oestrogen receptor (ER) antagonist to attenuate the effects of ßE2, suggesting that ßE2 exerted its effects on RP models via the classic ERs. In addition, we performed a bioinformatics analysis, and the results indicated that the reported oestrogen response element (ERE) sequence is present in the promoter region of the mouse NDRG2 gene. Overall, our results suggest that ßE2 attenuated the apoptosis of photoreceptor cells in RP models by maintaining NDRG2 expression via a classic ER-mediated mechanism.

Expression profiling of long noncoding RNAs and the dynamic changes of lncRNA-NR024118 and Cdkn1c in angiotensin II-treated cardiac fibroblasts.

A growing body of evidence shows that long non-coding RNAs (lncRNAs) are involved in multiple human diseases than previously realized. However, no information is available now about lncRNAs in cardiac fibroblasts. The expression profile of lncRNAs was analyzed in Ang II-treated cardiac fibroblasts using lncRNAs arrays. The analysis showed that 282 of 4376 detected lncRNAs demonstrated >2-fold differential expression in response to the treatment with Ang II (100 nm) for 24 h. Among of them, 22 lncRNAs showed a greater than 4-fold changes. Meanwhile, Ang II also induced a widely expression changes in protein-coding genes in cardiac fibroblasts. Quantitative real time PCR confirmed the changes of six lncRNAs (AF159100, BC086588, MRNR026574, MRAK134679, NR024118, AX765700) and mRNAs (IL6, RGS2, PRG4, TIMP1, Cdkn1c, TIMP3, Col I, Col III and Fibronectin) in cardiac fibroblasts. Bioinformatic analysis indicated the process of cell proliferation. Further studies revealed that the down-regulating of Ang II on the expression of lncRNA-NR024118 was time-dependent, that the level of NR024118 was lowest at 24 h and back at 48 h. Ang II also dynamically down regulated the expression of Cdkn1c in cardiac fibroblasts. Ang II at a range from 10(-9) M to 10(-6) M induced a decrease of NR024118 and Cdkn1c in cardiac fibroblasts. In conclusion, the expression profile of lncRNAs was significantly altered in the Ang II-treated cardiac fibroblasts and Ang II dynamically regulated the expression of lncRNA-NR024118 and Cdkn1c in cardiac fibroblasts, indicating the potential role of NR024118 in cardiac fibroblasts.

[A family-based association study of the EGR3 gene polymorphisms and schizophrenia].

Previous studies showed that EGR3 gene located in chromosome 8p21.3 was involved in the etiology of schizophrenia. However, the finding failed to be replicated in several case-control studies. To investigate the genetic role of the EGR3 gene in Chinese psychiatric patients, we genotyped five single nucleotide polymorphisms (SNPs) in EGR3 gene locus using 93 nuclear families in Han Chinese, and performed transmission disequilibrium test (TDT). In this study, two SNPs (rs1996147 and rs3750192) showed significant association with schizophrenia (c2>4.40, P<0.05). In the linkage disequilibrium analysis, the significant association was also found in two- (rs3750192-rs35201266), three- (rs1877670- rs3750192-rs7009708) and four-SNP (rs1996147-rs1877670-rs3750192-rs7009708) tests of haplotype analyses (c2>7.10, global P<0.05). Overall, the results suggested that EGR3 gene may play an important role in schizophrenia susceptibility in the Han Chinese population, and further functional exploration of the EGR3 gene will contribute to the underlying molecular mechanism for schizophrenia pathogenesis.

[Construction and expression of recombinant adenovirus containing human catalase gene in vitro].

OBJECTIVE: To construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro. METHODS: Total RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods. RESULT: High expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity. CONCLUSION: Ad/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

[Expression of ghrelin and its receptor GHS-R in the hypothalamus and gastrointestinal tract in rats with chronic renal failure].

OBJECTIVE: To investigate the expression of ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), in the hypothalamus and gastrointestinal tract in rats with chronic renal failure (CRF) and explore their relationship with the disorder of gastrointestinal tract motility. METHODS: SD rats were randomly divided into sham-operated group (n=8) and CRF group (n=16), and in the latter group, the rats were subjected to 5/6 nephrectomy to induce CRF. Real-time PCR and immunohistochemical staining were used to detect the distribution of mRNA and protein of ghrelin and GHS-R in the gastric fundus, duodenum, and hypothalamus. RESULTS: The rats in the CRF group showed a significantly higher expression of ghrelin mRNA and protein in the gastric fundus but a lower expression in the hypothalamus than those in the sham-operated group (P<0.01), but the expression in the duodenum was similar between the two groups (P>0.05). The expression of GHS-R mRNA and protein in the gastric fundus was significantly higher in the CRF group than in the sham-operated group (P<0.01), while in the hypothalamus and duodenum, the expression was significantly lower in the CRF group (P<0.01). CONCLUSION: The different distribution patterns of ghrelin and GHS-R in the tissues may be an important pathological basis of gastrointestinal motility disorder in CRF.

Analysis of correlation between blood biochemical indicators and bone mineral density of post-menopausal women.

Osteoporosis is a degenerative disease of the skeletal system, and its major complication is fracture that severely influences the living quality of the middle-aged and the aged. The purpose of this study was to investigate the significance of sex hormones and some biochemical indicators related to bone metabolism in the genesis and development of osteoporosis. The plasma samples were collected from 244 post-menopausal women of Xi'an urban area, and their plasma contents of testosterone, estradiol, calcitonin, osteocalcin and N-terminal propeptide of type I procollagen were detected by ELISA. The activity of tartrate-resistant acid phosphatase was determined by spectrophotometric method, and the content of nitric oxide was measured by Griess method. Bone mineral density (BMD) in lumbar vertebrae (L1-L4) and hips was measured by QDR-2000 dual energy X-ray absorptiometry. The concentrations of the biochemical indicators were compared among the three groups (normal bone mass group, osteopenia group and osteoporosis group), and Pearson correlation analysis was used to verify the correlations between the indicators and BMD. The comparison results of blood biochemical indicators of BMD-based groups showed that the plasma contents of estradiol (P = 0.006), testosterone (P = 0.038) and calcitonin (P = 0.042) decreased more significantly in the osteoporosis group, but the content of osteocalcin (P = 0.008) increased significantly in osteoporosis group than those in the other groups. The correlation analysis between BMD of different parts and the blood biochemical indicators showed that there was a significant positive correlation between estradiol and the BMD of lumber vertebra (r = 0.200, P = 0.002), femoral neck (r = 0.160, P = 0.013), and great trochanter (r = 0.204, P = 0.001). Significant positive correlations between calcitonin and BMD of lumber vertebra (r = 0.166, P = 0.018) and femoral great trochanter (r = 0.152, P = 0.041), and between testosterone and BMD of femoral great trochanter (r = 0.158, P = 0.014) were also observed. In addition, there existed significant negative correlations between osteocalcin and BMD of lumber vertebra (r = -0.220, P = 0.001), femoral neck (r = -0.259, P < 0.000), and great trochanter (r = -0.221, P = 0.001), and between the activity of tartrate-resistant acid phosphatase and BMD of femoral great trochanter (r = -0.135, P = 0.037). The partial correlation analysis also showed that there were significant correlations between estradiol (r = 0.160, P = 0.014), calcitonin (r = 0.240, P = 0.013), osteocalcin (r = -0.226, P = 0.023) and BMD when the influence of age was excluded. The Pearson correlation analysis of biochemical indicators showed there were positive correlations between the contents of testosterone and calcitonin, testosterone and osteocalcin, calcitonin and osteocalcin, calcitonin and PINP, calcitonin and NO, osteocalcin and NO, and PINP and NO, but negative correlations between the contents of testosterone and PINP, estradiol and calcitonin, estradiol and osteocalcin, and estradiol and NO. The blood contents of sex hormones and calcitonin significantly influence BMD and osteoporosis development, and the increase of osteocalcin contents could be used as a biomarker to indicate the degree of osteoporosis in post-menopausal women.

[Expression of protein arginine N-methyltransferases in E3 rat models of acute asthma].

OBJECTIVE: To observe the expression of protein arginine N-methyltransferase (PRMT) genes in the lung and spleen of E3 rats with acute asthma. METHODS: E3 rats with ovalbumin-induced pulmonary inflammation were divided into two groups (n=10), and the validity of the acute asthma model was evaluated by histological observation with HE and PAS staining and by measurement of NO production. Semi-quantitative RT-PCR was employed to detect the expressions of PRMT1-PRMT6 genes in the lung and spleen tissues of the rats. RESULTS: In the lung tissue of the asthmatic rats, the gene expressions of PRMT1 (P<0.01), PRMT2 (P<0.01), PRMT3 (P<0.05) and PRMT5 (P<0.05) were significantly increased, but the expression of PRMT4 gene (P<0.05) was significantly decreased as compared with those in the control tissue. In the spleen tissue of the asthmatic rats, the expressions of PRMT2 (P<0.05) and PRMT5 genes (P<0.05) showed a significant increase as compared with those in the control rat tissue. CONCLUSION: The gene expressions of PRMTs vary significantly between asthmatic rats and control rats, suggesting that PRMTs play an important role in the post-translational modification process of asthma-related genes.

[Identification of differentially expressed genes in rat asthma model by suppression subtractive hybridization technology].

OBJECTIVE: To identify differentially expressed genes related to asthma by using a rat model. METHODS: Total RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI). RESULTS: There were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments. CONCLUSION: The forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.

Evidence for transmission disequilibrium at the DAOA gene locus in a schizophrenia family sample.

Recently, the DAOA gene locus on chromosome 13q32-q34 has been implicated in the etiology of schizophrenia. We genotyped three single-nucleotide polymorphisms (SNPs: rs778294, rs779293 and rs3918342) in this region in 126 Chinese family trios. In this study, we have identified statistically significant transmission disequilibrium in two markers rs778293 (P=0.01) and rs3918342 (P=0.02), and a highly significant under-transmission between haplotype CAT (P=0.0005) and schizophrenia. The results provide further evidence to support that DAOA gene locus is involved in conferring susceptibility to schizophrenia.

[KCNE2 protein S98 phosphorylation in heart of old SHR rats detected by point mutagenesis].

OBJECTIVE: To investigate the phosphorylation of KCNE2 protein in heart of old SHR rats. METHODS: The membrane proteins from ventricular myocardium of old SHR were extracted, treated with or without alkaline phosphatase and tested binding with Ab2 (an anti-KCNE2 polyclonal antibody) by Western blot. A KCNE2 fusion protein with c-myc was obtained from in vitro translation system and treated with or without alkaline phosphatase. A series of mono- and double-point mutated fusion KCNE2 proteins with c-myc were obtained from an in vitro translation system, and Western blots with Ab2 or anti-myc antibody were performed. RESULTS: After alkaline phosphatase treatment, Ab2 significantly attenuated its binding with KCNE2. In vitro translation system confirmed that after alkaline phosphatase treatment, Ab2 weakened binding ability to KCNE2 while binding to c-myc was not changed. Point mutation experiments showed that serine residue in position 98 of KCNE2 proteins might be phosphorylated. CONCLUSION: KCNE2 protein in heart of old SHR rats is phosphorylated and this phosphorylation takes place in serine residue of position 98.