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B J Nicholson

Publications and source records attributed to B J Nicholson.

50 records · Page 3Linked to original sources
PubMed1990

Retinoid-enhanced gap junctional communication is achieved by increased levels of connexin 43 mRNA and protein.

Natural and synthetic retinoids are potent inhibitors of experimental carcinogenesis in animals and cause reversion of premalignant lesions in humans. In the model C3H 10T1/2 cell system, retinoids enhance postconfluent growth control, reversibly inhibit carcinogen-induced transformation, and enhance gap junctional intercellular communication. These effects are highly correlated. 10T1/2 cells were found to express low levels of connexin 43, a gap junctional protein first found in the heart. After treatment of confluent 10T1/2 cells with the synthetic retinoid tetrahydrotetramethylnapthalenylpropenylbenzoic acid (TTNPB), levels of connexin 43 mRNA and protein increased within 6 h of treatment, while elevation of junctional communication was detected within 12-18 h. The maximally effective concentration of TTNPB (10(-8) M) caused an approximate 10-fold elevation of connexin 43 gene transcripts after 72 h. Indirect immunofluorescence microscopy using a polyclonal antibody to the synthetic C-terminal region of connexin 43 demonstrated that TTNPB induced many fluorescent plaques in regions of cell-cell contact. These results provide a molecular basis for the retinoid-enhanced junctional communication in 10T1/2 cells. It is proposed that one action of retinoids is to modulate the intercellular transfer of signal molecules. These could mediate many of the physiological actions of retinoids on growth control and carcinogenesis.

Animals↗
PubMed1989

Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.

While a number of different gap junction proteins have now been identified, hepatic gap junctions are unique in being the first demonstrated case where two homologous, but distinct, proteins (28,000 and 21,000 Mr) are found within a single gap junctional plaque (Nicholson, B. J., R. Dermietzel, D. Teplow, O. Traub, K. Willecke, and J.-P. Revel. 1987. Nature [Lond.]. 329:732-734). The cDNA for the major 28,000-Mr component has been cloned (Paul, D. L. 1986. J. Cell Biol. 103:123-134) (Kumar, N. M., and N. B. Gilula. 1986. J. Cell Biol. 103:767-776) and, based on its deduced formula weight of 32,007, has been designated connexin 32 (or Cx32 as used here). We now report the selection and characterization of clones for the second 21,000-Mr protein using an oligonucleotide derived from the amino-terminal protein sequence. Together the cDNAs represent 2.4 kb of the single 2.5-kb message detected in Northern blots. An open reading frame of 678 bp coding for a protein with a calculated molecular mass of 26,453 D was identified. Overall sequence homology with Cx32 and Cx43 (64 and 51% amino acid identities, respectively) and a similar predicted tertiary structure confirm that this protein forms part of the connexin family and is consequently referred to as Cx26. Consistent with observations on Cx43 (Beyer, E. C., D. L. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629) the most marked divergence between Cx26 and other members of the family lies in the sequence of the cytoplasmic domains. The Cx26 gene is present as a single copy per haploid genome in rat and, based on Southern blots, appears to contain at least one intron outside the open reading frame. Northern blots indicate that Cx32 and Cx26 are typically coexpressed, messages for both having been identified in liver, kidney, intestine, lung, spleen, stomach, testes, and brain, but not heart and adult skeletal muscle. This raises the interesting prospect of having differential modes of regulating intercellular channels within a given tissue and, at least in the case of liver, a given cell.

Amino Acid Sequence↗
PubMed1987

The cardiac gap junction protein (Mr 47,000) has a tissue-specific cytoplasmic domain of Mr 17,000 at its carboxy-terminus.

The molecular weight of the heart gap junctional protein subunit was, until recently, believed to be about Mr 28,000-30,000, similar to that of other previously characterized gap junctional proteins. A larger polypeptide of about Mr 44,000-47,000, which undergoes proteolysis during isolation, has recently been proposed as the form of the heart junction protein in vivo. We show here that this entity has the same amino-terminal sequence as the previously characterized Mr 29,000-30,000 component. Thus, the cardiac junctional protein has, at its carboxy-terminus, cytoplasmic domain of Mr 17,000; this domain is absent in the liver protein. These observations provide further evidence that gap junction proteins form a highly diversified family.

Amino Acid Sequence↗
PubMed1985

The Mr 28,000 gap junction proteins from rat heart and liver are different but related.

The sequence of the amino-terminal 32 residues of the rat heart Mr 28,000 gap junction protein presented here allows, for the first time, a sequence comparison of gap junctional proteins from different tissues (heart and liver). Comparison of the rat heart gap junction protein sequence and that available from rat liver reveals 43% sequence identity and conservative changes at an additional 25% of the positions. Both proteins exhibit a hydrophobic domain which could represent a transmembrane span of the junction. This result unequivocally demonstrates the existence of at least two forms of the gap junction protein. As yet, no homology is evident between the gap junctional proteins of either heart or liver and main intrinsic protein from rat eye lens.

Amino Acid Sequence↗
PubMed1984

Molecular organization of gap junctions.

Highly purified gap junction fractions from heart and liver contain a single major protein component. The proteins isolated from different organs have apparent molecular weights of 26,000-30,000. Peptide mapping and partial sequencing show close homology of the hepatic junctional protein of different species. In contrast, no homologies can be detected when polypeptides from different tissues of the rat were compared by peptide mapping. Preliminary results from partial sequencing, however, show that the amino terminal regions of the liver and heart proteins are related to one another. Sequencing has not yet revealed any such homologies between the lens and the other junction proteins.

Animals↗
PubMed1983

Major intrinsic polypeptide of lens membrane. Biochemical and immunological characterization of the major cyanogen bromide fragment.

A protein of Mr 26000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the Mr 26000 polypeptide from bovine lenses yields a major fragment of Mr 15000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the Mr 26000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the Mr 26000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the Mr 26000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the Mr 26000 polypeptide is buried within the lipid bilayer.

Amino Acid Sequence↗
PubMed1983

Differences between liver gap junction protein and lens MIP 26 from rat: implications for tissue specificity of gap junctions.

Liver gap junctions and gap-junction-like structures from eye lenses are each comprised of a single major protein (Mr 28,000 and 26,000, respectively). These proteins display different two-dimensional peptide fingerprints, distinct amino acid compositions, nonhomologous N-terminal amino acid sequences and different sensitivities to proteases when part of the intact junction. However, the junctional protein of each tissue is well conserved between species, as demonstrated previously for lens and now for liver in several mammalian species. The possiblity of tissue-specific gap junction proteins is discussed in the light of data suggesting that rat heart gap junctions are comprised of yet a third protein.

Amino Acid Sequence↗
PubMed1983

Comparative analysis of the gap junction protein from rat heart and liver: is there a tissue specificity of gap junctions?

Gap junctions have been isolated from both rat heart and liver, tissues where junctions are typical in appearance and physiology. The purity of the fractions obtained was monitored by electron microscopy (thin-sectioning and negative staining) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The myocardial gap junctions are comprised of a single polypeptide of Mr 28,000, apparently derived from a protein of Mr 30,000. Hepatic gap junctions are also comprised of a single native protein of Mr 28,000 as previously reported. Exhaustive trypsin digestion of the isolated junctions cleaves both of these proteins similarly, while leaving their characteristic junctional lattice structures intact. However, comparison of heart and liver junctional proteins by two-dimensional peptide mapping of tryptic and alpha-chymotryptic fragments, followed by high pressure liquid chromatography, reveals no homology between these proteins.

Animals↗
PubMed1981

The dynamic state of liver gap junctions.

By the use of a simple, rapid method for the isolation of gap junctions from small amounts of rat liver (2-3 g), we have followed the incorporation of the radiolabeled amino acid precursors 3H-leucine and 35S-methionine into the gap junction protein. In timed studies with 35S-methionine as precursor, the specific activity in the protein is maximal by 4 h after a single injection of 300 microCi/100 g body weight. From the decay in the specific activity with time after a single injection, the gap junction protein has an apparent half-life of about 19 h. Because of problems of reutilization of radiolabeled amino acid with 35S-methionine as precursor, this apparent half-life probably overestimates the true half-life and indicates a surprisingly rapid turnover of the gap junction protein. This short half-life suggests that, in rat liver, the gap junctions may be very responsive to alterations in physiological demands.

Animals↗
PubMed1981

Rat liver gap junction protein: properties and partial sequence.

Gap junctions, strongly implicated as channels for direct cell-to-cell communication, have been isolated from rat liver in high yield and purity. These gap junction fractions contain few morphologically recognizable contaminants, but NaDodSO4/polyacrylamide gel electrophoresis reveals a number of polypeptides. With the exception of a nonjunctional component of Mr 38,000 and some poorly soluble material, including collagen, all the polypeptides have very similar or identical two-dimensional peptide maps and arise from proteolytic cleavage of the COOH-terminus or aggregation of a Mr 28,000 protein. We report the sequence of the NH2-terminal 52 amino acids of this protein. The polypeptide (Mr approximately equal to 10,000) characteristic of trypsin-treated gap junction preparations is shown to be two distinct polypeptides, both derived from the Mr 28,000 protein.

Amino Acid Sequence↗
PubMedDate not supplied

Connexin expression and cell coupling fail to reverse the v-src transformed growth characteristics of a Cx43-/- cell line.

Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60v-src was confirmed in all transfectants as well as the ability of pp60v-src to phosphorylate Cx43 in several clones. Despite the documented effect of pp60v-src on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.

Animals↗