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1.
Neuropsychol Rehabil ; 29(1): 144-159, 2019 Jan.
Article in English | MEDLINE | ID: mdl-28051902

ABSTRACT

This study examined the use of the Hong Kong version of the Rivermead Behavioral Memory Test-Third Edition (RBMT-3) for older adults, and by presenting the optimal cut-off scores for patients with cognitive impairments, and for a group of peers who have functional everyday cognition. Hundred older adults residing in community dwellings were recruited from three non-government organisations and completed the RBMT-3: 29 patients with mild to moderate dementia, 34 persons at risk for MCI, and 37 matched older adults with everyday functional cognition for a healthy control group (NC). The test has excellent inter-rater (ICC [2, 1] = 0.997), intra-rater (ICC [3, 1] = 0), and parallel version (ICC [3, 1] = 0.990) reliabilities, as well as satisfactory internal consistency (Cronbach's alpha: 0.643-0.832). The scores of the MCI group were significantly lower than those of NC group in four subtests. The optimal cut-off scaled scores of ≤ 41.5, ≤ 102.5, and ≤ 131.5 are suggested for the RBMT-3 to discriminate between patients with mild and moderate dementia, mild dementia and MCI, and MCI and NC, with sensitivities 73%, 100% and 94.1%, respectively. This version is useful to differentiate those with or without risk of cognitive impairments.


Subject(s)
Cognition Disorders/psychology , Cognition Disorders/rehabilitation , Memory and Learning Tests/standards , Occupational Therapy/methods , Outcome Assessment, Health Care/methods , Aged , Aged, 80 and over , Analysis of Variance , Female , Hong Kong , Humans , Male , Middle Aged , Photic Stimulation/methods , Psychometrics , Reproducibility of Results
2.
Clin Microbiol Infect ; 24(1): 82.e1-82.e4, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28506784

ABSTRACT

OBJECTIVES: This study aims to assess the association between patient contact and intestinal carriage of multidrug-resistant organisms (MDRO) by sampling healthcare personnel (HCP) and staff without patient contact. METHODS: For this observational study, we recruited 400 HCP who worked in our 200-bed research hospital and 400 individuals without patient contact between November 2013 and February 2015. Participants submitted two self-collected perirectal swabs and a questionnaire. Swabs were processed for multidrug-resistant Gram-negative bacteria and vancomycin-resistant enterococci (VRE). Questionnaires explored occupational and personal risk factors for MDRO carriage. RESULTS: Among 800 participants, 94.4% (755/800) submitted at least one swab, and 91.4% (731/800) also submitted questionnaires. Extended spectrum ß-lactamase-producing organisms were recovered from 3.4% (26/755) of participants, and only one carbapenemase-producing organism was recovered. No VRE were detected. The potential exposure of 68.9% (250/363) of HCP who reported caring for MDRO-colonized patients did not result in a rate of MDRO carriage among HCP (4.0%; 15/379) significantly higher than that of staff without patient contact (3.2%; 12/376; p 0.55). CONCLUSIONS: This is the largest US study of HCP intestinal MDRO carriage. The low colonization rate is probably reflective of local community background rates, suggesting that HCP intestinal colonization plays a minor role in nosocomial spread of MDROs in a non-outbreak setting. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01952158.


Subject(s)
Bacterial Infections/transmission , Carbapenem-Resistant Enterobacteriaceae/isolation & purification , Carrier State/microbiology , Health Personnel , Intestines/microbiology , Vancomycin-Resistant Enterococci/isolation & purification , Adult , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Bacterial Proteins/analysis , Cross Infection/microbiology , Drug Resistance, Multiple, Bacterial , Female , Humans , Male , Middle Aged , Prevalence , Risk Factors , Surveys and Questionnaires , beta-Lactamases/analysis
3.
Int J Rehabil Res ; 26(4): 251-5, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14634358

ABSTRACT

The present study sought to identify the factors as well as what would predict discharge of persons with brain injury. Demographics (age, pre-injury educational level, pre-injury occupational status) and Disability Rating Scale (DRS) scores during admission and upon discharge were used for discharge status prediction. A multiple discriminant analysis (MDA) revealed that the DRS scores at admission and upon discharge were significant predictors that correctly classified 72% of grouped cases.


Subject(s)
Brain Injuries/rehabilitation , Disability Evaluation , Patient Discharge , Discriminant Analysis , Female , Hong Kong , Humans , Male , Middle Aged , Outcome Assessment, Health Care , Prospective Studies , Reproducibility of Results , Sampling Studies
4.
J Cell Biol ; 154(4): 815-27, 2001 Aug 20.
Article in English | MEDLINE | ID: mdl-11514593

ABSTRACT

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.


Subject(s)
Connexin 43/metabolism , Gap Junctions/physiology , Oncogene Protein pp60(v-src)/metabolism , Tyrosine , Cell Communication , Cell Membrane , Clone Cells , Connexin 43/genetics , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Mutagenesis, Site-Directed , Phosphorylation , Serine
6.
Cell Commun Adhes ; 8(4-6): 265-9, 2001.
Article in English | MEDLINE | ID: mdl-12064600

ABSTRACT

It is not clear how the v-Src oncoprotein disrupts gap junctional communication (GJC) established by connexin43 (Cx43) in mammalian cells. In this study, an experimental system was established to stably express v-Src and wild type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout (KO) mouse cell line. When co-expressed with v-Src, the levels of phosphotyrosine (pTyr) from Y247F, Y265F, and Y247F/Y265F Cx43 mutants were reduced to approximately 57%, 10%, and 2% of the level of pTyr from wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. These data also implied that phosphorylation of Cx43 at Y265 was required for efficient phosphorylation of Cx43 at Y247. Most importantly, our measurements of GJC demonstrated that, in contrast to the wt Cx43 gap junction channels, the Y247F, Y265F, and Y247F/Y265F Cx43 channels were resistant to the disruption by v-Src. In conclusion, our studies support a model for processive phosphorylation of Cx43 on tyrosine, at the Y265 site followed by the Y247 site, in mediating the disruption of GJC induced by v-Src in mammalian cells.


Subject(s)
Cell Communication/physiology , Connexin 43/metabolism , Gap Junctions/metabolism , Oncogene Protein pp60(v-src)/metabolism , Tyrosine/metabolism , Animals , Cell Line , Connexin 43/genetics , Mice , Mice, Knockout , Models, Biological , Mutagenesis, Site-Directed , Oncogene Protein pp60(v-src)/genetics , Phosphorylation , Rats
7.
Cancer Epidemiol Biomarkers Prev ; 9(11): 1199-204, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11097227

ABSTRACT

Tobacco smoking is a strong cause of lung cancer. However, because only a small proportion of smokers develop the disease, other factors, including genetic susceptibility, may be important in determining lung cancer risk. Polymorphisms in the TP53 tumor suppressor gene and HRAS1 proto-oncogene have been associated in some studies with this cancer; we sought to replicate these associations in an ethnically diverse population in Hawaii. We conducted a population-based case-control study among 334 incident lung cancer cases and 446 controls of Caucasian, Japanese, or Native Hawaiian origin. In-person interviews collected detailed information on lifestyle risk factors. DNA was extracted from peripheral blood leukocytes, and genotyping was performed using a PCR-based assay for the TP53 codon 72 polymorphism and Southern blot analysis and PCR for allelic polymorphisms in the HRAS1 minisatellite. Logistic regression analyses were used to compute odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for smoking and other risk factors. The presence of two rare HRAS1 alleles was associated with a 2.2-fold (95% CI, 1.0-5.0) increased lung cancer risk for all ethnic groups combined. The association was present in Native Hawaiians (OR, 5.2; 95% CI, 1.1-24.4) and was suggested for Japanese (OR, 2.8; 95% CI, 0.6-12.5); no association was observed in Caucasians (OR, 0.8; 95% CI, 0.2-3.6). This association was also observed for each lung cancer cell type. The presence of only one rare allele did not increase risk for any ethnic group or cell type. No significant association was found between the TP53 codon 72 polymorphism and lung cancer [OR, 1.4 (95% CI, 0.8-2.4) for the Pro/Pro genotype compared with the Arg/Arg genotype]. This study suggests that the presence of two rare HRAS1 alleles confers an increased lung cancer risk in Native Hawaiians and Japanese but possibly not in Caucasians. The amino acid replacement of arginine by proline at codon 72 of TP53 appears not to be important in determining lung cancer risk in this population.


Subject(s)
Genes, p53/genetics , Genes, ras/genetics , Lung Neoplasms/ethnology , Lung Neoplasms/genetics , Polymorphism, Genetic , Adult , Aged , Asian People/genetics , Case-Control Studies , Ethnicity , Female , Humans , Lung Neoplasms/etiology , Male , Middle Aged , Odds Ratio , Polymerase Chain Reaction , Proto-Oncogene Mas , Risk Factors , White People/genetics
8.
J Cell Biol ; 149(7): 1503-12, 2000 Jun 26.
Article in English | MEDLINE | ID: mdl-10871288

ABSTRACT

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.


Subject(s)
Cell Communication/physiology , Connexin 43/metabolism , Gap Junctions/metabolism , Protein Kinase C/metabolism , Animals , Cells, Cultured , Gap Junctions/physiology , Mice , Mice, Knockout , Mutagenesis, Site-Directed/physiology , Neoplasms/metabolism , Neoplasms/physiopathology , Phosphorylation , Sequence Analysis, Protein , Serine/metabolism
9.
Mol Cell Biol Res Commun ; 3(2): 115-21, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10775509

ABSTRACT

To study the gap junction protein connexin37 (Cx37), we stably transfected cell lines with constructs of human Cx37 containing the epitope tag FLAG (DYKDDDDK). A Cx37 construct containing the FLAG moiety at the carboxyl terminus (Cx37F) was expressed in BWEM cells, and did not substantially alter the levels of endogenous Cx43 in these cells. Immunostaining showed that Cx37F colocalized with Cx43 at cell-cell contacts. Pulse-chase metabolic labeling and immunoprecipitation with anti-FLAG antibodies indicated that Cx37F was synthesized as a protein that ran at 35.9 +/- 0.9 kDa on reducing SDS-PAGE but chased into a slower migrating band at 38.0 +/- 1.0 kDa. This shift in mobility was due to phosphorylation on serine residues, based on [(32)P]-metabolic labeling, immunoprecipitation, and phosphoamino acid analyses. The transition to the phosphoCx37F correlated with a loss of solubility in 1% Triton X-100. Based on the [(35)S]-methionine pulse-chase experiments, the half-life of the labeled Cx37F was approximately 3 h, which is within the range reported for other connexins. Analysis of dye injection experiments indicated that dye transfer was reduced in Cx37-transfected cells in comparison to parental BWEM cells, suggesting that formation of heteromeric Cx37-Cx43 channels reduced the molecular permeability of communication between these cells. Moreover, the similarities of previously demonstrated kinetic details and modification of Cx43 to our new data regarding Cx37 provide evidence for a commonality in processing and assembly steps of these two connexins.


Subject(s)
Connexins/metabolism , Epitopes/metabolism , Animals , Blotting, Western , Cell Line , Connexins/biosynthesis , Connexins/genetics , Humans , Kinetics , Oligopeptides , Peptides/chemistry , Protein Processing, Post-Translational , Rats , Transfection , Gap Junction alpha-4 Protein
10.
Methods ; 20(2): 219-31, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10671315

ABSTRACT

Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy.


Subject(s)
Bacterial Proteins/metabolism , Connexin 43/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/metabolism , Animals , Bacterial Proteins/genetics , Connexin 43/genetics , DNA-Binding Proteins , Embryo, Mammalian , Escherichia coli/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gap Junctions/physiology , Gene Library , Gene Transfer Techniques , Mice , Plasmids , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Repressor Proteins , Restriction Mapping , Serine Endopeptidases/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
11.
Arch Biochem Biophys ; 384(2): 205-15, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11368307

ABSTRACT

Gap junctions are a unique type of intercellular junction found in most animal cell types. Gap junctions permit the intercellular passage of small molecules and have been implicated in diverse biological processes, such as development, cellular metabolism, and cellular growth control. In vertebrates, gap junctions are composed of proteins from the "connexin" gene family. The majority of connexins are modified posttranslationally by phosphorylation, primarily on serine amino acids; however, phosphotyrosine has also been detected in connexin from cells coexpressing nonreceptor tyrosine protein kinases. Connexins are targeted by numerous protein kinases, of which some have been identified: protein kinase C, mitogen-activated protein kinase, and the v-Src tyrosine protein kinase. Phosphorylation has been implicated in the regulation of a broad variety of connexin processes, such as the trafficking, assembly/disassembly, degradation, as well as the gating of gap junction channels. This review examines the consequences of connexin phosphorylation for the regulation of gap junctional communication.


Subject(s)
Connexins/metabolism , Gap Junctions/metabolism , Amino Acid Sequence , Animals , Connexin 43/metabolism , Molecular Sequence Data , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Gap Junction beta-1 Protein
12.
Mol Carcinog ; 25(3): 187-95, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10411145

ABSTRACT

v-src-transformed fibroblasts have significantly reduced levels of gap junction-mediated intercellular communication. This observed downregulation of cellular communication has been associated with tyrosine phosphorylation of the gap-junction protein connexin 43 (Cx43). Previously, we demonstrated that purified, kinase-active pp60src phosphorylates Cx43 in vitro (J Biol Chem 1995; 270:12751-12761). More recently, we reported that this association is mediated by the SH2 and SH3 domains of pp60v-src (J Biol Chem 1997;272:22824-22831). In this report, we present in vivo evidence supporting the hypothesis that Cx43 is an endogenous substrate of pp60v-src in v-src-transformed fibroblasts. Cytological localization studies with confocal microscopy demonstrated that pp60v-src and Cx43 were partially co-localized in regions of the plasma membrane. Cx43 and pp60v-src co-immunoprecipitated from v-src-transformed fibroblasts, indicating that the two proteins were associated, and form a stable complex. Furthermore, pp60v-src could phosphorylate co-immunoprecipitated Cx43 in an immune-complex kinase assay. Two-dimensional phosphopeptide mapping of the immune-complexed Cx43 phosphorylated in vitro demonstrated that the sites of tyrosine phosphorylation were consistent with previously identified sites of pp60v-src phosphorylation. These results provide additional in vivo evidence that Cx43 is a direct substrate of pp60v-src in v-src-transformed fibroblasts. The ability of pp60v-src to alter gap junction-mediated cellular communication may serve as one mechanism by which pp60v-src initiates and/or maintains aspects of cellular transformation.


Subject(s)
Connexin 43/metabolism , Gap Junctions/metabolism , Oncogene Protein pp60(v-src)/metabolism , Animals , Cell Line, Transformed , Fibroblasts/metabolism , Fluorescent Antibody Technique , Microscopy, Confocal , Oncogene Protein pp60(v-src)/physiology , Peptide Mapping , Phosphorylation , Precipitin Tests , Protein Binding , Rats
13.
Cancer Lett ; 147(1-2): 163-73, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10660102

ABSTRACT

Antisense phosphorothioate oligodeoxynucleotides (ODNs) are increasingly used to target specific proteins for inhibition. Previous reports of antisense inhibition of the inducible nitric oxide synthase (iNOS) gene suggested its utility in defining the role of nitric oxide (NO) in carcinogenesis, as NO is mutagenic and chemical inhibitors of iNOS block neoplastic transformation in C3H 10T1/2 fibroblasts. Treatment with ODNs (0.025-25 microM) directed against 15mer sequences in the iNOS coding region decreased NO production consistent with a reduction of iNOS protein and iNOS mRNA, however, control ODNs (2.5 microM) also showed considerable nonspecific inhibition of NO synthesis. Treatment with both iNOS antisense and missense ODNs during the promotional phase of the C3H10T1/2 transformation assay significantly increased the number of neoplastic foci in 3-methylcholanthrene (MCA) treated cells which corresponded with the ability of the ODN to inhibit NO production. Enhanced neoplastic transformation and non-specific inhibition of NO synthesis resulting from exposure to antisense ODNs suggest limitations to their long-term use in humans at higher doses.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cell Transformation, Neoplastic/drug effects , Fibroblasts/drug effects , Oligonucleotides, Antisense/pharmacology , Thionucleotides/pharmacology , Animals , Base Sequence , Blotting, Western , Carcinogens , Cell Line , Cell Transformation, Neoplastic/chemically induced , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Fibroblasts/cytology , Fibroblasts/enzymology , Interferons/pharmacology , Lipopolysaccharides/pharmacology , Methylcholanthrene , Mice , Mice, Inbred C3H , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Oligonucleotides, Antisense/genetics , RNA, Messenger/metabolism , Thionucleotides/genetics , Transfection , Tumor Stem Cell Assay
14.
Cancer Res ; 58(21): 4858-63, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9809991

ABSTRACT

The dramatic shift in the pathological presentation of lung cancer [the proportional decrease in squamous cell carcinoma (SCC) and increase in adenocarcinoma (AC)] observed in the United States after the 1950s may have taken place as the result of the reduction in polycyclic aromatic hydrocarbons (PAHs) and the increase in N-nitrosamines in inhaled smoke from filtered low-yield cigarettes. The predominant mutation patterns of these tumors also suggest differences in their etiology. We tested the hypothesis that genetic susceptibility to PAHs, as determined by polymorphisms in CYP1A1 and GSTM1, predominantly causes lung SCCs, and susceptibility to nitrosamines, as determined by polymorphisms in CYP2E1, predominantly causes lung ACs. CYP1A1 and GSTM1 play a major role in the metabolic activation and detoxification of PAHs, respectively, and CYP2E1 plays a major role in the metabolic activation of nitrosamines. We conducted a population-based case-control study among 341 incident lung cancer cases and 456 controls of Caucasian, Japanese, or Hawaiian origin. In-person interviews collected detailed information on lifestyle risk factors, and DNA extracted from peripheral leukocytes was used in PCR-based genotyping assays. Logistic regression analyses were used to compute odds ratios and 95% confidence intervals (CIs) for each cell type, adjusting for smoking and dietary variables. The presence of at least one copy of the CYP1A1 MspI variant allele was found to be associated with a 2.4-fold (95% CI, 1.2-4.7) increase in the risk of SCC when this gene was considered singly and a 3.1-fold (95% CI, 1.2-7.9) increase in the risk of SCC when combined with a GSTM1 deletion. No significant association was found between MspI and all lung cancers or other cell types or with the CYP1A1 exon 7 polymorphism. In contrast, the CYP2E1 RsaI and DraI polymorphisms were not clearly related to SCC risk, but these homozygous variant genotypes were associated with a 10-fold (95% CI, 0.0-0.5) decrease in the risk of overall lung cancer (RsaI variant) and AC (DraI variant) compared to the homozygous wild-type genotypes. Inverse associations with these two closely linked CYP2E1 polymorphisms were also suggested for small cell carcinoma. In agreement with past experimental and epidemiological data, the associations found in this study between CYP1A1 and lung SCC and between CYP2E1 and lung AC suggest a certain specificity of tobacco smoke PAHs for lung SCC and tobacco-specific nitrosamines for lung ACs.


Subject(s)
Carcinogens/metabolism , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP2E1/genetics , Genetic Predisposition to Disease , Glutathione Transferase/genetics , Isoenzymes/genetics , Lung Neoplasms/genetics , Nicotiana , Plants, Toxic , Polycyclic Aromatic Hydrocarbons/metabolism , Adult , Aged , Alleles , Carcinogens/toxicity , Genotype , Humans , Middle Aged
15.
Circ Res ; 83(1): 27-32, 1998 Jul 13.
Article in English | MEDLINE | ID: mdl-9670915

ABSTRACT

Connexin43(Cx43) channels can be regulated by a variety of factors, including low pHi. Structure/function studies from this laboratory have demonstrated that pH gating follows a particle-receptor mechanism, similar to the "ball-and-chain" model of voltage-dependent inactivation of ion channels. The question whether the particle-receptor model is applicable only to pH gating or to other forms of Cx43 regulation as well remains. To address this question, we looked at the uncoupling effects of insulin and of insulin-like growth factor-1 (IGF) on Cx43 channels expressed in Xenopus oocytes. These agonists do not induce changes in pHi. Junctional conductance (Gj) was measured by the dual 2-electrode voltage-clamp technique. Control studies showed that relative Gj did not change spontaneously as a function of time. Continuous exposure of Cx43-expressing oocytes to insulin (10 micro/L) led to a decrease in Gj. After 80 minutes, Gj was 54+/-5% from control (n= 12). Exposure of oocytes to IGF (10 nmol/L) caused an even more pronounced change in Gj (37+/-4% of control, n=6). The time course of the IGF-induced uncoupling was similar to that observed after insulin exposure. The effect of insulin was abolished by truncation of the carboxyl-terminal domain of Cx43 at amino acid 257 (M257). Interestingly, as in the case of pH gating, coexpression of the carboxyl-terminal domain (amino acids 258 to 282) together with M257 rescued the ability of insulin to reduce coupling (Gj, 39+/-12% from control; n=6). Structure/function experiments using various deletion mutants of the carboxyl-terminal domain showed that insulin treatment does not modify Gj if amino acids 261 to 280 are missing from the Cx43 sequence. Our results suggest that a particle-receptor (or ball-and-chain) mechanism, similar to that described for pH gating, also applies to chemical regulation of Cx43 by other factors.


Subject(s)
Connexin 43/metabolism , Insulin/pharmacology , Ion Channels/drug effects , Ion Channels/metabolism , Amino Acid Sequence , Animals , Connexin 43/genetics , Electric Conductivity , Female , Gap Junctions/drug effects , Gap Junctions/physiology , Gene Deletion , Hydrogen-Ion Concentration , Insulin-Like Growth Factor I/pharmacology , Oocytes/metabolism , Structure-Activity Relationship , Xenopus laevis
16.
J Cell Sci ; 111 ( Pt 6): 833-41, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9472011

ABSTRACT

The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacrylamide gel electrophoresis. The electrophoretic mobility of connexin43 from mitotic cells was distinctly reduced to a form (P3) that migrated slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-treated and untreated cultures. Mitotic FT210 cells, which contain a temperature-sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 connexin43 when maintained at the permissive temperature where p34(cdc2) is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 connexin43. These results indicated that generation of the P3 connexin43 was dependent upon active p34(cdc2)/cyclin B kinase. Although the p34(cdc2)kinase phosphorylated connexin43 in vitro on peptides containing serine 255, the major phosphotryptic peptides in P3 connexin43 from mitotic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase. The P3 connexin43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures. These events may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis. Results of initial studies using inhibitors of protein degradative and synthetic pathways suggested the likelihood that protein degradation and synthesis participate in the disappearance of the P3 connexin43 and restoration of the pattern of connexin43 isoforms observed in nonmitotic cells.


Subject(s)
CDC2 Protein Kinase/physiology , Connexin 43/metabolism , Mitosis , Animals , Cell Line , Connexin 43/chemistry , Connexin 43/drug effects , Epithelial Cells , Fibroblasts , Intracellular Fluid/metabolism , Isomerism , Mitosis/drug effects , Nocodazole/pharmacology , Phosphorylation , Rats
17.
Circ Res ; 82(9): 929-35, 1998 May 18.
Article in English | MEDLINE | ID: mdl-9598590

ABSTRACT

Structure/function analysis shows that the carboxyl terminal (CT) domain of connexin43 (Cx43) is essential for the chemical regulation of cell-cell communication. Of particular interest is the region between amino acids 260 and 300. Structural preservation of this region is essential for acidification-induced uncoupling (ie, pH gating). In this study, we report data showing that a 17mer peptide of the same sequence as amino acids 271 to 287 of Cx43 (CSSPTAPLSPMSPPGYK) can prevent pH gating of Cx43-expressing oocytes. Experiments were carried out in pairs of Xenopus oocytes previously injected with connexin38 antisense and expressing wild-type Cx43. Junctional conductance was measured electrophysiologically. pHi was determined from the light emission of the proton-sensitive dye dextran-seminaphthorhodafluor. Intracellular acidification was induced by superfusion with a bicarbonate-buffered solution gassed with a progressively increasing concentration of CO2. Injection of water alone into both oocytes of a Cx43-expressing pair or injection of a peptide from region 321 to 337 of Cx43 did not modify pH sensitivity. However, injection of a polypeptide corresponding to amino acids 241 to 382 of Cx43 interfered with the ability of gap junctions to close on acidification. Similar results were obtained when a 17mer peptide (region 271 to 287) was injected into both oocytes of the pair. Normal Cx43 pH gating was observed if (1) the amino acid sequence of the 17mer peptide was scrambled or (2) the N and the C ends of the 17mer peptide were not included in the sequence. This is the first demonstration of a molecule that can interfere with the chemical regulation of connexin channels in a cell pair. The data may lead to the development of small molecules that can be used in Cx43-expressing multicellular preparations to study the role of gap junction regulation in normal as well as diseased states.


Subject(s)
Connexin 43/physiology , Gap Junctions/physiology , Oligopeptides/pharmacology , Amino Acid Sequence , Animals , Cell Communication/drug effects , Connexin 43/chemistry , Hydrogen-Ion Concentration , Ion Channel Gating , Molecular Sequence Data , Oocytes , Peptide Fragments/pharmacology , Structure-Activity Relationship , Xenopus laevis
18.
J Biol Chem ; 273(15): 9188-96, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9535909

ABSTRACT

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Communication/physiology , Connexin 43/physiology , Gap Junctions/physiology , Signal Transduction , Animals , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Communication/drug effects , Cell Line , Connexin 43/deficiency , Connexin 43/genetics , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Flavonoids/pharmacology , Gap Junctions/drug effects , HeLa Cells , Humans , Kinetics , Lysophospholipids/pharmacology , Mice , Mice, Knockout , Models, Biological , Phosphorylation , Rats , Recombinant Proteins/biosynthesis , Serine , Transfection
19.
J Biol Chem ; 272(36): 22824-31, 1997 Sep 05.
Article in English | MEDLINE | ID: mdl-9278444

ABSTRACT

Reduction of gap junctional communication in v-src transformed cells is accompanied by tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). Cx43 is phosphorylated on tyrosine by v-Src. The Src homology 3 (SH3) and Src homology 2 (SH2) domains of v-Src mediate interactions with substrate proteins. SH3 domains interact with proline-rich peptide motifs. SH2 domains associate with short amino acid sequences containing phosphotyrosine. We present evidence that the SH3 and SH2 domains of v-Src bind to proline-rich motifs and a phosphorylated tyrosine residue in the C-terminal tail of Cx43. Cx43 bound to the SH3 domain of v-Src, but not c-Src, in vitro. Tyrosine-phosphorylated Cx43 bound to the SH2 domain of v-Src in vitro. v-Src coprecipitated with Cx43 from v-src-transformed Rat-1 fibroblasts. Mutations in the SH3 and SH2 domains of v-Src, and in the proline-rich region or tyrosine 265 of Cx43, reduced interactions between v-Src and Cx43 in vivo. Tyrosine phosphorylation of Cx43 was dependent on the association of v-Src and Cx43. These results provide further evidence for the direct involvement of v-Src in tyrosine phosphorylation of Cx43 and inhibition of gap junctional communication in v-src-transformed cells.


Subject(s)
Connexin 43/metabolism , Oncogene Protein pp60(v-src)/metabolism , Tyrosine/metabolism , src Homology Domains , Amino Acid Sequence , Animals , Cell Line , Connexin 43/chemistry , Humans , Molecular Sequence Data , Phosphorylation , Precipitin Tests , Proline/metabolism , Rats
20.
Cell Growth Differ ; 8(9): 1015-27, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9300183

ABSTRACT

Immortalized cells from embryonic connexin43 knockout mice (Cx43-/-) and homozygous littermates (Cx43+/+) were cloned and characterized to determine whether the absence of Cx43 function would induce observable phenotypic changes. Cells of the Cx43+/+ clones expressed Cx43 and engaged in gap junctional communication with 10-12 neighboring cells. The Cx43-/- cells were devoid of Cx43 and communicated to less than 1 cell. Electrophysiological analysis indicated that the Cx43-/- cells communicated through Cx45 channels from 8-80-fold less than did the Cx43+/+ subclones, which seemed to communicate through Cx43 and Cx45 channels. The Cx43-/- clones grew at faster rates and to higher saturation densities, had a more spindly morphology, were more refractile, and adhered less well to the substratum than did the Cx43+/+ clones. Reintroducing the Cx43 gene into the Cx43-/- clones resulted in three subclones that communicated to 3-4 cells. Partial restoration of gap junctional communication in the three subclones was accompanied by reduced growth rates and saturation densities (2-fold compared to that of parental Cx43-/- clones) but no reversions in morphology or cell-substratum adhesion. The increased growth rates and saturation densities, altered morphology, and decreased cell adhesion displayed by the Cx43-/- clones reflect a subset of the properties of transformed cells. These studies advance the hypothesis that loss of Cx43 function during development may cause cells to acquire a preneoplastic condition.


Subject(s)
Connexin 43/genetics , Animals , Cell Division/genetics , Cell Line, Transformed , Clone Cells , Connexin 43/biosynthesis , Gap Junctions/genetics , Gap Junctions/pathology , Mice , Mice, Knockout , Phenotype , RNA/analysis , RNA/biosynthesis , Time Factors , Transfection
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