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)

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.
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PMID:The effects of isolation on chondrocyte gene expression. 1699 90

Royal jelly (RJ) has diverse physiological and pharmacological functions. We observed its weak estrogenic activity in the previous study. RJ stimulated the proliferation of mouse osteoblast-like MC3T3-E1 cells at 0.1 mg/ml, and the effect was blocked by the specific estrogen receptor antagonist ICI 182,780. The addition of 0.1-1.0 mg/ml RJ enhanced collagen production in culture medium. Oral administration of RJ to normal female mice for 9 weeks increased the ash content of their tibiae. DNA microarray analysis revealed significant changes in gene expression related to extracellular matrix formation when the femurs of mice fed RJ were analyzed. Quantitative reverse transcriptase-PCR (RT-PCR) confirmed up-regulation of procollagen I alpha1 gene expression. These data suggest that RJ as a whole or some of its individual components stimulates production of type I collagen and other activities for bone formation through action on osteoblasts.
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PMID:Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines. 1703 Oct 45

Eicosapentaenoic acid (EPA) has been reported to have beneficial effects on the progression of various renal diseases including diabetic nephropathy; however, the precise mechanisms are not completely understood. We examined the effects of EPA on the early stage of type 2 diabetic nephropathy in KKA(y)/Ta mice and the possible role of inflammation, oxidative stress, and growth factor in this process. KKA(y)/Ta mice were divided into 2 groups. The treatment group was injected with EPA ethyl ester at 1 g/kg per day intraperitoneally from 12 to 20 weeks of age and the control group was injected with saline. Renal morphologic examinations were performed after 8 weeks of treatment. Glomerular macrophage infiltration and expression of monocyte chemoattractant protein 1, malondialdehyde (MDA), nitrotyrosine, transforming growth factor beta1 (TGF-beta1), and type I collagen were evaluated. Eicosapentaenoic acid decreased the levels of urinary albumin, serum triglyceride and MDA, and improved glucose intolerance in KKA(y)/Ta mice. Morphometric analysis showed that accumulation of extracellular matrix and the tubulointerstitial fibrosis area were significantly decreased after treatment. Immunohistochemistry revealed that glomerular macrophage infiltration and the expression of MDA and nitrotyrosine in KKA(y)/Ta mice were increased and were inhibited by EPA treatment. Protein and gene expression levels of monocyte chemoattractant protein 1, TGF-beta1, and type I collagen, which were evaluated by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction, were down-regulated in the EPA treatment group. In conclusion, EPA improves type 2 diabetic nephropathy in KKA(y)/Ta mice. This beneficial effect might be mediated by attenuation of metabolic abnormalities and inhibition of renal inflammation, oxidative stress, and TGF-beta expression.
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PMID:Effects of eicosapentaenoic acid on the early stage of type 2 diabetic nephropathy in KKA(y)/Ta mice: involvement of anti-inflammation and antioxidative stress. 1714 29

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been investigated for biomedical applications due to its many biologically favorable properties. However, to explore its application in bone tissue engineering, the poorly bioactive surface property of PHBV must be improved. To engineer PHBV to achieve a biologically active surface, in this study each porous PHBV matrix was prepared by solute leaching of salt/PHBV cast film and was treated with ozone followed by dip coating with type I collagen. The biological responses of osteoblast-like UMR-106 cells after being grown on the engineered PHBV matrix were evaluated. Confocal microscopy and the MTT assay were used to map and quantify the viable cell proliferation on the PHBV matrix, respectively. The cells were cultivated in osteogenic media containing beta-glycerophosphate and later stained with alizarin red to visualize mineralization of the matrix. RNA was extracted from the UMR-106 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to detect expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a house keeping gene) and bone sialoprotein (BSP) (marker of the osteoblastic phenotype). The results showed that the UMR-106 cells after cultivation on the engineered PHBV matrix retained the osteoblastic phenotype characteristics, indicating that the porous PHBV matrix after ozone treatment and collagen dip coatings are a promising scaffold for bone tissue engineering applications.
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PMID:Evaluation of the biological responses of osteoblast-like UMR-106 cells to the engineered porous PHBV matrix. 1718 85

Under normal growth conditions, in vitro dermal microvascular endothelial cells (HDMEC) retain an epithelioid morphology and do not synthesize matrix proteins found increased in scar tissue. When injured by a standard scratch, cells at the wound edge and within the culture transform into spindle-shaped, myofibroblast-like cells. To determine if the transformed cells synthesize matrix proteins, expression of type I collagen and alpha smooth muscle actin (alpha-SMA) was investigated by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve hours following injury, a major upregulation in expression of alpha-SMA and type I collagen was observed both in cells proximal and distal to the wound edge. Cells with the typical morphology of myofibroblasts and displaying intracellular alpha-SMA positive fibrils were observed in HDMEC throughout the culture. In contrast, type IV collagen, a basement membrane protein, was not detected in migrating cells. Following completion of wound repair (24-36 h), type I collagen was no longer expressed and type IV collagen synthesis increased to prewound levels. Quantitative RT-PCR confirmed the changes in gene expression for both type I collagen and alpha-SMA at each time point during repair. These results demonstrate that normal skin microvascular endothelial cells retain an ability to transform into myofibroblast-like cells when injured and to synthesize matrix proteins not expressed in noninjured cells. The synthesis of matrix proteins by injured endothelial cells suggests a direct role for the endothelium in the pathology of scar formation.
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PMID:Mechanisms of microvascular wound repair II. Injury induces transformation of endothelial cells into myofibroblasts and the synthesis of matrix proteins. 1731 65

In chronic renal disease, the progressive accumulation of collagen and other extracellular matrix proteins in the mesangium results in fibrosis, glomerulosclerosis, and eventual renal failure. Mice deficient in proalpha2(I) collagen are not only a model of osteogenesis imperfecta but also accumulate fibrillar homotrimeric type I collagen in the mesangium. This accumulation spreads to the subendothelial space in the peripheral capillary loops. Picosirius red staining of kidney sections demonstrates that in comparison to wild-type mice, Col1a2-deficient homozygous and heterozygous mice exhibit abnormal glomerular collagen deposition in a gene dosage-dependent manner. The glomerulopathy initiates during the first postnatal week, appears progressive following the pattern of glomerular maturation and results in albuminuria in severely affected animals. In situ hybridization revealed no gross differences in steady-state proalpha1(I) and proalpha2(I) collagen mRNA levels among the three genotypes. Quantitative reverse transcriptase-polymerase chain reaction, however, using whole kidney sections showed a twofold increase in steady-state proalpha1(I) collagen mRNA in 1-month homozygous Col1a2-deficient animals compared with wild-type and heterozygous animals. We suggest that glomerular collagen deposition seen in the osteogenesis imperfecta model mice is, in part, owing to pretranslational mechanisms and may represent an over compensation of wound healing.
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PMID:Type I collagen glomerulopathy: postnatal collagen deposition follows glomerular maturation. 1736 Nov 18

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

To investigate the use of a scaffold seeded with genetically modified meniscal cells or mesenchymal stem cells (MSCs) for the healing of meniscal lesions, primary meniscus cells and bone marrow-derived MSCs were isolated from bovine calves and transduced with first-generation adenoviral vectors encoding green fluorescent protein, luciferase, or transforming growth factor (TGF)-beta1 complementary deoxyribonucleic acid (cDNA). The genetically modified cells were seeded in type I collagen-glycosaminoglycan (GAG) matrices and transplanted into tears of the avascular zone of bovine menisci. After 3 weeks of in vitro culture, constructs and repair tissues were analyzed histologically, biochemically, and using reverse transcriptase polymerase chain reaction. Recombinant adenovirus readily transduced meniscal cells and MSCs, and transgene expression remained high after the cells were incorporated into collagen-GAG matrices. Transfer of TGF-beta1 cDNA increased cellularitiy and the synthesis of GAG/DNA [microg/microg]. It also led to stronger staining for proteoglycans and type II collagen and enhanced expression of meniscal genes. Transplantation of the TGF-beta1 transduced constructs into meniscal lesions of the avascular zone resulted in filling of the lesions with repair tissue after 3 weeks of in vitro culture. These results indicate that TGF-beta1 cDNA delivery may affect cell-based meniscus repair approaches in vivo.
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PMID:Genetically enhanced engineering of meniscus tissue using ex vivo delivery of transforming growth factor-beta 1 complementary deoxyribonucleic acid. 1756 2

The relationship between radiographic measures of severity of osteoarthritis (OA) and changes in gene expression in joints are not well characterised. In this study, the expression of 11 candidate genes was characterised by quantitative reverse transcriptase polymerase chain reaction in normal and OA cartilage and bone from the elbows of dogs with fragmented coronoid disease. The levels of expression of type I collagen alpha2 chain (COL1A2), type III collagen alpha1 chain (COL3A1), lumican (LUM), matrix metalloproteinase-2 (MMP2), -9 (MMP9) and -13 (MMP13) genes were increased and the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and cathepsin D (CTSD) genes were decreased in OA cartilage relative to normal cartilage. All differences correlated with radiographic measures of severity of OA. Levels of expression of COL1A2, MMP2, MMP9, MMP13 and TIMP1 were increased, whereas expression of TIMP2 was decreased in OA bone relative to normal bone. Cartilage gene expression may be correlated with the radiographic severity of OA.
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PMID:Cartilage gene expression correlates with radiographic severity of canine elbow osteoarthritis. 1793

The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase-polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment.
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PMID:Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels. 1856 50


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