Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro. Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT/Algipore), deproteinized bovine HA (Bio-Oss) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I. We found that cells cultured on C GRAFT/Algipore) and Pep Gen P-15 showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT/Algipore and Pep Gen P-15 in contrast to cells grown on Bio-Oss, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three materials throughout the whole culture period. The results of our in vitro study show that the differences in metabolic activity of cells grown on HA materials are directly related to the substrate on which they are grown. They confirm the excellent properties of HA carrying the cell binding peptide P-15 and HA calcified from red algae as used in maxillofacial surgery procedures.
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PMID:Invitro study of adherent mandibular osteoblast-like cells on carrier materials. 1605 76

One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.
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PMID:Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. 1621 Apr 10

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.
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PMID:Regulation of cementoblast gene expression by inorganic phosphate in vitro. 1646 74

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.
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PMID:Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro. 1646 78

We investigated gene expression patterns and functional classifications regarding the clusters of human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs)--which possess a multipotent ability--because little is known about the precise moleculobiological clues by which these cells activate their differentiating ability or functionality to eventually form dentin and bone, respectively. We first verified the expressions of the alkaline phosphatase (ALP) gene, dentin matrix protein 1 (DMP-1), and dentinsialophosphoprotein (DSPP) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and consequently discovered the high expressions of these genes. Total RNA was also followed by hybridization with a human microarray system consisting of 12,814 genes. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors, and transcription regulators. Functional and clustering analyses of differences in gene expression levels revealed cell signaling, cell communication, or cell metabolism. In the future, information on the gene expression patterns of hDPSCs and hMSCs might be useful in determining the detailed functional roles of the relevant genes and applicable to stem cell therapies, and these cells could also be used as multipotent cell sources for gene technology and tissue engineering technology.
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PMID:Cluster analysis and gene expression profiles: a cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy. 1656 96

To study the effects of Icariin on expression of osteopontin (OPN) mRNA and type I collagen in rat osteoblasts in vitro and to explore its possible mechanisms in preventing osteoporosis. OB was isolated from calvaria of new-born new-born fetal Sprague-Dawley (SD) rats by means of modified sequential collagenase digestion and incubated in MEM medium and the cell morphology was observed under inverted phase contrast microscope, OB was identified by alkaline phosphatase (ALP) staining. Different concentration (0.1 microg/mL, 1.0 microg/ml, 10 microg/mL) of Icariin was added to the OB and incubated. The effect of Icariin on the proliferation and osteogenesis of OB was monitored by MTT analysis. The expression of type I collagen was estimated with immunohistochemistry techniques. The expression levels of mRNA of OPN in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction (RT-PCR). The expression of OPN mRNA and type I collagen was strengthened gradually with the increase of Icariin concentration and peaked with 10 microg/mL Icariin on the 5th day. Icariin could significantly promote the expression of OPN mRNA and type I collagen in rat osteoblasts in vitro. The levels of expression of OPN mRNA and type I collagen were changed with different concentration of Icariin. Icariin could effectively prevent and treat osteoporosis and promote the bone formation.
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PMID:Effects of Icariin on expression of OPN mRNA and type I collagen in rat osteoblasts in vitro. 1669 27

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.
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PMID:BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. 1706 Mar 21

Noggin is a secreted protein that inhibits the binding of bone morphogenetic proteins (BMPs) to their cognate receptor. Its role in human mesenchymal stem cell differentiation has not been well studied. Here, we studied the effect of noggin on human mesenchymal stem cell differentiation induced by inflammatory cytokines (activated T-cell conditioned medium (ACTTCM) or the combination of four T-cell cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII)), BMPs, or dexamthasone (DEX). HMSC treated with TTII alone rapidly induced alkaline phosphatase (AlkP) activity. Inclusion of noggin resulted in an additive effect. Noggin acted additively with DEX to induce a significantly higher level of AlkP induction than either noggin or DEX alone. Noggin was examined for its ability to inhibit mineralization in long-term cultures of HMSC stimulated with BMP-2, BMP-6, BMP-7, DEX, or TTII. Surprisingly, noggin alone induced mineralization while it did not inhibit mineralization induced by TTII or BMP-2, BMP-6, or BMP-7. Interestingly, when HMSC were treated with both noggin and DEX they acted synergistically to induce mineralization nearly 3-fold over DEX alone and 30-fold over noggin alone. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that T-cell cytokines induced noggin, Runx2, BMP-2, and osteocalcin gene expression, while noggin alone induced BMP-2 and osteocalcin gene expression, but not Runx2, although it increased the expression of ActRII, a receptor for BMP-2. These results suggest that in HMSC, the anabolic action of inflammation on bone formation occurs through the induction of noggin, which then induces BMP-2 receptor and BMP-2 leading to the activation of the differentiation process.
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PMID:The role of noggin in human mesenchymal stem cell differentiation. 1713 53

sigma(S) is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of sigma(S) causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, sigma(70). phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by sigma(S). Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of sigma(S) in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpoS (+) and rpoS mutants, as well as between high and low-sigma(S) producers. Using this method, we provide evidence that sigma(S) contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in sigma(S) levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of sigma(S) on agar plates.
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PMID:Alkaline phosphatase as a reporter of sigma(S) levels and rpoS polymorphisms in different E. coli strains. 1766 71

Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of Streptomyces coelicolor by homology with the amino acid sequence obtained from the PhoA protein of Streptomyces griseus. PhoA and PhoC correspond to broad-spectrum alkaline phosphatases whereas PhoD is similar to a Ca(2+)-dependent phospholipase D of Streptomyces chromofuscus. The phoA and phoD genes were efficiently expressed in R5 medium under phosphate-limited conditions, as shown by studies using the xylE reporter gene, whereas phoC was poorly transcribed under the same conditions. Expression of phoA was clearly PhoP-dependent since it was not transcribed in the S. coelicolor DeltaphoP mutant and was strongly activated under low phosphate concentrations. Similarly, expression of phoD was PhoP-dependent and highly sensitive to phosphate availability. By contrast, expression of phoC was not PhoP-dependent. Electrophoretic mobility shift assays showed that PhoP binds to the phoA and phoD promoters, but not to that of phoC. Footprinting studies with GST-PhoP revealed the presence of a PHO box (two direct 11 nt repeats) in the phoA promoter and two PHO boxes in the promoter of phoD. The transcription start points of the three promoters were identified by primer extension. The transcription start point of phoD coincides with the G of its translation start codon, indicating that this gene is transcribed as a leaderless mRNA. The deduced -10 and -35 regions of phoD (but not those of phoA) overlapped with the PHO boxes in this promoter, suggesting that an excess of PhoP interferes with binding of the RNA polymerase to this promoter. In summary, the three promoters showed clear differences in the modulation of their expression by PhoP.
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PMID:Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions. 1790 50


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