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)

The effect of standard orthopaedic implant materials on osteoblast proliferation and differentiation was investigated using a human osteoblast cell culture system. Human fetal osteoblasts 1.19 were cultured on stainless steel, cobalt-chrome-molybdenum, and commercially pure titanium for 12 days. Tissue culture polystyrene was used as a control. Cell proliferation was measured by electronic cell counting and by a colorimetric proliferation assay. To assess the degree of differentiation, levels of alkaline phosphatase activity, collagen Type I, and osteocalcin production were measured. Osteocalcin gene expression was measured by reverse transcriptase-polymerase chain reaction. Electronic cell counting and proliferation assays showed lower cell numbers and delayed proliferation on stainless steel and cobalt-chrome-molybdenum compared with titanium and polystyrene. Alkaline phosphatase and osteocalcin were measured higher on titanium than on stainless steel or cobalt-chrome-molybdenum. Differences in collagen Type I production were not found. Reverse transcriptase-polymerase chain reaction showed the highest osteocalcin gene expression on titanium. The human fetal osteoblast cell line 1.19 provides a rapidly proliferating and differentiating system for testing biomaterials in which differences in osteoblast proliferation and differentiation on orthopaedic implant materials could be revealed, suggesting that the chemistry of biomaterials has a dynamic effect on proliferation and differentiation of human osteoblasts.
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PMID:Testing of skeletal implant surfaces with human fetal osteoblasts. 1179 45

We describe the use of non-traditional methods of probe synthesis and quantification and detection of hybridization that appreciably improved non-radioactive in situ hybridization (ISH) in human airway tissue. To avoid the problems of bacterial cloning, plasmid digestion, and probe hydrolysis, we synthesised complementary RNA probes (riboprobes) for ISH from PCR-generated DNA. DNA template was produced by nested PCR incorporation of T7 and SP6 RNA polymerase promoters. We then compared the efficiency of in vitro transcription from PCR-generated template with traditional plasmid template by quantifying the relative probe fluorescence in denaturing gels. Transcription with SP6 or T7 polymerase in either orientation produced TNF riboprobes from a single PCR-generated template more efficiently than from plasmid, providing there were no primer hairpin loops. Fluorescence quantification enabled equal amounts of probe label to be used in ISH, eliminating signals from the sense probe and demonstrating that probes transcribed from PCR templates were as sensitive as hydrolyzed probe transcribed from plasmid. Detection of ISH by a conventional anti-hapten, alkaline phosphatase-based technique was found to cause tissue damage due to extended substrate incubation at high pH. We therefore developed a four-layer, avidin-biotin-peroxidase technique that afforded greater sensitivity, allowing brief substrate incubation and resulting in structural preservation of tissue.
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PMID:Improvement of non-radioactive in situ hybridization in human airway tissues: use of PCR-generated templates for synthesis of probes and an antibody sandwich technique for detection of hybridization. 1189 7

Two subforms of RNA polymerase III-IIIa and IIIb--were identified in human placenta nuclei. These subforms differed in molecular weight of one subunit, and in buoyant density in glycerol concentration gradient. Protein kinase activity, which phosphorylates at least four subunits of RNA polymerase IIIa and three subunits of RNA polymerase IIIb in vitro, was copurified with both the subforms. Protein kinase activity was inhibited by wortmannin, a specific PI3-kinase inhibitor. RNA polymerase III dephosphorylation by alkaline phosphatase in vitro decrease the transcription level on specific Alu-template. The associated protein kinase was not able to phosphorylate dephosphorylated RNA polymerase IIIa and to restore the transcription level up to the control one.
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PMID:[Phosphorylation and dephosphorylation of rna polymerase III holoenzyme are modifications regulating the level of transcription in vitro]. 1209 66

Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the result of enhanced adipogenesis and decreased osteoblastogenesis from the MSCs. Thus, cultures of MSCs were established from young donors (age 18-42, n = 34), elderly healthy donors (age 66-78, n = 20), and patients with OP (age 58-76, n = 15). Cells were cultured for 2 weeks in an adipogenic medium (containing 15% horse serum and 100 nM dexamethasone), osteogenic medium (containing 10% fetal calf serum [FCS] and 10 nM calcitriol), or control medium (10% FCS). The MSCs were identified by their abilities to form colonies. Total number of colonies, osteoblastic colonies stained positive for alkaline phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated. In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR). The adipogenic and osteogenic media induced cell differentiation and the expression of adipocytic and osteoblastic lineage-specific markers, respectively. We found no age-related changes in the osteoblastic or adipocytic colony formation or the steady state levels of mRNA of the adipogenic or osteogenic gene markers. Cells obtained from patients with OP showed a pattern of differentiation similar to those of age-matched controls. In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP. Other mechanisms responsible for age-related decrease in bone formation need to be determined.
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PMID:Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures. 1220 Jun 57

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.
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PMID:Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute. 1265 9

The purpose of this investigation was to examine by reverse-transcriptase polymerase chain reaction analysis the osteogenic differentiation of twice-passaged Sprague-Dawley rat bone marrow stromal cells in type I collagen gel cultured for 3 weeks. Two culture media were used here, namely Dulbecco's modified Eagle (DME) medium supplemented with vitamin C [Dex (-)] and those with vitamin C, dexamethasone and beta-glycerophosphate [Dex (+)]. Culture with Dex (-) medium in collagen gel for 3 weeks brought about the well-developed cell network and middle-stage osteogenic phenotype expression characterized by mRNA for alkaline phosphatase, osteonectin and osteopontin while those for bone sialo protein and osteocalcin were not detected. On the contrary, culture with Dex (+) medium in collagen gel for 3 weeks lead to necrosis of the cells. These results indicate that culture in collagen gel with Dex (-) DME medium containing vitamin C was useful for three-dimensional culture and middle-stage osteogenic differentiation of twice-passaged bone marrow stromal cells. This study might contribute to tissue engineering therapy to fix bone and periodontal defects in the future.
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PMID:Studies on osteogenic differentiation of rat bone marrow stromal cells cultured in type I collagen gel by RT-PCR analysis. 1288 Apr 3

8,5'-cyclopurine-2'-deoxynucleosides in DNA are repaired by nucleotide-excision repair, and act as strong blocks to DNA polymerases, RNA polymerase II and transcription factor binding. Thus, it is important to accurately determine the level of these lesions in DNA. There is controversy in the literature regarding the ability of different enzymes to release these compounds from oligodeoxynucleotides or DNA. We used liquid chromatography/mass spectrometry (LC/MS) to investigate the ability of several enzymes to release (5'S)-8,5'-cyclo-2'-deoxyadenosine [(5'S)-cdA] from dinucleotides and oligodeoxynucleotides and from DNA. The data show that (5'S)-cdA is completely released from DNA by hydrolysis with nuclease P1, snake venom phosphodiesterase and alkaline phosphatase. The identity of the normal nucleoside 5' to the (5'S)-cdA had a significant effect on its release. Using LC/MS, we also showed that the levels of (5'S)-cdA were within an order of magnitude of those of 8-hydroxy-2'-deoxyguanosine, and three times higher than those of 8-hydroxy-2'-deoxyadenosine in pig liver DNA. Different DNA isolation methods affected the levels of the latter two lesions, but did not influence those of (5'S)-cdA. We conclude that (5'S)-cdA can be completely released from DNA by enzymic hydrolysis, and the level of (5'S)-cdA in tissue DNA is comparable to those of other oxidatively induced DNA lesions.
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PMID:Complete release of (5'S)-8,5'-cyclo-2'-deoxyadenosine from dinucleotides, oligodeoxynucleotides and DNA, and direct comparison of its levels in cellular DNA with other oxidatively induced DNA lesions. 1521 37

The RNA polymerase core associated with sigma(S) transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by sigma(D)-associated RNA polymerase. A mutation in the gene that encodes sigma(S), rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of sigma(S) inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by sigma(S). In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by sigma(S). The effect of sigma(S) on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position -13 is important for the positive effect of sigma(S) on pst. The interpretation of these observations is based on the competition between sigma(S) and sigma(D) for the binding to the core RNA polymerase.
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PMID:A differential effect of sigmaS on the expression of the PHO regulon genes of Escherichia coli. 1534 56

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.
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PMID:Homogeneous fluorescent assay for RNA polymerase. 1595 Jan 66

The orphan nuclear hormone receptor estrogen-related receptor alpha (ERRalpha, NR3B1) is a constitutive transcription factor that is structurally and functionally related to the classic estrogen receptors. ERRalpha can recognize both the estrogen response element and its own binding site (ERRE) in either dimeric or monomeric forms. ERRalpha is also a phosphoprotein whose expression in human breast tumors correlates with that of the receptor tyrosine kinase ErbB2, suggesting that its transcriptional activity could be regulated by signaling cascades. Here, we investigated growth factor regulation of ERRalpha function and found that it is phosphorylated in MCF-7 breast cancer cells in response to epidermal growth factor (EGF), an event that enhances its DNA binding. Interestingly, treatment with alkaline phosphatase shifts ERRalpha from a dimeric to a monomeric DNA-binding factor, and only the dimeric form interacts with the coactivator PGC-1alpha. In vitro, the DNA-binding domain of ERRalpha is selectively phosphorylated by protein kinase Cdelta (PKCdelta), which increases its DNA-binding activity, whereas expression of constitutively active PKCdelta enhances TFF1 promoter activity via the ERRE. However, whereas treatment of MCF-7 cells with the phorbol ester phorbol-12-myristate 13-acetate also enhances ERRalpha activation of the TFF1 promoter reporter, it does not affect ERRalpha activity on its own promoter. In agreement, chromatin immunoprecipitation analysis shows that ERRalpha and RNA polymerase II are preferentially recruited to the TFF1 promoter after EGF treatment, whereas recruitment of these factors to its own promoter is not affected. These results reveal a mechanism through which growth factor signaling can selectively activate ERRalpha target genes in breast cancer cells.
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PMID:Epidermal growth factor-induced signaling in breast cancer cells results in selective target gene activation by orphan nuclear receptor estrogen-related receptor alpha. 1602 13


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