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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) have been suggested to play a role in dental pulp destruction. This study examined the effects of interleukin (IL)-1 alpha on pulp fibroblasts. The ability of these cells to degrade collagen was determined with or without IL-1 alpha utilizing a cell-mediated collagen degradation assay. Reverse transcriptase-polymerase chain reaction was utilized to examine the mRNA expression of multiple MMPs and TIMPs with and without IL-1 alpha, while Western blot analyses and zymography were utilized to examine their protein expression. The collagen degradation mediated by these cells was stimulated by IL-1 alpha and inhibited by MMP inhibitors. IL-1 alpha increased the mRNA expression of MMP-1 and MMP-3, as well as induced MMP-7. Western blot analyses confirmed these results. IL-1 alpha increased the secreted protein level of TIMP-1, while only slightly affected the level of TIMP-2. These results suggest that IL-1 alpha can induce pulp destruction by differentially regulating MMPs and TIMPs.
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PMID:Interleukin-1 alpha alters the expression of matrix metalloproteinases and collagen degradation by pulp fibroblasts. 1650 Feb 23

We investigated the effect of transforming growthfactor beta (TGFbeta1) short hairpin RNA (shRNA) mediated by pcDU6 plasmid on TGFbeta1 expression in human peritoneal mesothelial cells (HPMCs) and compared that effect with the effect of antisense TGFbeta1 RNA. We designed two pairs of oligonucleotides for two selectedfragments of coding sequence containing a 21-nucleotide (nt) TGFbeta1 sequence starting with GGCC. After annealing, double-stranded DNA was formed and separately ligated to plasmid pcDU6 [pcDNA3.1(-) with U6 promoter). The inverted motif contained six spacers and four Ts, which made it possible to form shRNA (TGFbgeta1 shRNA1 and TGFbeta1 shRNA2). We generated recombinant human TGFbeta1 antisense mammalian expression vector, and we isolated HPMCs from human greater omentum by pancreatin disaggregation to establish a stable cell-culture model. We used Lipofectamine 2000 to transfect third-passage HPMCs with plasmid pcDU6 mediating the expression of TGFbeta1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFbeta1 messenger RNA (mRNA). The resulting transfected cells were then stimulated with 4.25% D-glucose and 10 microg/mL lipopolysaccharide (GS+LPS). We used semi-quantitative reverse-transcriptase polymerase chain reaction to detect the expression of TGFbeta1, fibronectin (FN), collagen 1, and plasminogen activator inhibitor type 1 (PAI-1) mRNA by the stimulated cells. The TGFbeta1, FN, and PAI-1 protein levels in the culture supernatant were measured with a sandwich enzyme-linked immunosorbent assay. Expression of TGFbeta1 was significantly upregulated in HPMCs stimulated with GS+LPS (p < 0.01). As compared with control HPMCs in serum-free F12 medium, HPMCs transfected with TGFbeta1 antisense RNA showed inhibited expression of FN, collagen 1, and PAI-1 mRNA (17%, 26%, and 9.6% respectively after 24 hours). Forty-eight hours after transfection, the FN and PAI-I proteins were inhibited by 54.55% and 61.13% respectively (p < 0.05). In the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups, TGFbeta1 expression was obviously downregulated as compared with the GS+LPS group and the pcDU6 void vector group (p < 0.01). No significant difference was observed between the pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups (p > 0.05). No significant difference was observed between the pcDNA3.1(-) vector-mediated antisense RNA group and the pcDU6 void vector group (p > 0.05). The expression of TGFbeta1 in pcDU6 plasmid vector-mediated TGFbeta1 shRNA groups was obviously downregulated as compared with the pcDNA3.1(-) plasmid vector-mediated antisense RNA group (p < 0.01). In HPMCs stimulated with GS+LPS, pcDU6 plasmid vector-mediated shRNA can significantly inhibit the induced expression of TGFbeta1. These results suggest the possible application of pcDU6 plasmid vector-mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.
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PMID:Inhibition of transforming growth factor beta (TGFbeta1) expression and extracellular matrix secretion in human peritoneal mesothelial cells by pcDU6 vector-mediated TGFbeta1 shRNA and by pcDNA3.1(-)-mediated antisense TGFbeta1 RNA. 1668 83

To analyze the molecular mechanisms of coronary vessel formation, we performed in vitro experiments on explant cultures of proepicardial organs (PEOs) excised from embryos taken from 9.5-day pregnant mice. When plated on coverglasses coated with rat tail collagen I, fibronectin, or laminin, PEO cells spread and formed an epithelial sheet. When PEOs were cultured on collagen gel in the presence of fetal calf serum (FCS), small projections were seen around the explants 3 days after plating. Around day 6, cord-like structures began to grow from the explants, gradually elongating, increasing in number, and forming a branching network. Histological sections demonstrated that the cells migrated into the gel and formed tube-like structures similar to the vascular channels of the embryonic heart. The cells lining the lumen of the tube-like structures were positive for platelet endothelial cell adhesion molecule (PECAM). Reverse transcriptase-polymerase chain reaction analyses demonstrated that the expression of PECAM, basic fibroblast growth factor (bFGF), and smooth muscle 22-alpha (SM22alpha) was upregulated in association with the tube formation, whereas the expression of Flk-1, Flt-1, and hepatocyte growth factor (HGF) was gradually downregulated. Vascular endothelial growth factor (VEGF) was continuously expressed during the culture. These changes were not observed when PEOs were explanted without FCS. Furthermore, addition of any one or combinational addition of the growth factors, including bFGF, VEGF, or HGF, did not induce tube formation. These results suggest that PEOs contain precursor cells of coronary vasculature and that vasculogenesis may be simultaneously regulated by multiple factors.
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PMID:In vitro model for mouse coronary vasculogenesis. 1676 Dec 83

Dental pulp destruction is believed to be regulated, in part, by the matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Cytokines are believed to be important in the pathogenesis of pulpitis. This study examined the effects that TNF-alpha, IL-1beta, IL-6, and TGF-beta1 have on the collagen degradation mediated by pulp fibroblasts utilizing a cell-mediated collagen degradation assay. Reverse transcriptase-polymerase chain reaction, Western blot analyses, and zymography were utilized to examine multiple MMPs and TIMPs. The collagen degradation mediated by these cells was stimulated by these cytokines. TNF-alpha, IL-1beta, and IL-6 increased the mRNA and/or protein expression of MMP-1, MMP-2, and MMP-3. TGF-beta1 decreased MMP-1 mRNA expression, while only slightly affecting the MMP-2 and MMP-3 mRNA and/or protein. These cytokines did not affect the expression of TIMP-1 or TIMP-2. These results suggest that these cytokines affect pulp destruction, in part, by differentially regulating the MMPs and TIMPs.
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PMID:The effects of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and transforming growth factor-beta1 on pulp fibroblast mediated collagen degradation. 1693 28

We have developed a photopolymerizable styrenated gelatin that can cross-link through polymerization induced by irradiation with visible light. The purpose of this study was to investigate the feasibility of using photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation. As visible light activates camphorquinone added as a photoinitiator, free radicals induce the polymerization of the gelatin macromer; the styrenated gelatin then becomes cross-linked. Rabbit articular chondrocytes were cultured in styrenated gelatin hydrogels and also in collagen gels as a control. After being cultured in the gels, the cells were collected from both gels and counted. Reverse transcriptase-polymerase chain reaction, histological examination, and quantification of the synthesized glycosaminoglycan were performed. On average, 26% of the embedded cells were collected from the gelatin hydrogel immediately after the crosslinking reaction. The surviving chondrocytes expressed the mRNA of type II collagen and aggrecan core protein and produced a cartilaginous matrix throughout the gelatin after 3 weeks. A slightly insufficient accumulation of the matrix was found in the internal region of the gelatin hydrogels, suggesting that less permeability for nutrients due to the high concentration and closely packed structure resulted in less cell viability. Although some limitations became evident, these results indicate that it may be possible to use photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation.
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PMID:Encapsulation of chondrocytes in photopolymerizable styrenated gelatin for cartilage tissue engineering. 1696 73

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal-epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal-epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and alpha6 integrin at the dermal-epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase-polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal-epidermal junction by increasing the production and the further arrangement of dermal-epidermal junction components.
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PMID:Mesenchymal-epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal-epidermal junction formation in a skin equivalent model. 1712 54

Alport disease is caused by mutations in genes encoding the alpha3, alpha4, or alpha5 chains of type IV collagen, which form the collagenous network of mature glomerular basement membrane (GBM). In the absence of alpha3, alpha4, alpha5 (IV) collagen, alpha1, alpha2 (IV) collagen persists, which ordinarily is found only in GBM of developing kidney. In addition to dysregulation of collagen IV, Alport GBM contains aberrant laminins, which may contribute to the progressive GBM thickening and splitting, proteinuria, and renal failure seen in this disorder. This study sought to characterize further the laminin dysregulation in collagen alpha3(IV) knockout mice, a model of Alport disease. With the use of confocal microscopy, laminin alpha1 and alpha5 abundance was quantified, and it was found that they co-distributed in significantly large amounts in areas of GBM thickening. In addition, labeling of entire glomeruli for laminin alpha5 was significantly greater in Alport mice than in wild-type siblings. Reverse transcriptase-PCR from isolated glomeruli demonstrated significantly more laminin alpha5 mRNA in Alport mice than in wild-type controls, indicating upregulated transcription of Lama5. For testing glomerular barrier function, ferritin was injected into 2-wk-old Alport and control mice, and GBM was examined by electron microscopy. Highest ferritin levels were seen in Alport GBM thickenings beneath effaced podocyte foot processes, but morphologically normal GBM was significantly permeable as well. We concluded that (1) ultrastructurally normal Alport GBM residing beneath differentiated podocyte foot processes is inherently and abnormally permeable, and (2) upregulation of Lama5 transcription and concentration of laminin alpha1 and alpha5 within Alport GBM thickenings contribute to abnormal permeabilities.
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PMID:Laminin compensation in collagen alpha3(IV) knockout (Alport) glomeruli contributes to permeability defects. 1769 9

The surface properties of a biomaterial are fundamental to determine the response of the host tissue. In the present study, we have followed a particular biomimetic strategy where electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built a calcified extracellular matrix on a titanium fiber-mesh surface. In comparison with control conditions, the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation and increased surface coating with type-I collagen, decorin, and osteopontin (9.8-fold, 11.3-fold, and 9.5-fold, respectively). Reverse transcriptase-polymerase analysis revealed the electromagnetically upregulated transcription specific for the foregoing matrix proteins and for the growth factor TGF-beta1. The immunofluorescence of type-I collagen, decorin, and osteopontin showed their colocalization in the cell-rich areas. The use of an electromagnetic bioreactor aimed at obtaining the surface modification of the biocompatible metallic scaffold in terms of cell colonization and coating with calcified extracellular matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.
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PMID:Electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto titanium fiber-mesh scaffolds. 1820 May 42

Mutations in the extracellular matrix molecule collagen VI underlie the congenital muscular dystrophy types Ullrich and Bethlem. Establishing the origin of collagen VI in muscle is important for understanding the pathophysiology of these diseases and for developing future treatment approaches involving cell-specific delivery. Because the cells that produce collagen VI cannot be identified by histologic analysis, we examined the production of collagen VI in pure cultures of primary myogenic cells and muscle interstitial fibroblasts from limb muscle of neonatal mice. Immunofluorescence staining and Western blot analysis revealed secretion and matrix deposition of collagen VI by interstitial fibroblasts but not by myogenic cells in vitro. Using Northern blot and real-time reverse-transcriptase-polymerase chain reaction analysis for the collagen VI genes col6a1, col6a2, col6a3, transcript levels for the 3 mRNAs were high in interstitial fibroblasts, whereas in primary myogenic cells, they were indistinguishable from background. Furthermore, retention of mutant collagen VI in muscle from 3 patients with collagen VI mutation was identified in interstitial fibroblastic cells but not in their myofibers. These results suggest that interstitial fibroblasts but not myogenic cells contribute significantly to the deposition of collagen VI in the extracellular matrix in skeletal muscle and imply major roles of this cell type and the extracellular matrix in the pathogenesis of these diseases.
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PMID:Muscle interstitial fibroblasts are the main source of collagen VI synthesis in skeletal muscle: implications for congenital muscular dystrophy types Ullrich and Bethlem. 1821 55

We previously demonstrated that a high dose of tacrolimus (1 mumol/L) induced expression of matrix metalloproteinase (MMP) proteins in human cultured gingival fibroblasts, suggesting a molecular mechanism maintaining gingival collagen homeostasis in tacrolimus-treated patients. Herein we have analyzed whether the effect on collagen turnover might be influenced by a therapeutic tacrolimus dose. Human gingival fibroblasts were incubated for 72 hours with 10 nmol/L, 100 nmol/L, and 1 mumol/L tacrolimus, or left untreated (CT). Collagen type I and III (COL-I, COL-III), lysyl hydroxylase 2b (LH2b), MMP-1 and -2, tissue inhibitor of MMP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA levels were assayed by reverse-transcriptase polymerase chain reaction, collagen protein levels by dot blot, and MMP activity by sodium dodecyl sulfate zymography. Tacrolimus did not affect COL-I, COL-III, or MMP gene expression, while LH2b and TGF-beta1 tended to be down-regulated after 1 mumol/L FK506. Conversely, protein levels of MMP-1 (P = NS) and MMP-2 (P < .05 vs CT, 10 nmol/L, 100 nmol/L) were up-regulated after 1 mumol/L tacrolimus. Our findings confirmed that a high dose of tacrolimus does not induce interstitial collagen overexpression by gingival fibroblasts and induces up-regulation of MMPs protein levels. Interestingly, at doses corresponding to whole blood trough levels, tacrolimus did not exert any evident effect on collagen turnover pathways, suggesting that tacrolimus is likely to not affect collagen homeostasis in the gingival connective tissue compartment of FK506-immunosuppressed subjects. This effect did not seem to be dose-dependent.
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PMID:A therapeutic dose of FK506 does not affect collagen turnover pathways in healthy human gingival fibroblasts. 1858 21


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