Human PIH1 associates with histone H4 to mediate the glucose-dependent enhancement of pre-rRNA synthesis.

Ribosome biogenesis is critical in the growth of eukaryotic cells, in which the synthesis of precursor ribosomal RNA is the first and rate-limiting step. Here, we show that human PIH1 domain-containing protein 1 (PIH1) interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex via SNF5 to human rRNA genes. This process is likely involved in PIH1-dependent DNase I-hypersensitive chromatin remodeling at the core promoter of the rRNA genes. PIH1 mediates the occupancy of not only the Brg1 complex but also the Pol I complex at the core promoter and enhances transcription initiation of rRNA genes. Additionally, the interaction between PIH1 and H4K16 expels TIP5, a component of the silencing nucleolar remodeling complex (NoRC), from the core region, suggesting that PIH1 is involved in the derepression of NoRC-silenced rRNA genes. These data indicate that PIH1 is a positive regulator of human rRNA genes and is of great importance for the recovery of human cells from nutrient starvation and the transition to glucose-induced exponential growth in vivo.

The association between chronic periodontitis and hypertension in rural adult Uygur residents / 中华心血管病杂志

<p><b>OBJECTIVE</b>To explore the association between chronic periodontitis and hypertension in rural adult Uygur residents.</p><p><b>METHODS</b>A total of 1415 Uygur residents aged 18 and over were selected by random multistage and probability proportional to size from 364 villages in Moyu county of Xinjiang Uygur autonomous region, all subjects received questionnaire, physical examination and biochemical analysis and oral examination. The subjects were categorized as periodontitis group and no periodontitis group, the periodontitis group was further categorized as mild, moderate and severe periodontitis subgroup. The relationship between chronic periodontitis with hypertension was analyzed by Spearman correlation. Binary logistic regression was used to calculate the influential factors for hypertension.</p><p><b>RESULTS</b>The prevalence rates of chronic periodontitis and hypertension were 66.0% (934/1415) and 33.8% (478/1415), respectively. The prevalence rates of hypertension were 18.7% (90/481), 35.1% (131/373), 32.3% (62/192), 52.8% (195/369) in no periodontitis, mild, moderate and severe periodontitis groups, respectively. Spearman correlation showed an association of chronic periodontitis with hypertension (r(s) = 0.273, P < 0.01). After adjustment for age, gender, body mass index, waist circumference, glycometabolism disorder, hyperlipidemia, chronic kidney disease, multiple logistic regression analysis showed that periodontitis was significantly associated with hypertension (OR = 1.75, 95%CI: 1.30 - 2.36, P < 0.01). Compared with no periodontitis, mild (OR = 1.76, 95%CI: 1.26 - 2.48, P < 0.01) and severe (OR = 2.26, 95%CI: 1.57 - 3.26, P < 0.01) periodontitis were significantly associated with hypertension while moderate periodontitis was not significantly associated with hypertension (OR = 1.21, 95%CI: 0.80 - 1.84, P > 0.05).</p><p><b>CONCLUSION</b>This study showed an independent association of periodontitis with hypertension in this study cohort.</p>

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

OBJECTIVE: To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). METHODS: HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. RESULTS: Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. CONCLUSION: The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

[Fusion expression, purification and bioactivity detection of human defensinalpha (HDalpha)].

AIM: To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research. METHODS: The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection. RESULTS: After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria. CONCLUSION: The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.

Construction and activity assay of the activating transcription factor 3 reporter vector pATF/CRE-luc.

Activating transcription factor 3 (ATF3), a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. To investigate the activity of ATF/CREB in cells with physiological stresses, we developed a practical reporter vector, the plasmid pATF/CRE-luc, bearing activating transcription factor/cAMP responsive element (ATF/CRE) binding sites. This plasmid was constructed by inserting three repeats of the ATF/CRE binding element into the plasmid pG5luc, replacing the GAL-4 binding sites. The plasmids pACT/ATF3 and pATF/CRE-luc were transfected into HeLa and NIH3T3 cells, respectively, and the results showed that the expression of luciferase was increased in a dose-dependent manner on plasmid pACT/ATF3. The data suggested that the plasmid pATF/CRE-luc could be used as a sensitive and convenient reporter system of ATF3 activity.

[Cloning and expression of a novel gene restin and preparation of antisera against the recombinant restin].

AIM: To clone a new human gene, restin, from retinoic acid-treated promyelocytic cell line HL-60, express the protein in E.coli and prepare the antisera against the protein. METHODS: The restin gene was amplified from retinoic acid-treated promyelocytic cell line HL-60 by RT-PCR and cloned into a prokaryotic expression vector. The recombinant restin was induced to express in E. coli by temperature. After preliminary purification by SDS-PAGE, the restin protein was used to immunize rabbits to obtain antisera. The titers and specificity of the rabbit anti-restin antisera were tested by Western blot and immunofluorescence analysis. RESULTS: Recombinant restin with M(r) being about 26,000 was highly expressed in E. coli. The titers of the anti-sera to restin ranged from 1:100 to 1:800. Immunofluorescence analysis showed that restin distributed mainly in the nuclei of COS-7 cells. CONCLUSION: We successfully prepared the antisera against restin, which are useful for further investigation of biological functions of restin.

Cloning expression in E.coli and biological activity of human thymosin beta(4).

The cDNA thymosin beta(4) was synthesized by combining of chemical and enzymatic methods. First, two complement fragments of thymosin beta(4) cDNA were synthesized by DNA synthesizer, and then denatured, annealed and extended by DNA polymerase. This fragment of thymosin beta(4) was then inserted into the EcoRV and HindIII restriction endonuclease site of an expression plasmid pLDH4 (a kind of E.coli plasmid) by blunt and cohesive ligations. Finally, the recombinant plasmid which expressed thymosin beta(4) was screened by digestion and DNA sequencing. This recombinant plasmid highly expressed the thymosin beta(4), which accounted for 30% of total bacteria proteins. By salting out and chromatography, a 95% purity of recombinant thymosin beta(4) was obtained. Biological assay indicated that the recombinant thymosin beta(4) could induce lymphocyte proliferation and differentiation.

[High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding].

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.

Cloning and Optimized Expression of Human Angiogenin in E.coli.

Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli. By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure. The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99. After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE. Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro. Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.