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O Traub

Publications and source records attributed to O Traub.

At least 91 records · Page 5Linked to original sources
PubMed1991

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.

Animals↗
PubMed1991

Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.

Animals↗
PubMed1989

Differential expression of three gap junction proteins in developing and mature brain tissues.

By using antibodies directed against gap junction proteins of liver (connexins 26 and 32) and heart (connexin 43), we have localized immunoreactivity to specific cell types in frozen sections of adult rodent brains. Connexin 32 reactivity was found in oligodendrocytes and also in a few neurons, whereas reactivity to connexins 26 and 43 was localized to leptomeningeal cells, ependymal cells, and pineal gland. Immunoreactivity with antibodies to connexin 43 also occurred in astrocytes. Furthermore, during embryonic and postnatal maturation of brain tissues, gap junction proteins were differentially expressed. Connexins 43 and 26 predominated in the neuroepithelium of embryonic brains, whereas connexin 32 was virtually absent. Between 3 and 6 weeks after birth, connexin 26 largely disappeared from immature brain; this time course corresponded to the increased expression of connexin 32. Expression of connexin 43 remained high throughout embryonic and postnatal development. These findings demonstrate that gap junction expression in the brain is diverse, with specific cell types expressing different connexins; this cell-specific distribution may imply differences in the function of these intercellular channels in different loci and developmental stages.

Aging↗
PubMed1989

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.

Animals↗
PubMed1987

The Ha-ras-induced transformed phenotype of rat-1 cells can be suppressed in hybrids with rat embryonic fibroblasts.

Somatic cell hybrids were isolated from fusions of diploid embryonic rat fibroblasts with transformed Rat-1 cells which contained 4 to 5 copies of the transforming human Ha-ras 1 gene. In contrast to their transformed parental cells four hybrid clones showed normal morphology, long latency periods of tumorigenicity in newborn rats, anchorage requirement of proliferation, and an eightfold-reduced amount of secreted transforming growth factor activity. Thus these hybrids are called suppressed with regard to expression of the Ha-ras-induced transformed phenotype. Tumorigenic derivatives of the suppressed hybrids that had segregated chromosomes were isolated. Since two of the tumorigenic hybrid clones showed the similar low level of secreted transforming growth factors as the suppressed hybrids, decreased production of transforming growth factor activity is unlikely to be a sufficient criterion for suppression of malignancy. Whereas one of the suppressed hybrids expressed the transforming gene product p21 at a level similar to that of the transformed parental cells, other suppressed hybrids expressed less p21. This suggests that the suppressed phenotype can be regulated at the posttranslational level of p21 but that additional controls of expression of p21 are likely to exist. DNA of the suppressed hybrids transformed Rat-1 cells to proliferation in the presence of semisolid agar. Thus the activated human Ha-ras gene in the suppressed hybrids retained its biological activity even though it did not transform these cells to tumorigenicity.

Animals↗
PubMed1987

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.

Animals↗
PubMed1987

Regulation of pancreatic exocrine function: a role for cell-to-cell communication?

The effect of heptanol, an alkanol which decreases gap junctional permeability, was investigated in the perfused rat pancreas. Under basal conditions, heptanol (3.5 mM) caused a three- to fourfold increase of pancreatic juice, protein, and amylase outputs. The effect on enzyme secretion was fully reversible upon removal of the alkanol and was not secondary to the release of acetylcholine from nerve endings, since it was not prevented by addition of atropine (10(-6) M) to the perfusate. By contrast, another alkanol, hexanol (3 mM), which does not decrease coupling between acinar cells in spite of anesthetic properties analogous to those of heptanol, did not alter pancreatic secretion. The effect of heptanol was not mediated by a significant stimulation of cyclic AMP, nor did the alkanol increase the secretion of lactic dehydrogenase, a cytosolic marker. Analysis of the numerical density of freeze-fractured and immunolabeled gap junctions between acinar cells did not show differences between heptanol-perfused and control pancreases. In addition, heptanol did not alter carbachol (10(-6) M and 10(-5) M)-evoked amylase release. Since heptanol blocks cell coupling, apparently without interfering with the main intracellular pathways triggering enzyme release, we suggest that downregulation of direct cell-to-cell communications increases pancreatic exocrine secretion. Thus, cell coupling is probably involved in the regulation of the secretory activity of acinar cells.

Alcohols↗
PubMed1987

Simultaneous light and electron microscopic observation of immunolabeled liver 27 KD gap junction protein on ultra-thin cryosections.

We report on immunolabeling of gap junction protein in rat liver. Simultaneous light and electron microscopic immunolabeling of ultra-thin frozen sections was performed to confirm that the antigenic targets of polyclonal antibodies and a monoclonal 27 KD antibody (12/1 C5) are the gap junctions. Our results clearly demonstrate that the immunoreactive sites determined by indirect immunofluorescence correspond to immunogold-labeled gap junctions identified in the same section according to electron microscopic criteria. Our results also support the concept that the 27 KD protein is a major constituent of gap junctions.

Animals↗
PubMed1987

Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes.

Hepatocytes prepared from 18-day-old mouse embryos were grown in serum-free medium and reached confluence after two days in culture. The total amount of the 26 kDa gap junction protein decreased in these cells during the first 24 h in culture and increased again between day 1 and day 3 more than 10-fold. At day 3 a half-life time of 2.5 to 3 h was determined for the 26 kDa protein by [35S]methionine incorporation and immunoprecipitation using affinity-purified anti-26 kDa. Incorporation of [32P]orthophosphate into the 26 kDa protein of cultured hepatocytes was found at serine residues (98%) and tyrosine residues (about 2%). The addition of dibutyryl cyclic adenosine monophosphate (db cAMP) to the culture medium at day 2 had two effects: After 15 min the extent of phosphorylation of the 26 kDa protein increased 2.7-fold whereas the total amount of the 26 kDa protein increased only 1.2-fold. After 3 h of incubation with db cAMP, a 2.5-fold increase of the 26 kDa protein was noticed which was accompanied by a 3.2-fold increase in phosphorylation of serine residues. The effects of db cAMP on phosphorylation of the 26 kDa protein could be augmented or mimicked by the addition of isoproterenol, theophylline or forskolin to the culture medium of hepatocytes. In extracts of rat hepatocarcinoma MH1C1 cells and dog kidney MDCK cells, a phosphorylated 26 kDa protein can be immunoprecipitated using anti-liver 26 kDa. These results demonstrate that the gap junction 26 kDa protein can be posttranslationally modified by cAMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Suppression and re-expression of transformed phenotype in hybrids of HA-ras-1-transformed rat-1 cells and early-passage rat embryonic fibroblasts.

Rat-1 cells which had been transformed with the activated Ha-ras-1 gene from human EJ bladder carcinoma cells were fused with diploid embryonic rat fibroblasts. Four selected cell hybrids expressed the human transforming gene product p21 at levels of 10 to 30% compared to 100% in the transformed parental cells. The hybrid cells, however, exhibited normal morphology, anchorage requirement for proliferation, and largely extended latency periods of tumorigenicity in newborn rats. Tumorigenic hybrid derivatives contained lower numbers of chromosomes than the tetraploid parental hybrids. DNA of the non-tumorigenic cell hybrids transformed Rat-1 cells to anchorage-independent proliferation as expected for the transforming human Ha-ras gene present in the donor DNA. We conclude that the transforming properties of the activated Ha-ras gene in Rat-1 cells can be suppressed at the post-translational level by the presence of the genome from diploid embryonic rat fibroblasts but additional controls of expression of the transforming gene are likely to exist. Normal cells contain suppressor gene(s) which safeguard these cells against transformation by the product of the transforming Ha-ras-1 oncogene.

Animals↗
PubMed1986

Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein.

By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions.

Amino Acid Sequence↗
PubMed1986

Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody.

A new rat monoclonal antibody was characterized which recognized the 26K protein in gap junctions of mouse, rat and human liver as shown by immunoblot, indirect immunofluorescence, and immunogold electron microscopy. This monoclonal antibody was used to investigate the abundance of gap junctions in chemically induced rat hepatocarcinomas. In comparison with livers of control animals we found in hepatocarcinomas an average decrease of 71% in the number of gap junctional immunofluorescent spots. A corresponding decrease of the total amount of the 26 K protein was detected by quantitative immunoblot. Changes in the proliferative state as well as in intercellular adhesion of hepatocarcinoma cells in contrast to normal hepatocytes might have contributed to cause this decrease of gap junctions in tumor tissue. Possibly the partial loss of gap junctions provided a selective advantage for those preneoplastic liver cells which developed into rapidly proliferating tumor cells.

Animals↗
PubMed1985

Transforming activity of DNA fragments from normal human lymphocytes results from spontaneous activation of a c-Ha-ras1 gene.

An activated human Ha-ras gene was present in a secondary NIH 3T3 transformant isolated after serial transfection of originally low-molecular-weight DNA fragments from normal human cells. This gene appeared to have acquired its transforming properties by a spontaneous mutation in codon 12 by substitution of a deoxythymidine residue for a deoxyguanosine residue. DNA rearrangements in the flanking sequences of the transferred Ha-ras gene were not involved in the activation of the protooncogene.

Animals↗
PubMed1984

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.

Animals↗
PubMed1984

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying.

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.

Animals↗
PubMed1983

Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes.

Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.

Animals↗
PubMed1983

Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis.

Changes in the total amount of the gap junction protein (M(r) 26,000) after partial hepatectomy or bile duct ligation and recanalization were investigated in rat liver membranes by quantitative immunoblot with rabbit antiserum to the M(r) 26,000 protein. The loss and reappearance of the M(r) 26,000 protein roughly paralleled loss and reappearance of gap junction plaques analyzed previously under similar physiological conditions by freeze-fracture of hepatocyte surfaces. The total amount of the hepatic M(r) 26,000 protein in liver plasma membranes and the total area of the hepatocyte surface occupied by gap junction plaques appeared to be proportional under these conditions. However, at the minimum, 28-35 hr after partial hepatectomy we still find about 15% of the M(r) 26,000 protein, in contrast to <1% of gap junction plaques, determined by morphometric analysis. This discrepancy is probably due to the fact that very small gap junction plaques, single connexons, and free M(r) 26,000 gap junction subunits are missed by the morphometric analysis. At the times of the minimal amount of the M(r) 26,000 protein in hepatic plasma membranes after partial hepatectomy or bile duct ligation we found that crude hepatic lysosomal membranes of these rats contained less M(r) 26,000 protein than lysosomal membranes of nonoperated control animals. Thus, we conclude that the decrease and increase of the total amount of the M(r) 26,000 protein cannot be explained only by dispersal and reuse of gap junction subunits but are largely due to degradation and resynthesis of the M(r) 26,000 protein. No significant change in the amount of the M(r) 21,000 protein that had been isolated with gap junction plaques was observed in liver plasma membranes after partial hepatectomy. This confirms our previous conclusion that the M(r) 26,000 and M(r) 21,000 proteins are independent of each other.

Bilirubin↗