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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three-dimensional (3D) culture of cells could closely mimic the in vivo situation with regard to cell function and microenvironment compared with plane monolayer cultured cells. In this paper, we established 3D culture of rat WB-F344 cells with rotary cell culture system (RCCS) to simulate microgravity environment, and examined cells proliferation, morphology, microstructure, E-cadherin protein quantity and mRNA expression of adhesion molecules by count the number of cells, optical microscope, transmission electron microscope and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that cells were polyhedron with lots of micovilli and mitochondria, which grow well and packed together densely to form irregular aggregates. Adjacent cells were connected with desmosome and tight junction. With the regard, the aggregates behaved 3D growth characteristics. Moreover, compared with control, mRNA level of Fibronectin and E-cadherin protein were increased, the changes maybe is the part mechanism in this microgravity simulated cells culture models which strengthened cells junction. This rotating 3D model might facilitate the study of interactions of cell-cell, cell-matrix and the mechanisms.
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PMID:[Three-dimensional spheroid model for cultivating WB-F344 cells in simulated microgravity]. 1689 8

As breast cancer remains the most common cause of cancer death in women, there is a continuing need not only to further characterise the processes of cancer progression, but also to improve accuracy of prognostic markers. Breast epithelial cells express components of the renin angiotensin system and studies suggest that these may be altered in disease progression. In addition, altered integrin expression correlates with lymph node metastasis. The aim of this study was to investigate the relationship between angiotensin II (AII) and integrins in breast tissue and, in particular, their role in breast cancer cell metastasis. Using in vitro assays, AII (10(-6) M)-treated MCF-7 and T47D breast cancer cells both show reduced adhesion to extracellular matrix proteins collagen-, fibronectin- and laminin-coated wells (P<0.001) and reduced invasion through collagen-, fibronectin- and laminin-coated membranes (P<0.05). This action was inhibited by co-treatment with the angiotensin type 1 receptor (AT1R) antagonist losartan (10(-5) M). The addition of the AT2R inhibitor PD123319 (10(-5) M) to AII-treated cells had no significant effect. Semi-quantitative reverse transcriptase-PCR and western blotting revealed that cells treated with AII (10(-6) M) expressed lower levels of both integrin alpha3 and beta1. Using specific inhibitors, this was shown to occur through protein kinase C signalling. These data suggest that AII reduces cell adhesion and invasion through the type 1 receptor and that this effect may be due to reduced expression of integrins, and in particular alpha3 and beta1.
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PMID:The role of angiotensin II in the regulation of breast cancer cell adhesion and invasion. 1695 38

Tenascin C (Tnc) is an alternatively spliced, multimodular extracellular matrix glycoprotein present in the ventricular zone of the developing brain. Pax6-deficient small eye (sey) mouse mutants show an altered Tnc expression pattern. Here, we investigated the expression of Tnc isoforms in neural stem/progenitor cells and their regulation by the paired-box transcription factor Pax6. Neural stem/progenitor cells cultured as neurospheres strongly expressed Tnc on the protein level. The Tnc isoform expression in neural stem/progenitor cells was analyzed by reverse transcriptase-PCR and dot blot Southern hybridization. In total, 20 different Tnc isoforms were detected in neurospheres derived from embryonic fore-brain cell suspensions. The Tnc isoform containing the fibronectin type III domains A1A4BD is novel and might be neural stem/progenitor cell-specific. Transient overexpression of Pax6 in neurospheres of the medial ganglionic eminence did not alter the total Tnc mRNA expression level but showed a pronounced regulative effect on different Tnc isoforms. The larger Tnc isoforms containing four, five, and six additional alternatively spliced fibronectin type III domains were up-regulated, whereas the small Tnc isoforms without any or with one additional domain were down-regulated. Thus, Pax6 is a homeodomain protein that also modulates the splicing machinery. We conclude that the combinatorial code of Tnc isoform expression in the neural stem/progenitor cell is complex and regulated by Pax6. These findings suggest a functional significance for individual Tnc isoforms in neural stem/progenitor cells.
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PMID:Neural stem/progenitor cells express 20 tenascin C isoforms that are differentially regulated by Pax6. 1726 84

Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post-flight analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, alpha-tubulin, actin, vimentin, fibronectin (FN), and beta1-integrin. The mRNA levels of OP and alpha-tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or beta1-integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity.
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PMID:Microgravity signal ensnarls cell adhesion, cytoskeleton, and matrix proteins of rat osteoblasts: osteopontin, CD44, osteonectin, and alpha-tubulin. 1738 75

An improved understanding of molecular response in the vocal folds to a synthetic extracellular matrix (sECM) during early wound repair is essential for understanding functional improvement of the tissue and implementation of future tissue-engineering strategies. The present study used real-time reverse transcriptase polymerase chain reaction to measure transcript expression of selected markers (procollagen alpha 2 type I, fibronectin, fibromodulin, hyaluronan synthase 2, and hyaluronidase 2) in 20 rabbits that underwent vocal fold biopsy bilaterally. After the biopsy, Carbylan-GSX 5% was injected immediately into the left vocal fold, and saline was injected into the right vocal fold. Two unwounded normal rabbit larynges were also harvested. Animals were sacrificed at days 1, 3, 5, and 10 post-surgery. At days 1, 3, and 10, no significant differences were found between the Carbylan-GSX-treated and saline-treated groups. At day 5, significant differences in procollagen (p = 0.02), fibronectin (p = 0.02), and transforming growth factor beta1 (p = 0.02) between the Carbylan-GSX-treated and saline-treated groups were measured. The presence of a sECM in the wound bed during the early stages of repair amplified the normal rabbit vocal fold wound-healing response over a short period of time. This amplification provided an optimal environment for tissue regeneration and may lead to the recovery of the functional biomechanical properties of the vocal folds necessary for voice production.
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PMID:Effect of a synthetic extracellular matrix on vocal fold lamina propria gene expression in early wound healing. 1751 34

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
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PMID:Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair. 1751 15

Cardiomyocytes selected from murine embryonic stem cells (ESCs) using the cardiac-specific promoter alpha-myosin heavy chain were embedded into collagen and fibronectin scaffolds. A custom-built device was used to expose these constructs to mechanical loading (10% stretch at 1, 2, or 3 Hz) or no loading. Constructs were evaluated using reverse transcriptase polymerase chain reaction, histology, and immunohistochemistry. Mechanical loading significantly affected gene expression, and these changes were dependent on the frequency of stretch. A 1 Hz cyclical stretch resulted in significantly lower gene expression, whereas a 3 Hz cyclical stretch resulted in significantly greater gene expression than in unstretched controls. These constructs also developed cardiac-specific cell structures similar to those found in vivo. This study describes a 3-dimensional model to examine the direct effect of mechanical loading on the differentiation of ESC-derived cardiomyocytes embedded in a defined extracellular matrix scaffold. A technique was also developed to isolate the areas within the constructs undergoing the most homogeneous strain so that the effect of mechanical loading on gene expression could be directly evaluated. These experiments emphasize that ESC-derived cardiomyocytes are actively responding to cues from their environment and that those cues can drive phenotypic control and cardiomyocyte differentiation.
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PMID:Effect of mechanical loading on three-dimensional cultures of embryonic stem cell-derived cardiomyocytes. 1833 4

Systemic sclerosis (SSc)-related pulmonary fibrosis, for which there are few effective therapies, is the most common cause of SSc-related mortality. We examined insulin-like growth factor (IGF)-II expression in explanted lung tissues from control and SSc patients to determine its role in the pathogenesis of fibrosis. IGF-II levels in vivo were detected using immunohistochemistry. Primary lung fibroblasts were cultured from lung tissues, and IGF-II mRNA was measured using reverse transcriptase-polymerase chain reaction. Western blot analysis measured extracellular matrix (ECM) production and phosphorylated signaling molecules. Immunostaining revealed increased IGF-II expression in fibroblastic foci of SSc lungs. Furthermore, primary SSc lung fibroblasts had a fourfold increase in IGF-II mRNA and a twofold increase in IGF-II protein compared with normal lung fibroblasts. IGF-II mRNA in SSc lung fibroblasts was expressed primarily from the P3 promoter of the IGF-II gene, and IGF-II induced both a dose- and time-dependent increase in collagen type I and fibronectin production. IGF-II triggered the activation of both phosphatidylinositol-3 kinase and Jun N-terminal kinase signaling cascades, the inhibition of which diminished IGF-II-induced ECM production. Our study demonstrates increased local IGF-II expression in SSc-associated pulmonary fibrosis both in vitro and in vivo as well as IGF-II-induced ECM production through both phosphatidylinositol-3 kinase- and Jun N-terminal kinase-dependent pathways. Our results provide novel insights into the role of IGF-II in the pathogenesis of SSc-associated pulmonary fibrosis.
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PMID:Insulin-like growth factor-II is increased in systemic sclerosis-associated pulmonary fibrosis and contributes to the fibrotic process via Jun N-terminal kinase- and phosphatidylinositol-3 kinase-dependent pathways. 1846 8

Hydroxyapatite (Ca(10)(PO(2))(6)(OH)(2), HA) coatings are widely used in un-cemented total hip arthroplasties because it increases implant fixation and bone in-growth. HA adsorbs high levels of proteins (e.g., fibronectin) and adhering macrophages release higher levels of BMP-2 compared to non-coated surfaces. Inflammation, however, can block the BMP-2 production from macrophages and it has been suggested that roughened surfaces activate macrophages. Therefore, the aim of the present study was to investigate whether commercially available 3D surfaces would generate increased BMP-2 and TGF-beta production and establish how this production is affected by macrophage activation. A mouse macrophage cell line (J774A.1) was incubated with 3D coatings resembling clinically used protestic surfaces, i.e., plasmaspray TiAlV coating with or without HA and porous TiAlV coating with or without HA. BMP-2 and TGF-beta production was determined after 24 h with ELISA and real time-reverse transcriptase-PCR (real time RT-PCR). Changes in pro-inflammatory response were established with ELISA after 6 and 24 h. The increased surface roughness of HA-coated surfaces was characterized with confocal microscopy. HA coating on plasmasprayed TiAlV coating was not found to induce BMP-2 and TGF-beta secretion in the J774A.1 macrophage cell line. In contrast, LPS-activated macrophages increased their TGF-beta, but not BMP-2, secretion when exposed to the prosthetic surfaces. These results imply that the well-known bone-inductive effects of HA may not be dependent on macrophage BMP-2 and TGF-beta secretion. Surface topography and protein adhesion may play a more pivotal role.
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PMID:Hydroxyapatite coatings did not increase TGF-beta and BMP-2 secretion in murine J774A.1 macrophages, but induced a pro-inflammatory cytokine response. 1922 47

Neovascularization can be improved using polymer scaffolds supporting endothelial progenitor cells (EPCs). The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured in a hyaluronan-based non-woven mesh (HYAFF-11). eEPCs were seeded on HYAFF-11 at the density of 1 x 10(6)/cm(2) and cultured with endothelial differentiating factors for 3 weeks. After 24 h, nearly 90% of EPCs were adherent. Cell viability, evaluated by methyltetrazolium test, was greater in HYAFF-11 than on the most commonly used fibronectin-coated dishes, even if a progressive decline in viability was observed starting from approximately the second week of culture. eEPCs easily migrated to and aggregated on the scaffold. Evidence of active protein synthesis and features of endothelial differentiation, including cellular transcytotic channels and micropinocytotic vesicles, was revealed using electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction analysis. eEPCs cultured in the scaffold also showed a certain angiogenic activity, as demonstrated by hepatocyte growth factor transcription and vascular endothelial growth factor secretion. In conclusion, eEPCs can migrate and adhere inside HYAFF-11, maintain their pre-endothelial phenotype, and express angiogenic factors, especially within the first week of growth. These results indicate that non-woven HYAFF-11 could be a promising candidate as a vehicle for eEPCs for regenerative medicine applications.
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PMID:Architectural organization and functional features of early endothelial progenitor cells cultured in a hyaluronan-based polymer scaffold. 1943 99


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