Biomedical subjects

O Traub

Publications and source records attributed to O Traub.

At least 55 records · Page 3Linked to original sources

PubMed1997

Coexpression of connexin45 and -32 in oligodendrocytes of rat brain.

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.

Animals↗

PubMed1996

The neural adhesion molecule L1 is phosphorylated on tyrosine and serine residues.

The neural cell adhesion molecule L1 is highly homologous in its extracellular domain between species and completely identical in its cytoplasmic domain. We report here that tyrosine residues of L1 are phosphorylated in addition to serine residues, as determined by monoclonal phosphotyrosine antibodies and phosphoamino acid analysis. This result supports the suggestion that the cytoplasmic domain of L1 might be involved in signal transduction.

Animals↗

PubMed1996

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Animals↗

PubMed1996

Regulation of connexin31 gene expression upon retinoic acid treatment in rat choriocarcinoma cells.

The controlled invasiveness of the trophoblast is based on the balance between invasive properties at implantation and the differentiation program of the developing placenta. During placental development in rats a switch of connexin gene expression has been observed in parallel to the switch from the invasive to the differentiated phenotype of trophoblast cells. To investigate the role of connexin expression for trophoblast invasion, proliferation, and differentiation, we studied one rat trophoblast (HRP-1) and one rat choriocarcinoma cell line (Rcho-1). The choriocarcinoma cells were characterized by expression of cx31 and a lack of E-cadherin, corresponding to the invasive trophoblast in vivo, whereas HRP-1 cells expressed cx43, normally found in the spongiotrophoblast and in late giant cells, and E-cadherin. Upon retinoic acid treatment, Rcho-1 cells irreversibly lost cx31 expression, accompanied by a loss of functional coupling. No changes in regard to connexin expression and cell-cell communication could be observed in HRP-1 cells. In addition, treatment of Rcho-1 cells with retinoic acid for 7 days upregulated expression of cx43 transcript, but no protein could be found. Proliferation was clearly reduced and the mean volume of cells doubled from Day 4 to Day 7 of retinoic acid treatment in Rcho-1 cells, while both parameters were not affected in HRP-1 cells. Both cell lines showed a similar invasion rate using a Matrigel invasion assay, and invasion was equally suppressed upon retinoic acid treatment. Thus the different connexin expression appears more likely to play a role in regulating proliferation and differentiation along the multilineage pathway than invasiveness of rat trophoblast cells.

Animals↗

PubMed1996

Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat.

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.

Animals↗

PubMed1996

Upregulation of gap junction protein connexin43 in alveolar epithelial cells of rats with radiation-induced pulmonary fibrosis.

The degree of immunoreactive connexin43 (C x 43) in rat lung was evaluated during the development of radiation-induced pulmonary fibrosis in rat by a double immunofluorescence technique using polyclonal antisera to Cx43 and monoclonal antibodies to cytokeratins on cryostat sections. In normal rat lungs, Cx43 was detected in pneumocytes type II and I, in large blood vessel endothelia, in peribronchial smooth muscle cells, and in some peribronchial and perivascular interstitial cells. As early as 1 week after irradiation, enhanced immunoreactivity for Cx43 in the epithelial cells was detected. In severely injured lungs (about 3 months after irradiation), Cx43 was found also in the cytoplasm of type II pneumocytes. These findings were confirmed by western blot data. Western blot analysis also revealed increased phosphorylation of Cx43. It remains to be investigated whether the increased content of Cx43 in irradiated rat lung may be due to an enhanced number of gap junctions between type I and II alveolar epithelial cells.

Actins↗

PubMed1996

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

Animals↗

PubMed1996

Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively.

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

Animals↗

PubMed1996

Connexin expression and intercellular communication in two- and three-dimensional in vitro cultures of human bladder carcinoma.

The identification of gap-junctional proteins (connexins) and the preparation of related antibodies provides new tools to study patterns of intercellular communication in tumors. Focusing on the biology of human bladder carcinoma, we compared the expression of gap-junctional proteins (connexins Cx26, Cx32, and Cx43) with a dye-coupling assay for gap-junctional intercellular communication in three cell lines with different urothelial differentiation. The cell lines HCV-29, RT4, and J82 were initially grown as monolayers of different ages. Connexin expression was found mostly positive over the time of culture and found constantly negative only in J82 cells for Cx26 and HCV-29 cells for Cx32. In HCV-29 cells, Cx26 increased in positivity over the time of culture. Western blotting with the antibodies confirmed the findings. Comparisons of dye transfer using Lucifer Yellow showed an increase of coupling in the normal urothelial cell line HCV-29 in contrast to a decrease of coupling in the tumor cell lines. Data were extended by multicellular spheroid (MCS) co-cultures with the stromal fibroblast line N1. In three-dimensional cultures as MCSs, Cx26 was increased in proximity of RT4 tumor cells to fibroblasts, and positivity was maintained in J82 cells. E-cadherin expression in cell lines showed no change in dependence of growth state. The data suggest that Cx26 plays a role in negative growth control or differentiation of urothelial cells. Preliminary comparative data on normal and neoplastic urothelium show all three connexins in normal urothelium, in contrast to varying amounts of Cx43 and low amounts of Cx32 in tumors and evident loss of Cx26 in low-grade tumors. Discrepancies between monolayer and MCS cultures are most likely due to higher differentiation in MCSs, and the continuation of systematic work with heterologous MCSs is indicated for more information on the role of gap-junctional proteins in human tumors.

Blotting, Western↗

PubMed1995

Negative growth control of HeLa cells by connexin genes: connexin species specificity.

In order to examine whether different connexin gene species exert different degrees of tumor-suppressing activity, we characterized growth characteristics of a gap junction-deficient human cancer cell line, HeLa cells, before and after transfection with cDNA for three different connexins, connexin (cx) 26, cx 40, and cx 43. All transfected cell lines (3 clones transfected with the cx 26 gene, 2 clones with cx 40, and 1 with cx 43) showed establishment of gap junctional intercellular communication (GJIC). Two of the cx 26-transfected clones showed significantly slower growth compared with the parental HeLa cells. When transfectants were grown in soft agar, the three cx 26-transfected clones grew much less than the other transfectants and parent HeLa cells. When injected into nude mice, the two cx 26 clones which exhibited the highest amount of cx 26 transcript induced almost no tumors, whereas other transfectants, including the cx 26 clone which exhibited the lowest amount of cx 26 transcript, were tumorigenic. Among transfectants of various connexin genes, there was no good inverse correlation between their GJIC and tumorigenicity. GJIC levels were significantly higher in tumors induced in nude mice by clone cx 26 A and E transfectants. These results suggest that all of the connexin genes examined could induce recovery of GJIC of HeLa cells, but only the cx 26 gene exerts strong negative growth control on HeLa cells; thus, this connexin gene may have different functions from other connexin genes.

Animals↗

PubMed1995

Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells.

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.

Animals↗

PubMed1995

Expression of gap junction genes, connexin40 and connexin43, during fetal mouse development.

The expression patterns of the gap junction genes connexin40 and connexin43 have been analyzed during late mouse fetal development, i.e., at embryonic days 14.5 and 16.5, by in situ hybridization and immunofluorescence. Connexin40 was found in endothelial cells of vessels, cardiomyocytes and in developing myoblasts and myotubes. Expression of connexin40 in developing muscle fibers was strong in the back muscles and weaker in the muscles of the limbs. The number of labeled cells in the back muscle decreased with ongoing differentiation of myoblasts, in accordance with the idea that connexin40 is only expressed in the early stages of muscle cell differentiation. Within a muscle bundle, connexin40 expression was predominantly found at the outermost side where myoblasts fuse to multinucleated myotubes. In contrast, connexin43 exhibits a wide and complex pattern of expression in fetal mouse development. It is found in organs originating from all three germ layers, such as epidermis, heart, lung, muscle, kidney and gut. Connexin43 transcript and protein were very abundant in tissues that had been undergoing inductive interactions, e.g., the inner enamel epithelium of the teeth, the glomeruli of the kidneys and the infundibulum forming the neural part of the pituitary gland. Very high connexin43 expression was found in the embryonic meninges (dura mater) and in the fetal adrenal cortex. During keratinocyte differentiation connexin43 mRNA expression decreased, being much stronger in the stratum basale than in stratum granulosum. No obvious discrepancy between the amount of mRNA and protein of either connexin was noticed, suggesting that there is no specific translational regulation at these developmental stages.

Animals↗

PubMed1995

Long-term effects of brief antihypertensive treatment on systolic blood pressure and vascular reactivity in young genetically hypertensive rats.

Recent studies have shown that angiotensin converting enzyme (ACE) inhibitor treatment in young spontaneously hypertensive rats (SHR) reduces blood pressure into adulthood. This study explored changes in vascular reactivity in adult normotensive (WKY) rats and stroke-prone SHR (SHRSP) receiving the following treatments at 6-10 weeks of age: (a) ACE inhibitor (ramipril); (b) hydralazine/hydrochlorothiazide (hydral/HCTZ); or (c) no treatment. The hypothesis tested was that vascular changes and blood pressure would be reduced in adult SHRSP treated with ramipril during development. At 17 weeks of age, rats were anesthetized and vascular tissue was excised. Isolated experiments in the aorta included characterization of initial phasic and tonic contractions to 0.1 microM angiotensin II (AII). A phenylephrine (PE) concentration-response curve was performed on carotid arteries, and threshold values were determined. All WKY groups showed lower systolic blood pressure (131 +/- 4 mmHg) and reduced phasic AII induced contraction (7.4 +/- 4.7%) compared with SHRSP (217 +/- 4 mmHg; 37.2 +/- 4%). Antihypertensive treatment reduced blood pressure (ramipril: 168 +/- 2; hydral/HCTZ: 198 +/- 6 mmHg) but not phasic AII responses in adult SHRSP; adult WKY rats were unaffected by treatment. Threshold values for PE in carotid arteries were lower in SHRSP than in WKY, indicating increased sensitivity. However, SHRSP treated with ramipril did not demonstrate increased sensitivity to PE. These data support the hypothesis that blood pressure and sensitivity to PE but not contractile responsiveness to AII in adult SHRSP are determined by an AII-sensitive mechanism during development.

Aging↗

PubMed1995

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Animals↗

PubMed1995

Expression of gap junction connexins in the human endometrium throughout the menstrual cycle.

Expression of connexins, the proteins which comprise gap junction channels, is regulated by ovarian hormones in the female reproductive tract of rodents. In order to determine if these hormones also affect connexin expression in the human uterus, the distribution patterns of different connexins (cx26, cx32, cx43) were investigated by immunohistochemistry in human endometrial tissue collected throughout the menstrual cycle. During the early proliferative phase of the cycle extremely low staining for connexin 43 was observed and connexin 26 antigens could not be detected. An increase in the amount of connexin 43 in stromal cells and of connexin 26 in glandular and luminal epithelial cells was seen from days 11-15 of the cycle. Following ovulation, the expression of both connexins was suppressed and was completely abolished in the late secretory phase. Weak staining for connexin 32 was found mainly in the late proliferative and the early secretory phase and was restricted to the basal membrane region of the glandular cells. These results suggest that the different connexins could represent cell biological markers for the proliferation and differentiation of the human endometrium throughout the menstrual cycle.

Connexin 26↗

PubMed1995

Sarcoplasmic reticulum Ca2+ uptake is not decreased in aorta from deoxycorticosterone acetate hypertensive rats: functional assessment with cyclopiazonic acid.

Ca2+ plays a major role in vascular contraction, and a defect in intracellular Ca2+ regulation has been associated with increased vascular reactivity in hypertension. To test the hypothesis that the sarcoplasmic reticulum does not adequately buffer Ca2+ in deoxycorticosterone acetate (DOCA) hypertension, contractile experiments were performed with a specific inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, cyclopiazonic acid (CPA). Contractile force in aortic strips from DOCA and control rats was measured, using standard muscle bath procedures, to evaluate (i) Ca2+ handling, assessing caffeine and serotonin (5HT) induced contractions in Ca(2+)-free buffer and (ii) relaxation rate after 5HT washout. Contractile responses elicited with 5HT (3 x 10(-6) mol/L) and caffeine (20 mmol/L) were greater in DOCA than in control arteries. CPA (1 x 10(-7) to 3 x 10(-5) mol/L) reduced phasic contractions to 5HT and caffeine in DOCA and control aorta, and no differences in the IC50 values were observed. Aortae from DOCA rats contracted when placed in normal buffer, subsequent to treatment with Ca(2+)-free buffer, but control aortae did not. CPA potentiated these responses in DOCA aorta and only caused a modest contraction in control aorta. CPA-induced contraction did not occur in Ca(2+)-free buffer, and it was inhibited by nifedipine (IC50 = 4 x 10(-9) mol/L). The relaxation rate, after 5HT washout (3 x 10(-6) mol/L), was increased in DOCA aorta (2.6 +/- 0.3 min) compared with control (1.7 +/- 0.2 min), and CPA (10(-5) mol/L) increased the relaxation rate in both groups. The results support the hypothesis of defective Ca2+ handling in DOCA hypertension. However, an increased Ca2+ influx, and not a decreased buffering ability of the sarcoplasmic reticulum, contributes to the enhanced vascular reactivity observed in DOCA hypertension.

Animals↗

PubMed1995

EDRF and norepinephrine-induced vasodilation in the canine coronary circulation.

Experiments were performed to characterize the role of endothelium-derived relaxing factor (EDRF) in coronary vasodilation caused by norepinephrine. The circumflex or left anterior descending coronary artery was cannulated and pump perfused with constant pressure or constant flow in closed-chest anesthetized dogs. Prostaglandin synthesis was blocked with ibuprofen. During constant-pressure perfusion, EDRF inhibition with intracoronary N omega-nitro-L-arginine (L-NNA) did not affect the vasodilation due to nitroglycerin (an endothelium-independent process). However, L-NNA did significantly inhibit (P < 0.001) the vasodilation due to acetylcholine (an endothelium-dependent process). In response to bolus injections of norepinephrine, EDRF inhibition with L-NNA significantly reduced coronary vasodilation (P < 0.001). This inhibition was partially reversed with L-arginine infusion. To determine whether an increase in shear stress due to an increase in flow was the stimulus for EDRF release, experiments were performed during constant-flow conditions. Vasodilation caused by nitroglycerin was not inhibited by L-NNA, but EDRF inhibition did blunt acetylcholine-induced vasodilation significantly (P < 0.001) during constant-flow perfusion. During EDRF inhibition with L-NNA, vasodilation due to norepinephrine was not significantly altered when coronary flow was held constant (P = 0.19). In conclusion, EDRF plays a role in norepinephrine-induced coronary vasodilation that is largely flow dependent.

Acetylcholine↗

PubMed1995

Role of nitric oxide in insulin-dependent diabetes mellitus-related vascular complications.

Patients with insulin-dependent diabetes mellitus are at high risk for vascular disorders such as hypertension, nephropathy, and retinopathy. The most common cause of morbidity and mortality in patients with insulin-dependent diabetes is vascular disease. Despite ongoing research, the pathogenesis of vascular disease in diabetes remains unclear. In recent years, numerous investigators have examined the role of the endothelium-derived relaxing factor, nitric oxide, in the disease state of hypertension and its complications. We review the role of nitric oxide in the development of diabetes-related vascular disease and discuss findings suggesting that nitric oxide metabolism and vascular responsiveness to nitric oxide are altered in diabetes. Patients with diabetes may benefit from therapy that addresses this pathogenic deficiency.

Diabetes Mellitus, Type 1↗