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1.
Oncogene ; 28(48): 4272-83, 2009 Dec 03.
Article in English | MEDLINE | ID: mdl-19767772

ABSTRACT

Transformation by the Src tyrosine kinase (Src) promotes nonanchored cell growth and migration. However, nontransformed cells can force Src-transformed cells to assume a normal morphology and phenotype by a process called 'contact normalization'. It has become clear that microRNA (miRNA) can affect tumorigenesis by targeting gene products that direct cell growth and migration. However, the roles of miRNA in Src transformation or contact normalization have not yet been reported. We examined the expression of 95 miRNAs and found 9 of them significantly affected by Src. In this study, we report that miR-218 and miR-224 were most significantly induced by Src, but not affected by contact normalization. In contrast, miR-126 was most significantly suppressed by Src and was induced by contact normalization in transformed cells. Mir-126 targets Crk, a component of the focal adhesion network that participates in events required for tumor cell migration. Accordingly, we show that miR-126 expression correlates inversely with Crk levels, motility and the invasive potential of human mammary carcinoma cells. Moreover, we show that miR-224 expression promotes nonanchored growth of nontransformed cells. These data reveal novel insights into how Src regulates miRNA expression to promote hallmarks of tumor cell growth and invasion, and how nontransformed cells can affect miRNA expression in adjacent tumor cells to inhibit this process.


Subject(s)
Cell Movement/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , MicroRNAs/pharmacology , src-Family Kinases/pharmacology , Animals , Cell Cycle , Cell Line, Tumor , Cell Movement/genetics , Cell Transformation, Neoplastic , Epithelial Cells , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/physiology , Homeodomain Proteins , Humans , Mice , Mice, Nude , MicroRNAs/genetics
2.
Cancer Res ; 61(4): 1334-7, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11245430

ABSTRACT

Anchorage-independent growth is a hallmark of tumor cells. We compared gene expression profiles of anchored and nonanchored human mammary carcinoma cells to study this phenomenon. In this study, we show that anchorage had striking effects on cell growth and morphology but altered transcript levels from a limited number of genes. Only about 1% of mRNA transcripts detected in these cells was altered by anchorage. These include genes related to amino acid and polyamine metabolism, apoptosis, ion channels, cytoskeletal and stress proteins, transcription factors, and growth factors. Some of these may be crucial for the survival of transformed cells. For example, clusterin and the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) were suppressed by anchorage, which could help prevent programmed cell death of these tumor cells. In addition to suppressing TRAIL expression, anchorage also decreased the susceptibility of these tumor cells to TRAIL-induced apoptosis as determined by poly(ADP-ribose) phosphorylase cleavage, annexin-V binding (P < 0.01), and cell cycle analysis (P < 0.0001). These data may help explain mechanisms by which anchorage prevents apoptosis of cells that would otherwise experience anoikis. Thus, genes found to be altered by this analysis could serve as potential targets for anticancer therapy. These findings suggest that TRAIL may be used as a means to target circulating epithelial tumor cells before their attachment and colonization at new sites.


Subject(s)
Anoikis/physiology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic/physiology , Membrane Glycoproteins/physiology , Tumor Necrosis Factor-alpha/physiology , Apoptosis Regulatory Proteins , Breast Neoplasms/metabolism , Cell Adhesion/genetics , Cell Division/physiology , Gene Expression Profiling , Humans , Membrane Glycoproteins/biosynthesis , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis
4.
Cancer Res ; 60(21): 6018-26, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11085522

ABSTRACT

Gap junction expression has been reported to control the growth of a variety of transformed cells. We undertook parallel analysis of connexins Cx32 and Cx43 in glioma cells, which revealed potential mechanisms underlying this phenomenon and led to several novel findings. Cx43, but not Cx32, suppressed C6 glioma cell growth. Paradoxically, Cx32 transfection resulted in severalfold more dye transfer than Cx43. However, Cx43 transfectants shared endogenous metabolites more efficiently than Cx32 transfectants. Interestingly, a significant portion of Cx43 permeants were incorporated into macromolecules more readily than those that transferred via Cx32. Cx43 induced contact inhibition of cell growth but in contrast to other reports, did not affect log phase growth rates. Cell death, senescence, or suppression of growth factor signaling was not involved because no significant alterations were seen in cell viability, telomerase, or mitogen-activated protein kinase activity. However, suppression of cell growth by Cx43 entailed the secretion of growth-regulatory factors. Most notably, a major component of conditioned medium that was affected by Cx43 was found to be MFG-E8 (milk fat globule epidermal growth factor 8), which is involved in cell anchorage and integrin signaling. These results indicate that Cx43 regulates cell growth by the modulation of extracellular growth factors including MFG-E8. Furthermore, the ability of a Cx to regulate cell growth may rely on its ability to mediate the intercellular transfer of endogenous metabolites but not artificial dyes.


Subject(s)
Antigens, Surface , Connexin 43/physiology , Gap Junctions/physiology , Glioma/pathology , Membrane Glycoproteins/antagonists & inhibitors , Milk Proteins , Amino Acid Sequence , Animals , Blotting, Northern , Cell Communication/physiology , Cell Division/physiology , Coloring Agents/pharmacokinetics , Connexin 43/biosynthesis , Connexin 43/genetics , Connexins/biosynthesis , Connexins/genetics , Connexins/physiology , Gap Junctions/metabolism , Glioma/genetics , Glioma/metabolism , Humans , MAP Kinase Signaling System/physiology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Molecular Sequence Data , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , Rats , Telomerase/metabolism , Transfection , Gap Junction beta-1 Protein
6.
Exp Cell Res ; 239(1): 82-92, 1998 Feb 25.
Article in English | MEDLINE | ID: mdl-9511727

ABSTRACT

Gap junctional communication has been implicated in numerous cellular processes. However, the repertoire of specific transjunctional substances which mediate these processes remains relatively unexplored. A few selected secondary messengers have been identified, at least indirectly (e.g., cAMP and IP3) and phenotypic complementation experiments have indicated that gap junctions enable communicating cells to distribute nucleotide pools as a shared resource. The latter would include high energy compounds such as ADP and ATP, allowing cells to share energy resources. We have utilized a nonbiased process to directly capture, identify, and quantify transjunctional compounds from C6 glioma cells, the transformed phenotype of which has been ameliorated by transfection with connexin43 (Cx43). This technique involves the direct isolation, identification, and quantitation of radioactive transjunctional molecules that travel from metabolically labeled "donor" cells to "receiver" cells. This report demonstrates that ADP and/or ATP represents over 6% of the transjunctional material derived from glucose in Cx43-transfected C6 glioma cells. Furthermore, equilibration of these high energy metabolites among first order neighbors is shown to occur in less than 20 min of communication.


Subject(s)
Adenosine Diphosphate/metabolism , Connexin 43/physiology , Gap Junctions/physiology , Animals , Cell Communication , Connexin 43/biosynthesis , Gap Junctions/ultrastructure , Glioma , Glucose/metabolism , Kinetics , Models, Biological , Phenotype , Rats , Recombinant Proteins/biosynthesis , Time Factors , Transfection , Tumor Cells, Cultured
7.
Exp Dermatol ; 6(4): 167-74, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9293388

ABSTRACT

The passage of specific growth modulating signals through gap junctions may regulate the proliferation and differentiation of human keratinocytes. To investigate this, we correlated the proliferation of normal human keratinocytes and a transformed squamous cell carcinoma cell line, SCC4, with the expression of the gap junctional proteins Cx43, 31 and 31.1, known to be expressed by keratinocytes. Proliferation was confined to preconfluent and confluent cultures of normal keratinocytes, falling to undetectable levels once postconfluency was achieved. Cx43, at both the message and protein levels, paralleled these changes, being elevated predominantly in preconfluent and confluent cultures, and downregulated in postconfluency. Similar results were found for Cx31 and 31.1 at the message level. In contrast, the proliferation of SCC4 cells cultured in media supplemented with 5.0% FCS was maintained at a substantial level from preconfluency through 2 weeks postconfluency. Cx43, 31, and 31.1 RNA and Cx43 protein expression mirrored the levels of proliferation within SCC4 cultures. Cx26 and 32 were not found in normal keratinocytes or SCC4 cells at any stage of differentiation. These data, illustrating a tight correlation between proliferation and Cx43, 31 and 31.1 expression, suggest that these connexins may represent proliferation-specific gap junctions within keratinocytes, and may therefore transmit signals that control keratinocyte division.


Subject(s)
Connexin 43/biosynthesis , Gap Junctions/chemistry , Keratinocytes/cytology , Keratinocytes/metabolism , Calcium/pharmacology , Cell Differentiation/physiology , Cell Division/physiology , Cell Line, Transformed , Connexin 26 , Connexin 43/genetics , Connexins/biosynthesis , Connexins/genetics , Gene Expression/physiology , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transglutaminases/drug effects , Transglutaminases/metabolism , Tumor Cells, Cultured
8.
J Public Health Policy ; 18(3): 302-24, 1997.
Article in English | MEDLINE | ID: mdl-9360347

ABSTRACT

Jobs for All at decent wages is not the only strategy for reducing poverty and economic inequality, but it is more desirable and more consonant with American values than a primary strategy of direct income redistribution through government benefits. To make jobs the primary strategy for people of working age, however, is not to overlook the need for certain types of income support in good times and in bad, and the important economic functions of the welfare state. Current welfare "reform" poses as a work strategy but is the very antithesis of jobs for all because it creates job seekers rather than jobs and will increase unemployment and lower wages. Economic and social benefits of full employment are identified, and criticisms of the strategy--that many current jobs are risky, boring and poorly paid--are addressed. The abiding and new obstacles to full employment are acknowledged, their seriousness assessed, and means for overcoming them proposed. The author concludes that the obstacles to jobs for all are primarily political rather than economic, and shows how the National Jobs for All Coalition is attempting to overcome them and to build a new movement for economic justice.


Subject(s)
Employment/economics , Public Policy , Social Welfare/legislation & jurisprudence , Social Welfare/trends , Female , Humans , Male , United States
9.
J Cell Biol ; 134(4): 1019-30, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8769424

ABSTRACT

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7-hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.


Subject(s)
Cell Membrane/physiology , Connexin 43/physiology , Gap Junctions/physiology , Alcohols/pharmacology , Animals , Biological Transport/drug effects , Calcium/pharmacology , Carcinoma, Hepatocellular , Cells, Cultured , DNA, Antisense , Enzyme Activation/drug effects , Fluoresceins , Fluorescent Dyes , HeLa Cells , Humans , Kidney , Magnesium/pharmacology , Oncogene Protein pp60(v-src)/physiology , Protein Kinase C/physiology , Rats , Signal Transduction/physiology , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Tumor Cells, Cultured
10.
Biol Reprod ; 54(4): 905-13, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8924512

ABSTRACT

Gap junctions form between rat endometrial stromal cells as they undergo decidualization. We have examined the steady-state levels of the gap junction transcripts, connexins 26 and 43 (cx26 and cx43), during artificially induced decidualization in vivo and found that they have a temporal pattern similar to that observed in pregnancy. An in vitro model of decidualization was then used. Endometrial stromal cells from rat uteri sensitized for decidualization were cultured for 24, 48, or 72 h before total RNA was extracted and subjected to Northern blot analyses to determine the steady-state levels of cx26 and cx43 transcripts. The analyses revealed that cx26 transcript steady-state levels decreased, whereas those for cx43 increased, from 24 to 72 h. Using an anti-cx43 antibody, punctate immunofluorescent signals were observed around the periphery of the cells, suggesting that cx43 had been assembled into membrane plaques. The presence of functional gap junctions between the cells was determined in vitro by two dye-coupling methods: preloading and scrape-loading. Calcein (995 Da) and a membrane-bound dye, dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (933 Da), were preloaded into 5% of the endometrial stromal cells before plating. The percentage of preloaded cells that transferred calcein to adjacent cells increased from 10% at 3 h after plating to 40% at 6 h. To determine whether or not cells maintain the ability to dye-couple throughout the culture period, carboxyfluorescein (CF; 376 Da) and rhodamine dextran (9.3 kDa) were introduced into cells by scraping the cells with a scalpel, and the distribution of dyes was determined 20 min later. In cells cultured for 24, 48, or 72 h, only CF was transferred to cells distal to the scrape line. The results from these experiments suggest that stromal cells can dye-couple throughout the culture period (3-72 h) and indicate that functional gap junctions form between endometrial stromal cells as they undergo decidualization in vitro.


Subject(s)
Connexins/biosynthesis , Decidua/physiology , Endometrium/metabolism , Gap Junctions/physiology , Gene Expression Regulation, Developmental/physiology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Stromal Cells/metabolism , Animals , Blotting, Northern , Connexins/genetics , Endometrium/ultrastructure , Epithelial Cells , Epithelium/physiology , Female , GAP-43 Protein , Immunohistochemistry , Pregnancy , Rats , Rats, Sprague-Dawley , Spectrometry, Fluorescence , Stromal Cells/ultrastructure
11.
Exp Cell Res ; 222(1): 48-53, 1996 Jan 10.
Article in English | MEDLINE | ID: mdl-8549672

ABSTRACT

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.


Subject(s)
Cell Communication/drug effects , Connexin 43/analysis , Gap Junctions/drug effects , Glycyrrhetinic Acid/analogs & derivatives , Succinates/pharmacology , Animals , Carbenoxolone , Connexin 43/biosynthesis , Electric Conductivity , Fluorescent Dyes , Gap Junctions/chemistry , Gap Junctions/ultrastructure , Glioma , Glycyrrhetinic Acid/pharmacology , Isoquinolines , Rats , Tumor Cells, Cultured
12.
Biotechniques ; 18(3): 490-7, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7779401

ABSTRACT

We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.


Subject(s)
Cell Communication/physiology , Fluorescent Dyes/metabolism , Gap Junctions/metabolism , Carbenoxolone , Carbocyanines/metabolism , Cell Communication/drug effects , Cell Line , Cell Line, Transformed , Connexin 43/genetics , Fluoresceins/metabolism , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhetinic Acid/pharmacology , Microscopy, Fluorescence , Oncogene Protein pp60(v-src)/genetics , Stereoisomerism , Succinates/pharmacology
13.
In Vivo ; 8(5): 745-54, 1994.
Article in English | MEDLINE | ID: mdl-7727722

ABSTRACT

There is increasing evidence that gap junctional communication plays an important role in the control of morphogenesis, differentiation and growth. Here we review the genetic diversity of connexins, structural proteins which form the gap junction, with emphasis on their tissue specific expression and present evidence that junctional communication is perturbed during the process of carcinogenesis. Finally we discuss the clinical implications of these findings in the light of recent experiments demonstrating that increased junctional communication, achieved by pharmacological or by molecular means, results in suppression of tumorigenicity or in enhanced growth control.


Subject(s)
Cell Communication/physiology , Cell Transformation, Neoplastic , Gap Junctions/physiology , Neoplasms/pathology , Neoplasms/physiopathology , Retinoids/pharmacology , Animals , Cell Communication/drug effects , Cell Differentiation/drug effects , Cell Transformation, Neoplastic/drug effects , Connexins/biosynthesis , Female , Gap Junctions/drug effects , Humans , Male , Neoplasms/prevention & control , Precancerous Conditions/pathology , Precancerous Conditions/physiopathology , Precancerous Conditions/prevention & control , Retinoids/therapeutic use , Skin Neoplasms/pathology , Skin Neoplasms/physiopathology
14.
Mol Carcinog ; 11(2): 106-14, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7916992

ABSTRACT

Antisense gene constructs have been very useful in the functional analysis of genes and their products. In this report we used a connexin 43 (Cx43) antisense gene construct to study the role that heterologous gap-junctional intracellular communication (GJIC) plays in the ability of untransformed fibroblasts to suppress the foci-forming ability of src oncogene-transformed cells. Untransformed Rat-1 fibroblasts transfected with the Cx43 antisense DNA construct showed marked decreases in Cx43 RNA and protein, which were accompanied by a corresponding decrease in GJIC. These Cx43 antisense-transfected cells maintained normal cell morphology, growth rates, and saturation densities and did not grow in soft-agar suspension. However, in coculture experiments, the Cx43 antisense cells were less effective than vector-alone-transfected, sense-transfected, and untransfected cells at inhibiting foci formation of pp60v-src-transformed cells. These effects of junctionally competent, normal cells were associated with the existence of heterologous GJIC with the transformed cells and did not appear to result from the elaboration of a stable, diffusible inhibitory factor. Thus, gap-junction-mediated transfer of putative regulatory molecules may play a role in the ability of untransformed cells to suppress the expression of certain properties of transformed cells.


Subject(s)
Cell Communication , Cell Division , Connexin 43/genetics , Gap Junctions/physiology , Animals , Antisense Elements (Genetics) , Blotting, Northern , Blotting, Western , Cell Line , Cell Transformation, Neoplastic , Fibroblasts , Genes, src , Rats , Transfection
15.
Cell Growth Differ ; 5(6): 687-96, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8086343

ABSTRACT

It has been proposed that the transfer of growth regulatory signals via gap junctions is important in the control of proliferation. In confluent 10T1/2 cells, growth control is enhanced by retinoids; this action is highly correlated with up-regulated gap junctional communication (GJC). Treatment of quiescent 10T1/2 cells with transforming growth factor (TGF) beta 1 and TGF-beta 2 resulted in elevated levels of proliferation together with increased expression of connexin43 protein and elevated GJC. Connexin43 was localized into plaques in regions of cell-cell contact; such plaques were found at high frequency in treated cells but only rarely in control cultures. These data illustrate that increased cell proliferation can occur in the presence of enhanced GJC, a result at variance with our previous results with retinoids. We suggest that either the proliferative stimulus of TGF-beta is sufficient to overwhelm any antiproliferative effect of GJC or that under conditions of TGF-beta stimulation, junctions convey net proliferative stimuli.


Subject(s)
Gap Junctions/physiology , Transforming Growth Factor beta/pharmacology , Animals , Cell Communication/drug effects , Cell Communication/physiology , Cell Line , Connexin 43/biosynthesis , Dose-Response Relationship, Drug , Drug Interactions , Gap Junctions/drug effects , Mice
16.
Dis Colon Rectum ; 37(5): 439-43, 1994 May.
Article in English | MEDLINE | ID: mdl-8181404

ABSTRACT

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19-59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent), Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas. Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months +/- 2.9 SEM (range, 1-94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.


Subject(s)
HIV Seropositivity/microbiology , Opportunistic Infections/microbiology , Rectal Diseases/microbiology , Acyclovir/therapeutic use , Adult , Anus Diseases/complications , Anus Diseases/diagnosis , Anus Diseases/microbiology , Anus Diseases/therapy , Chlamydia/isolation & purification , Combined Modality Therapy , Cytomegalovirus/isolation & purification , HIV Seropositivity/complications , HIV Seropositivity/diagnosis , HIV Seropositivity/therapy , Humans , Male , Middle Aged , Mycobacterium/isolation & purification , Neisseria gonorrhoeae/isolation & purification , Opportunistic Infections/complications , Opportunistic Infections/diagnosis , Opportunistic Infections/therapy , Prospective Studies , Rectal Diseases/complications , Rectal Diseases/diagnosis , Rectal Diseases/therapy , Risk-Taking , Simplexvirus/isolation & purification , Treatment Outcome
17.
Gene ; 133(2): 233-5, 1993 Nov 15.
Article in English | MEDLINE | ID: mdl-7901127

ABSTRACT

A chicken DNA fragment, which was isolated from a cosmid genomic library using a murine homeobox probe, is shown here to be a likely candidate for encoding a protein homologous to the bovine B15 subunit of NADH ubiquinone oxidoreductase. The genomic sequence suggests the presence of an intron separating two coding regions. An alignment between the bovine NADH B15 protein and its putative avian homolog implies the presence of conserved domains, including a transmembrane helix, tyrosine sulfatation site, and a phosphorylation consensus site.


Subject(s)
DNA-Binding Proteins/genetics , Genes, Homeobox , Homeodomain Proteins , NAD(P)H Dehydrogenase (Quinone)/genetics , Amino Acid Sequence , Animals , Base Sequence , Chickens , DNA , Mice , Molecular Sequence Data , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
18.
Biochem J ; 295 ( Pt 3): 735-42, 1993 Nov 01.
Article in English | MEDLINE | ID: mdl-7694570

ABSTRACT

Connexin43 phosphorylation was analysed in non-transformed and pp60v-src-transformed Rat-1 fibroblasts. Connexin43 appeared to be the primary connexin expressed in these cells. Although gap-junctional communication was disrupted in pp60v-src-transformed cells, they contained more connexin43 protein and RNA than their non-transformed counterpart. Connexin43 was phosphorylated within minutes of its synthesis in both cell types and appeared to be degraded while in the phosphorylated state. Phosphopeptide and phosphoamino acid analyses suggested that connexin43 in both cell types contained at least five fragments with serine phosphorylation. The major difference in connexin43 phosphorylation between the pp60v-src-transformed and non-transformed cells was that, whereas approx. 70% of the phosphorylated connexin43 in the former contained phosphotyrosine, this phosphoamino acid was not detected in connexin43 isolated from the latter cells. These data support the hypothesis that phosphorylation of connexin43 on tyrosine is critical for the blockade of gap-junctional communication which occurs concomitantly with transformation by the pp60v-src oncogene.


Subject(s)
Connexin 43/metabolism , Oncogene Protein pp60(v-src)/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Line, Transformed , Fibroblasts/metabolism , Immunosorbent Techniques , Oncogene Protein pp60(v-src)/genetics , Peptide Fragments/analysis , Phosphorylation , Phosphoserine/analysis , Phosphoserine/metabolism , Phosphotyrosine , RNA, Messenger/metabolism , Rats , Transformation, Genetic , Tyrosine/analogs & derivatives , Tyrosine/analysis
20.
J Invest Dermatol ; 99(6): 836-41, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1469298

ABSTRACT

Human keratinocytes synthesize interstitial collagenase, a 72-kDa gelatinase, and a recently described 92-kDa gelatinase/type IV collagenase. We examined the synthesis of this novel enzyme by basal keratinocytes apposed to plastic, basement membrane collagen (type IV), and interstitial dermal collagen (type I). Samples of conditioned medium were electrophoresed on a 10% polyacrylamide, gelatin-ladened zymogram. Protein bands with gelatin-cleaving properties were identified by clarification of the gel and quantified by densitometry. A 92-kDa band had marked gelatinolytic activity and increased in culture over 72 h. The identification of this 92-kDa band as type IV collagenase was demonstrated by Western immunoblotting using monospecific antibody to the 92-kDa type IV collagenase. Keratinocytes apposed to type I collagen exhibited a threefold increase in the synthesis of the 92-kDa enzyme compared to cultures apposed to type IV collagen and a 1.5-times increase compared to plastic. The specificity of this enhancement was shown by constant levels of other proteins (e.g., the 72-kDa gelatinase). This study demonstrates that cell-matrix interactions modulate the synthesis of a recently described, keratinocyte-derived, 92-kDa gelatinase and that specific collagen types (I versus IV) have opposite effects upon the synthesis of this enzyme.


Subject(s)
Collagenases/biosynthesis , Keratinocytes/enzymology , Collagen/pharmacology , Culture Media, Conditioned , Densitometry , Extracellular Matrix/physiology , Female , Humans , Infant, Newborn , Male , Matrix Metalloproteinase 9 , Tumor Cells, Cultured
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