Biomedical subjects

K Willecke

Publications and source records attributed to K Willecke.

At least 109 records · Page 6Linked to original sources

PubMed1992

Chromosomal assignments of mouse connexin genes, coding for gap junctional proteins, by somatic cell hybridization.

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse x Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.

Animals↗

PubMed1992

The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43.

This investigation was initiated in order to find out whether expression of the hepatocyte-specific phenotype is accompanied by expression of certain connexin genes coding for gap junctional protein subunits. Several clones of mouse embryonic hepatocytes immortalized in serum-free MX83 medium by infection with recombinant retrovirus-expressed transcripts for connexin32, connexin26, albumin, alpha-fetoprotein, tyrosine aminotransferase, as well as aldolase A and B, at more than half of the levels found in primary mouse hepatocytes. In addition the immortalized hepatocyte clones contained low levels of connexin43 mRNA of which only trace amounts were detected in primary embryonic mouse hepatocytes and in rat liver. Two of the immortalized hepatocyte clones were shifted from serum-free MX83 medium to Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum and, after 2, 14, or 180 days, back to MX83 medium. We found that expression of connexin32 and connexin26 mRNAs as well as transcripts of other liver-specific proteins was reversibly decreased in serum-containing medium, whereas the expression level of connexin43 transcripts was increased in serum-containing DMEM compared to serum-free MX83 medium. The expression levels of connexin26, connexin32, or connexin43 mRNAs were altered by the addition of fetal calf serum or arginine or by the absence of hydrocortisone in MX83 medium, all of which contributed to the shift in phenotype. Furthermore several dedifferentiated cell lines derived from rat or mouse liver and cultivated in serum-containing medium were found to express little connexin32 or connexin26 mRNA but relatively high levels of connexin43 mRNA.

Animals↗

PubMed1992

Suppression of transformed phenotype in hybrids of v-fgr and v-raf transformed rat-1 cells with rat embryonic fibroblasts is due to transcriptional inactivation of viral oncogenes.

Rat-1 cells that had been transformed to tumorigenicity by transfection with the retroviral oncogenes v-raf from 3611-murine sarcoma virus, or v-fgr from Gardner-Rasheed feline sarcoma virus were fused with rat embryonic fibroblasts at an early passage. In both fusion experiments hybrid cells were isolated that exhibited normal morphology, anchorage requirement for proliferation, and either no tumorigenicity (v-fgr) or extended latency periods for tumor growth (v-raf) in nude mice. Transcription of viral oncogenes is drastically reduced in hybrid cells (at least 30-fold compared to their transformed parental cells), while the half-life of the corresponding transcripts is not effected. In the chromatin of hybrid cells the integrated retroviral oncogenes are as sensitive to degradation with pancreatic DNase I as the endogenous actin gene. Thus the observed down regulation of proviral transcript levels does not correlate with changes in chromatin structure. We conclude that in hybrids of (v-fgr)- and (v-raf)-transformed Rat-1 cells with embryonic fibroblasts, transcription of the retroviral oncogenes appears to be repressed by trans-acting factors of the normal parental cell.

Animals↗

PubMed1992

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung.

From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (alpha subgroup of connexins) than to mouse Cx32 (beta subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lung-similar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two- to fourfold higher than in the corresponding adult tissues. Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +/- 35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (tau = 580 ms at Vj of +/- 50 mV). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

Amino Acid Sequence↗

PubMed1992

Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs.

In an attempt to characterize connexin genes expressed early in mouse development we screened a cDNA library from mouse F9 embryonic carcinoma cells with mouse connexin37 cDNA and mouse connexin31.1 genomic DNA under low stringency of hybridization. We detected 5 different connexin cDNAs coding for mouse connexins31, -31.1, -32, -43, and -45 (reviewed in Willecke et al., Eur. J. Cell Biol. 56, 1-7 (1991)). Here we describe characterization of mouse connexin31 cDNA coding for a protein of 270 amino acids (Mr 30,905) that shows 8 amino acid exchanges compared to its rat analog recently deduced from its genomic sequence. Mouse connexin45 cDNA codes for a protein of 396 amino acids (Mr 45,671) that exhibits 84% amino acid identity compared to its chick analog described. Cx31 and Cx45 are coded for by single genes in the mouse genome. After Northern blot hybridization, we detected two Cx31 transcripts of 1.9 and 2.3 kb in total mouse RNA from skin, keratinocyte-derived cell lines, and in testis. Cx45 cDNA hybridized to a 2.2 kb mRNA in lung, brain, skin, heart, and intestine. This transcript showed maximal expression in adult lung and in embryonic tissues tested (brain, skin, kidney) where it was at least 40-fold more abundant than in the corresponding adult tissues.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of mouse connexins26 and -32: similar genomic organization but distinct promoter sequences of two gap junction genes.

Connexins26 and -32 are subunit proteins of gap junctions that are coexpressed in hepatocytes and several tissues but individually expressed in other cells. Molecular cloning of both corresponding mouse genes revealed similar genomic organization, i.e., each gene consists of two exons with the complete coding region located in the second exon. The first exon of each gene is preceded by a TATA-less promoter region. The promoter of the mouse Cx26 gene has at least two transcription start sites and is located in a very GC-rich region which is reminiscent of promoters of house-keeping genes. Putative consensus sequences for a metal response element, the transcription factor NFkappaB, and several GC-boxes were found within 600 bp upstream of the Cx26 transcription start sites. The promoter region of the mouse Cx32 gene contains two putative binding sites for the transcription factor HNF-1 and consensus motifs for NF-1 as well as NFkappaB within 680 bp upstream of the main transcription start site. Thus the sequence comparison of mouse Cx26 and Cx32 promoter regions provides hints for possible consensus elements that could control individual expression as well as common regulation of these gap junction genes in various tissues. Cx26 mRNA is much more abundant in adult mouse skin than in adult kidney and liver where Cx32 transcripts are relatively strongly expressed.

Amino Acid Sequence↗

PubMed1991

Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions.

We have studied the proliferation of rat 208F cells (a derivative of Rat-1 cells) transformed by activated c-Ha-ras, v-fgr, v-raf, v-fms, or v-src oncogenes during cocultivation with an excess of early passage rat embryonic fibroblasts or immortal 208F cells. The total number and size of foci formed by oncogene-transformed 208F cells were strongly reduced by cocultured normal fibroblasts. The extent of growth suppression of transformed foci appears to be dependent on the type of transforming oncogene and on the type of normal fibroblasts rather than on the extent of gap-junctional communication between transformed and normal cells. Total inhibition of fluorescent dye transfer between normal and transformed cells by the 3 beta-O-hemisuccinate of 18 alpha-glycyrrhetinic acid (18 alpha-carbenoxolone), an inhibitor of gap-junctional communication in human fibroblasts, did not prevent growth inhibition of transformed cells in the cocultivation assay. Since adjacent cells remained electrically coupled in the presence of this inhibitor it is possible that the strongly reduced metabolic cooperation, as indicated by the lack of fluorescent dye transfer, is sufficient for mediating the growth-inhibitory effect of normal fibroblasts. 208F cell-conditioned medium, however, also caused strong growth inhibition of transformed derivatives, suggesting that the effect is in part mediated by release of stable growth inhibitor(s) from 208F cells.

Animals↗

PubMed1991

In vivo modulation of connexin 43 gene expression and junctional coupling of pancreatic B-cells.

We have explored the expression of gap junctional proteins and corresponding mRNAs by insulin-producing B-cells of native rat pancreas and of a transplantable rat insulinoma. By immunostaining cryostat sections (indirect immunofluorescence) and crude membrane preparations (Western blots) with antibodies against connexins 26, 32, and 43 and by hybridizing total islet and insulinoma RNA (Northern blot) with cRNAs for the latter two proteins, we have found that normal and tumoral B-cells express connexin 43 but do not show detectable levels of either connexin 32 or 26. By evaluating the conductance (dual patch-clamp whole-cell recording) and permeability of junctional channels (microinjection of Lucifer yellow), we have found that control B-cells show low levels of electrical and dye coupling in only a portion of the pairs studied. By studying B-cells of glibenclamide-treated rats, we have found that sustained stimulation of insulin release in vivo is associated with a two-fold increase in the level of connexin 43 gene transcripts and in the incidence of both ionic and dye coupling. These observations indicate that (1) connexin 43 is a major component of communicating channels between insulin-producing cells; (2) some but not all B-cells are electrically coupled by low conductance junctional channels; and (3) connexin 43 gene transcripts and incidence of junctional coupling are modulated in parallel during sustained stimulation of B-cell functioning in vivo.

Animals↗

PubMed1991

Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung.

The coding sequence (333 amino acids) of a new connexin protein, designated mouse connexin37 (Cx37 or Cx37.6) due to the deduced theoretical molecular mass of 37.600 kD, has been determined from cDNA and genomic clones. As seen in other connexins, its gene has no introns within the coding region and the deduced amino acid sequence is predicted to have similar topology to other connexins that form intercellular channels. The amino acid sequence of mouse Cx37 is most similar to rat connexin43 (59% identity) and Xenopus connexin38 (66% identity) when compared from the NH2 terminus to the end of the fourth putative transmembrane region. When expressed in Xenopus oocytes Cx37 forms functional intercellular channels that exhibit more sensitive and rapid gating in response to voltage than any previously characterized vertebrate gap junction. Under stringent conditions the Cx37 cDNA hybridizes to an mRNA of 1.7 kb that is found highly abundant in lung and to progressively lesser extents in brain, kidney, skin, spleen, liver, intestine, and heart. Embryonic brain, kidney, and skin express two to fivefold higher levels of the Cx37 transcript than the corresponding adult tissues. Cx37 transcripts were also found to increase two to threefold in response to retinoic acid treatment of cultured embryonic carcinoma F9 cells.

Amino Acid Sequence↗

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↗

PubMed1991

The diversity of connexin genes encoding gap junctional proteins.

The multigene family of connexins is larger than previously anticipated. Ten different connexin homologous sequences have been characterized in the mouse genome, five of which are probably the mouse analogues of the known rat connexins26, -31, -32, -43, and -46. Since the additional 5 sequences have been isolated as cDNAs or hybridize specifically to distinct mRNA species, they most likely represent functional connexin genes. Since seven of the genomic connexin sequences have been shown to contain no intron in the coding sequence, this may apply to all mammalian connexin genes. Some of the structural features based on amino acid sequences deduced from cDNA or genomic sequences and the RNA expression pattern of the new connexins are compared with previously described connexins. The structural diversity of the connexin genes suggests that they fulfill different functions coordinated with, and perhaps required for, different programs of cellular differentiation.

Amino Acid Sequence↗

PubMed1990

[Human lymphocyte transformation after immortalization by transfection with cytoplasmic DNA from mouse L cells of the cytokines IL-1alpha, IL-6 and TNF].

Human lymphocytes from peripheral blood were induced to proliferate indefinitely in vitro by transfection with cytoplasmic DNA isolated from transformed mouse L929 cells. Two cell lines analyzed proliferate in chemically defined, serum-free media without addition of cytokines. Using RIA and bioassays, the cells were found to secrete IL-1 alpha, IL-6, and TNF into the culture medium whereas no IL-2, IL-4, BCGF, CSF, or IFN-gamma were detected. Northern blot hybridizations revealed TNF alpha mRNA (1.5 kb) as well as TNF beta mRNA (1.4 kb) transcripts in RNA of the immortalized cells. Since the immortalized lymphoid cell lines neither form colonies in soft agar medium nor induce tumors after injection into immunodeficient nude mice, we suggest that these cytokines may be involved in autocrine growth stimulation of human lymphoid cell lines immortalized by transfection with cytoplasmic DNA from mouse L-cells.

Animals↗

PubMed1990

Six genes of the human connexin gene family coding for gap junctional proteins are assigned to four different human chromosomes.

Connexin genes code for proteins that form cell-to-cell channels known as gap junctions. The genes for the known connexins 26, 32, 43, and 46 have been assigned to human chromosomes, 13, X, 6, and 13, respectively, by analysis of a panel of human-mouse somatic cell hybrids using rat cDNA probes. A pseudogene of connexin 43 that lacks an intron of the cx43 gene has been located on human chromosome 5. Furthermore, the genes of the two new connexins 37 and 40 have both been assigned to human chromosome 1. Thus the human chromosomes 1 and 13 each carry at least two different connexin genes. Their exact location on these chromosomes is not yet known. From our results subchromosomal assignments can be deduced for the human cx32 gene to Xq13-p11, the human cx37 gene as well as the human cx40 gene to 1pter-q12, and the human cx43 gene to 6q14-qter. The generation of the connexin multigene family from a hypothetical ancestral connexin gene is discussed.

Blotting, Southern↗

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↗

PubMed1988

Immortalization of human lymphocytes by transfection with DNA from mouse L929 cytoplasts.

Transfection of human peripheral blood lymphocytes with DNA from mouse L929 cytoplasts induced proliferation of lymphocytes and the formation of B and T cell-derived cell lines with apparently unlimited growth potential. The cell lines could be grown in serum-containing media as well as in chemically defined serum-free media, have a nearly normal human karyotype, did not form colonies in soft-agar medium, and were not tumorigenic after injection into nude mice. For immortalization of human lymphocytes, DNA from L929 cytoplasts was 100-fold more efficient than L929 nuclear DNA. The ability of cytoplast DNA to immortalize lymphocytes could be consecutively transferred by using total cellular DNA from primary or secondary transfectants. Circular or linear mitochondrial DNA of L929 cells did not lead to immortalization of human lymphocytes. Since DNA with immortalizing activity could be isolated from cytoplasts, the Hirt supernatant, and a mitochondria-depleted cytoplasmic fraction of L929 cells, we conclude that the immortalizing DNA is located extramitochondrially in the cytoplasm of L929 cells.

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↗