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)

Patients with generalized atrophic benign epidermolysis bullosa often show decreased expression of type XVII collagen, a transmembrane hemidesmosomal protein encoded by COL17A1. This report documents a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa, and applies a new methodology to define and characterize the resulting mRNA splice variants. Mutational analysis of COL17A1 identified a maternally inherited G-to-T transversion at the -1 position of exon 32. This acceptor splice-site mutation led to the formation of aberrant transcripts present at extremely low levels. Based on our recent finding that cycloheximide stabilized mutant COL17A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effects of the splice-site mutation on splicing of COL17A1 transcripts were determined using reverse transcriptase polymerase chain reaction of total RNA from keratinocytes incubated for 2.5 h in the presence or absence of 10 microg cycloheximide per ml. Using this approach, an abnormally spliced transcript was identified that contains an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splice variants, including one derived from the skipping of exon 32, were also identified. These results indicate the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice-site mutations, because: (i) aberrant splicing often generates a premature termination codon, (ii) transcripts with premature termination codons can occur at low or undetectable levels due to nonsense-mediated mRNA decay, and (iii) the levels of these transcripts can be increased by cycloheximide.
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PMID:Cycloheximide facilitates the identification of aberrant transcripts resulting from a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa. 945 13

To investigate biological roles of human endogenous retroviruses (ERVs), the author examined the viral mRNA expression in the normal systemic organs in vivo and its regulation by cytokines in cultured cells. The following evidence suggesting biological activities of a human ERV, ERV3, was obtained. First, the ERV3 mRNA was demonstrated at different levels in organs, and at consistently high levels in adrenal glands from all individuals and in all adrenocortical adenomas examined, by Northern hybridization. In situ hybridization revealed that the ERV3 expression was localized in all three layers of the adrenal cortex, but not in the medulla. These results suggest that the ERV3 expression may relate to the cellular differentiation and/or steroid production of adrenocortical cells. Second, the amount of ERV3 mRNA in cultured endothelial cells from human umbilical vein was significantly increased with any of TNF-alpha, IL-1 beta or IL-1 alpha stimulation but decreased with IFN-gamma treatment, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with competitive PCR. The collective evidence suggests that the ERV3 expression may be upregulated at the inflammatory sites of vessels in vivo, and that the ERV3 expression may, therefore, play certain pathogenic roles in diseases, including collagen and vascular diseases in man.
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PMID:[Tissue-specific expression of human endogenous retrovirus mRNA and its regulation by cytokines in vitro]. 946 16

Our laboratory has developed a method for the repair of osteochondral defects by implanting cultured perichondrial cells attached to a biodegradable polylactic acid scaffold. The success of this approach depends in part on the proliferative characteristics and the phenotype of the implanted cells. Transforming growth factor-beta 1 has been reported to influence these parameters in several mesenchymal-derived tissues in vitro and in vivo. The chondrocytic phenotype is marked by an enhanced expression of the collagen type-II gene. In this study, cultures grown from explants of rabbit rib perichondrium were exposed to exogenously added transforming growth factor-beta 1 at concentrations of 0.1-10 ng/ml of media. Cell proliferation and collagen gene expression were measured. The expression of types I and II collagen genes was analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. The exogenous addition of transforming growth factor-beta 1 at a concentration of 0.1-10 ng/ml resulted in tritiated thymidine uptake by perichondrial cells, with optimum proliferative effects at 0.1 ng/ml. Transforming growth factor-beta 1 added at concentrations of 0.1 and 0.5 ng/ml significantly upregulated the expression of type-II collagen mRNAs. The results suggest that, when the chondrocytic phenotype is defined by markedly enhanced type-II collagen gene expression, the chondrocytic phenotype of explant cultures of perichondrium-derived cells is enhanced by the exogenous addition of transforming growth factor-beta 1.
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PMID:Chondrogenic phenotype of perichondrium-derived chondroprogenitor cells is influenced by transforming growth factor-beta 1. 949 3

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17beta-estradiol (E2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J Bone Miner Res 9:321-327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstrated by the appearance of osteoblastic markers such as alpha1(I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OP), and transforming growth factor-beta1 (TGFbeta1), which were detected by using reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation, including deposition of collagen fibers and matrix mineralization, was also studied at several time points of the 3-week culture period. The effect of E2 on the appearance of osteoblastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E2, the rate of cell proliferation was increased in the early phase of cultures, but not after day 6. The addition of E2 in subcultures resulted in an increase of levels of mRNA for COL1, ALP, OCN, OP, and TGF-beta1. ALP activity was also increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E2. All E2 concentrations used (0.01-10 nmol/L) were effective but the maximum response was obtained with 0.1 nmol/L E2. Addition of the antiestrogen ICI 182,780 abolished the E2-induced stimulation of proliferation and later an increase in ALP activity. Addition of ICI 182,780 without the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E2 stimulates sequential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner.
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PMID:Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. 951 12

Dermatofibroma (DF) is histologically characterized by proliferation of fibroblasts in the dermis. Multiple DFs occasionally develop in patients with autoimmune disorders under immunosuppressive therapy; however, the pathogenesis of DF is still unclear. To elucidate immunological involvement in the mechanism of the fibrosis in DF, we studied the role of interleukin-1 (IL-1), which has a number of biological functions, including proliferation and collagen production of fibroblasts, on DF-derived fibroblasts. 3H-thymidine incorporation was used to examine the effects of Il-1 alpha and IL-1 beta in 4 cultured fibroblast strains derived from DF and 5 fibroblast strains from normal skin. Expression of mRNA of IL-1 was also analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Basal 3H-TdR incorporation without stimulant of DF-derived fibroblasts showed a significantly greater growth activity than normal skin-derived fibroblasts (2, 632 +/- 525 vs. 762 +/- 144 dpm, p < 0.01). Both IL-1 alpha and IL-1 beta showed a stronger growth-stimulatory activity on DF-derived fibroblasts in a dose-dependent manner than normal fibroblasts, and the percent 3H-TdR uptake of DF was 1.4-fold (IL-1 alpha; 1,000 U/ml) and 1.3-fold (IL-1 beta; 1,000 U/ml) as compared with normal fibroblasts; however, the differences did not reach any significance. When increasing concentrations of IL-1 receptor antagonist (IL-1 alpha) were added to culture medium stimulated with IL-1 alpha, the proliferative response of fibroblasts was significantly reduced. Expression of IL-1 beta and RNA was detected on both DF-derived and normal skin-derived fibroblasts, while that of IL-1 alpha mRNA was detected only on DF-derived fibroblasts. Our results suggest that IL-1 may be involved in the fibrotic process in DF at the transcriptional level and play a role in the fibroblast proliferation in an autocrine manner.
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PMID:Possible involvement of interleukin-1 in the pathogenesis of dermatofibroma. 953 85

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.
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PMID:Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart. 968 29

Generalized atrophic benign epidermolysis bullosa (GABEB; OMIM no. 226650) is a rare hemidesmosomal variant of EB, inherited in an autosomal recessive fashion. In previous studies, mutations in the gene (COL17A1) encoding the type XVII collagen, a transmembrane component of hemidesmosomes, were detected in most patients with GABEB. However, evidence for genetic defects in the laminin 5 genes has also been presented. In the present investigation, we examined three patients, representing two families with GABEB, for mutations in the LAMB3 gene. Heteroduplex scanning of the gene, followed by direct automated sequencing, revealed that Patient 1 was a compound heterozygote for a missense mutation (C293S) and a premature termination codon-causing mutation (1367delAC). The latter mutation resulted in accelerated mRNA decay, which rendered the corresponding mRNA transcript undetectable by reverse transcriptase-PCR. Patients 2 and 3, siblings with slightly different clinical presentations, were homozygous for a G-->A transition affecting the last nucleotide of exon 7 (628G-->A). This mutation resulted in amino acid substitution (E210K), as well as in multiple aberrant splice variants affecting exons 6 to 8. These observations expand the repertoire of LAMB3 mutations in nonlethal variants of EB, and they illustrate the consequences of the mutations at the mRNA and protein levels.
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PMID:LAMB3 mutations in generalized atrophic benign epidermolysis bullosa: consequences at the mRNA and protein levels. 969 May 63

We examined the expression of selected growth factors, growth factor receptors, elements of extracellular matrix and cell adhesion molecules in the germinal matrix layer (GML) utilizing immunohistochemistry and reverse transcriptase polymerase chain reaction. At autopsy brain samples from 10 neonatal infants were used. Epidermal growth factor receptor (EGFR) was significantly expressed in the matrix cells. While transforming growth factor alpha and heparin-binding epidermal growth factor-like growth factor were found in the matrix cells or vascular wall as ligands, epidermal growth factor was not expressed. EGFR and its ligands are thought to be important factors for the maintenance of the matrix cells and cell-to-cell interactions. Insulin like growth factor I, its receptor Ibeta and tenascin were found in the stroma of the GML and periventricular region. Vascular endothelial growth factor and receptor Flk-1, laminin A and B2, fibronectin, collagen type IV and integrins such as beta3, alpha5beta1 and alphaVbeta3 were found mainly in or around the vascular wall indicating their important roles for vascularization. Transforming growth factor beta2 and its receptor II were expressed in the matrix cells and/or vascular wall suggesting a role in proliferation and/or regression of the vasculature. CD44 and Thy-1 were also expressed in the matrix cells.
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PMID:Growth factors in infant germinal matrix: relationship to extracellular matrix and cell adhesion molecules. 973 49

Lipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H] collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover.
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PMID:Lipodermatosclerosis is characterized by elevated expression and activation of matrix metalloproteinases: implications for venous ulcer formation. 1038 52

An orthodontically treated tooth is often destabilized in its newly corrected location and relapses towards its original position. Hitherto, the explanation for this phenomenon was that orthodontic force brings about "stretching" of gingival collagen fiber, which "pull back" the tooth towards its pretreatment position. A previous ultrastructural study showed that after force application the gingival collagen fibres were torn, laterally spaced and of increased diameter. Therefore, they could not "pull back" the tooth and be the cause of the relapse. In the present study, in order to find a more plausible explanation at the molecular level, the effect of pressure on the gene transcription of collagen type I and tissue collagenase was examined by semiquantitative, reverse transcriptase-polymerase chain reaction assay. Attached buccal gingiva was excised from anaesthetized dogs and gingival fibroblasts were grown in culture. Following application of pressure (0.167 kg/l g cell mass), the transcription of collagen type I was increased while that of tissue collagenase was decreased. These results corroborate the ultrastructural in vivo findings that orthodontic force is associated with larger amounts of collagen type I in the gingiva.
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PMID:The effect of centrifugal force on the transcription levels of collagen type I and collagenase in cultured canine gingival fibroblasts. 983 7


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