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)

Gap junctions (GJ) are aggregates of intercellular channels, composed of connexin (Cx) protein, between adjacent cells. The vertebrate ovarian follicle contains homocellular (granulosa cell-granulosa cell) and heterocellular (granulosa cell-oocyte) GJ. However, the function of GJ during final oocyte differentiation (maturation) is controversial. The objectives of this study are to reexamine the number and identity of Cx genes that are expressed in the Xenopus ovary, and to examine the potential role of GJ in oocyte maturation by determining the temporal association between changes in ovarian Cx mRNA content and the process of maturation. We used reverse transcriptase-polymerase chain reaction to amplify ovarian cDNA fragments using degenerate Cx primers. We amplified three Cx-like fragments: one was novel and two corresponded to known Cx of Xenopus ovaries (Cx38 and 43). The novel fragment was used to screen an ovarian cDNA library. One positive clone was identified and its nucleotide sequence determined. Its deduced amino acid sequence showed that it corresponded to a novel Cx, Cx41, belonging to the Group II class of Cx. Xenopus Cx41 showed the highest homology to rat Cx37 (65% identity, 80% similarity). Also, the last 10 C-terminal amino acids of Cx41 were identical to those of rat, mouse, and human Cx37. Cx41 transcripts were detected by riboprobe mapping in ovarian somatic cells, heart, leg muscle, liver and eye, but not in brain or in oocytes of any developmental stage. Full-grown follicles incubated in vitro with human chorionic gonadotropin became committed to mature within 1-4 hr, and physical signs of maturation (germinal vesicle breakdown) were seen at 4-5 hr. Significant reductions in the levels of Cx41 and 43, but not 38 transcripts were seen at 4 hr, after oocytes had committed to mature. Thus, if availability of Cx mRNA determines availability of Cx protein and GJ, our results would suggest that irreversible commitment to maturation occurred prior to major declines in follicular GJ during the periovulatory period. The present study is the first to report the presence of at least two hormone-responsive Cx gene transcripts (Cx41 and 43 in Xenopus) in ovaries of a single animal species.
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PMID:Molecular cloning, tissue distribution, and hormonal control in the ovary of Cx41 mRNA, a novel Xenopus connexin gene transcript. 856 53

Osteocytes have been proposed to be the cells primarily responsible for sensing the effects of mechanical loading in bone. Osteocytes respond to loading in vivo, and have been shown to express osteotropic agents and their receptors, and cell/matrix adhesion molecules in vitro, but the functional significance of such findings is not clear. One obstacle to increased understanding of the role of osteocytes in the regulation of bone mass is that the cells are not easily accessible for study. In situ studies are difficult, and although it is possible to extract and culture osteocytes from neonatal bones, the responses of such cells might be very different from those in older bones in situ. We have developed a technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), and have shown that they express mRNA for beta-actin (beta-ACT), osteocalcin (OC), connexin-43 (Cx43), insulin-like growth factor I (IGF-I), c-fos and c-jun, but not tumor necrosis factor alpha (TNF-alpha) or tartrate-resistant acid phosphatase (TRAP). The principle behind the method is that after removal of the periosteum, tangential cryostat sections of a tubular bone contain RNA only from osteocytes and a very small number of endothelial cells as long as the marrow cavity is not broached. Using this method, we have investigated gene expression in cells from rat ulnar cortical bone under forming and resorbing bone surfaces. In addition, we have investigated the effect on gene expression of mechanical loading which, if repeated daily, initiates new bone formation on quiescent or resorbing surfaces. Although the expression of the genes we have studied in osteocytes is different from those expressed by the periosteal surfaces overlying the cortex, we have not detected loading-related changes in osteocyte gene expression in any cortical bones. This may be because of the extreme sensitivity of the PCR technique which can only resolve large differences in expression. The use of quantitative methods in the future may allow demonstration of regulated gene expression in osteocytes.
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PMID:Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase. 885 45

1. Phenylephrine (10 microM) evoked rises in tension in isolated rings of endothelium-denuded rabbit superior mesenteric artery. These increases consisted of a tonic component with superimposed rhythmic activity, the frequency of which generally remained constant over time but whose amplitude exhibited cycle-to-cycle variability. 2. The amplitude, but not the frequency, of the rhythmic activity was affected by a series of short peptides possessing sequence homology with extracellular loops 1 and 2 of connexin 43 (Cx43). Oscillatory behaviour was abolished at concentrations of 100-300 microM (IC50 of 20-30 microM), without change in average tone. No synergy was evident between peptides corresponding to the extracellular loops, and cytoplasmic loop peptides were biologically inactive. 3. The putative gap junction inhibitor heptanol mimicked the action of the extracellular loop peptides and abolished rhythmic activity at concentrations of 100-300 microM without effects on frequency. However, in marked contrast to the peptides, heptanol completely inhibited the contraction evoked by phenylephrine (IC50, 283 +/- 28 microM). 4. The presence of mRNA encoding Cx32, Cx40 and Cx43 was detected in the rabbit superior mesenteric artery by reverse transcriptase-polymerase chain reaction. Western blot analysis showed that Cx43 was the major connexin in the endothelium-denuded vessel wall. 5. We conclude that intercellular communication between vascular smooth muscle cells via gap junctions is essential for synchronized rhythmic activity in isolated arterial tissue, whereas tonic force development appears to be independent of cell-cell coupling. The molecular specificity of the peptide probes employed in the study suggests that the smooth muscle relaxant effects of heptanol may be non-specific and unrelated to inhibition of gap junctional communication.
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PMID:Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries. 928 78

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.
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PMID:Downregulation of connexin 45 gene products during mouse heart development. 1038 88

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase-polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43-positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild-type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin-deficient mice to cells in different regions of the retina.
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PMID:Expression patterns of connexin genes in mouse retina. 1095 39

Although both Schwann cells and oligodendrocytes express connexin32 (Cx32), the loss of this connexin causes demyelination only in the PNS. To determine whether oligodendrocytes might express another connexin that can function in place of Cx32, we searched for novel CNS-specific connexins using reverse transcriptase-PCR and degenerate primers. We identified Cx29, whose transcript was restricted to brain, spinal cord, and sciatic nerve. Developmental expression of Cx29 mRNA in the CNS paralleled that of other myelin-related mRNAs, including Cx32. In the CNS, Cx29 antibodies labeled the internodal and juxtaparanodal regions of small myelin sheaths, whereas Cx32 staining was restricted to large myelinated fibers. In the PNS, Cx29 expression preceded that of Cx32 and declined to lower levels than Cx32 in adulthood. In adult sciatic nerve, Cx29 was primarily localized to the innermost aspects of the myelin sheath, the paranode, the juxtaparanode, and the inner mesaxon. Cx29 displayed a striking coincidence with Kv1.2 K(+) channels, which are localized in the axonal membrane. Both Cx29 and Cx32 were found in the incisures. Cx29 expressed in N2A cells did not induce intercellular conductances but did participate in the formation of active channels when coexpressed with Cx32. Together, these data show that Cx29 and Cx32 are expressed by myelinating glial cells with distinct distributions.
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PMID:Connexin29 is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems. 1215 25

In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 degrees C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 mm galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 degrees C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 mm galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase-polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.
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PMID:Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment. 1562 Dec 51

The effects of histone deacetylase inhibitor Trichostatin A (TSA) on connexin (Cx) expression and gap junctional intercellular communication (GJIC) were investigated in primary cultures of adult rat hepatocytes. GJIC was monitored by using the scrape-loading/dye transfer method. Immunoblotting and immunocytochemistry were used to investigate Cx protein levels and localization. Cx gene expression was studied by means of quantitative reverse transcriptase-polymerase chain reaction. TSA increased Cx32 protein levels and affected negatively the Cx26 protein levels. The latter was preferentially located in the cytosol of cultured cells. TSA also promoted the appearance of Cx43 in the nuclear compartment of primary cultured hepatocytes. Overall, this resulted in enhanced GJIC activity. It is important to note that the time of onset of TSA treatment was crucial for the extent of its outcome and that the effects of TSA on Cx protein levels occurred independently of transcriptional changes. TSA differentially affects Cx proteins in primary rat hepatocyte cultures, suggesting distinct regulation and/or distinct roles of the different Cx species in the control of hepatic homeostasis. TSA enhances GJIC between primary cultured rat hepatocytes, an interesting finding supporting its use to further optimize liver-based in vitro models for pharmacotoxicological purposes.
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PMID:Trichostatin a enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes. 1653 68

The expression patterns of connexin (Cx) genes, encoding gap junctional proteins, are tissue- and cell-specific and, as their expression is mostly suppressed during carcinogenic processes, they are appropriate for monitoring tumour development. In this study, using reverse transcriptase-coupled polymerase chain reaction (RT-PCR), the expression of Cx mRNAs was examined in seven normal canine mammary glands and in 31 mammary gland tumour samples. Cx26 and Cx43 gene expression was studied in all normal tissues using specific Cx26 and Cx43 primers. When the expression patterns of Cx26 and Cx43 genes were analyzed in several types of canine mammary gland tumours, it was noted that it was the loss of Cx26 expression rather than the occurrence of Cx43 expression that was associated with malignancy. These results suggest that Cx26 plays an important role in tumourigenesis of canine mammary gland.
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PMID:Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours. 1677 44

The rat cell line R3/1 displays several phenotypical features of alveolar epithelial type I cells. In order to evaluate this cell line as potential in vitro model for drug disposition studies, R3/1 cells were cultured on Transwell filters and the transepithelial electrical resistance (TEER) was measured to test the integrity of cell layers. The mRNA expression of cell junctional components including E-cadherin, occludin, ZO-1 and ZO-2 was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) and the corresponding proteins by immunofluorescence microscopy (IFM). Moreover, the expression pattern of catabolic peptidases, carboxypeptidase M, aminopeptidases (AP): A, B, N and P, gamma-glutamyltransferase (GGT), dipeptidylpeptidase IV, angiotensin-converting enzyme (ACE), and endopeptidases (EP) 24.11 and 24.15 was analysed in R3/1 cells and compared to rat alveolar epithelial I-like cells in primary culture. TEER peaked at 99+/-17Omegacm(2) after 5 days in culture. Addition of 0.1muM dexamethasone (DEX) with 20% foetal bovine serum further increased TEER by 65%. However, none of the culture conditions used in our study yielded monolayers with TEER values comparable to those of primary cultures of rat pneumocytes. No transcripts encoding for E-cadherin and occludin were detected by RT-PCR. However, ZO-1 and -2 mRNA transcripts were found. IFM using a monoclonal antibody against occludin confirmed the absence of the protein in R3/1 cells. Of the investigated proteolytic enzymes, mRNA transcripts encoding APA and APB as well as EP 24.11 and EP 24.15 were detected; a pattern similar to that of rat alveolar epithelial I-like cells in primary culture. Thus, although R3/1 cells express certain markers typical for type I pneumocytes (e.g., T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) they do not form electrically tight monolayers. This excludes R3/1 cells from being used as an in vitro model for alveolar absorption. However, the cell line may be suitable to study stability of inhaled and endogenous proteins.
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PMID:Characterisation of the R3/1 cell line as an alveolar epithelial cell model for drug disposition studies. 1910 87


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