Abstract. We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the threedimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten loci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3-strain, and similarly, Sir3p staining is no longer punctate in a sir4-strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.S EVERAL lines of evidence, including in situ hybridization with whole chromosome probes, suggest that the organization of chromosomes within the interphase nucleus is not random (for review see Cremer et al., 1993). Indeed, it is generally assumed that three-dimensional nuclear organization is likely to facilitate essential nuclear functions, such as transcription, the processing and transport of mRNA, replication, and recombination. Evidence for the organization of chromosomes in prophase nuclei was provided over a hundred years ago by Rabl's observation that salamander chromosomes are positioned in nuclei with centromeres clustered at one pole and the telomeres at the opposite pole (RAN, 1885). Work by Sedat and his colleagues later lent support to this notion through the study of the polytene salivary gland chromosomes of Drosophila melanogaster. They found that centromeres, fused into the chromocenter, abut the nuclear envelope within a restricted area while telomeres tended to cluster at the opposite pole (Mathog et al., 1984;Hochstrasser et al., 1986). A peripheral localization of telomeres has been also reported in Trypanosoma (Chung et

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Connexin-36 (Cx36) is a gap junction protein expressed by the insulin-producing ␤-cells. We investigated the contribution of this protein in normal ␤-cell function by using a viral gene transfer approach to alter Cx36 content in the insulin-producing line of INS-1E cells and rat pancreatic islets. Transcripts for Cx43, Cx45, and Cx36 were detected by reverse transcriptase-PCR in freshly isolated pancreatic islets, whereas only a transcript for Cx36 was detected in INS-1E cells. After infection with a sense viral vector, which induced de novo Cx36 expression in the Cx-defective HeLa cells we used to control the transgene expression, Western blot, immunofluorescence, and freeze-fracture analysis showed a large increase of Cx36 within INS-1E cell membranes. In contrast, after infection with an antisense vector, Cx36 content was decreased by 80%. Glucoseinduced insulin release and insulin content were decreased, whether infected INS-1E cells expressed Cx36 levels that were largely higher or lower than those observed in wild-type control cells. In both cases, basal insulin secretion was unaffected. Comparable observations on basal secretion and insulin content were made in freshly isolated rat pancreatic islets. The data indicate that large changes in Cx36 alter insulin content and, at least in INS-1E cells, also affect glucose-induced insulin release.

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Abstract-Connexin43 (Cx43), the predominant gap junction protein in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension induced by inhibition of nitric oxide synthase on the expression of Cx43 in aorta and heart as well as on the distensibility of the carotid artery. Administration of 0.4 g/L N G -nitro-L-arginine methyl ester (L-NAME) to rats in their drinking water for 4 weeks increased intra-arterial mean blood pressure, wall thickness of aorta and carotid artery (25%), and heart weight (17%). Analysis of heart mRNA demonstrated increased expression of the fetal skeletal ␣-actin and of atrial natriuretic peptide but not of Cx43. In contrast, Cx43 mRNA and protein were decreased by 50% in the aortas of L-NAME-treated rats that did not show increased carotid distensibility. Because these data contrasted with those obtained in the 2-kidney, 1 clip model of rat hypertension, which is characterized by increased arterial distensibility and Cx43 expression in aorta, we investigated by Western blot analysis the posttranslational modifications of Cx43. We found that Cx43 was more phosphorylated in the aorta of 2-kidney, 1 clip rats than in that of L-NAME or control rats, which indicated a differential regulation of Cx43 in different models of hypertension. The data suggest that the cell-to-cell communication mediated by Cx43 channels may help regulate the elasticity of the vascular wall. Key Words: connexin Ⅲ aorta Ⅲ heart Ⅲ elasticity Ⅲ hypertension Ⅲ nitric oxide G ap junctions comprise specialized channels that represent 1 pathway by which vertebrate cells communicate 1 and ensure the electrical and mechanical coupling of different types of muscle cells. 2,3 In vessels, gap junctions provide a pathway to modulate the contractile activity of smooth muscle cells. 4 -9 In the myocardium, junctional coupling ensures the same function and is also implicated in the propagation and synchronization of electrical activity. 10 Characterization of the proteins that form gap junctions is expected to provide insights on the possible involvement of junctional channels during cardiac and arterial hypertrophy associated with hypertension. [11][12][13] We have recently shown that in 2 hypertensive rat models that feature a comparable degree of hypertension, as a result of either the clipping of 1 renal artery or of a DOCA-salt treatment, increased levels of Cx43 were associated with a marked hypertrophy of smooth muscle cells in the aortic wall. 11,12 Under these conditions, the isobaric distensibility of the carotid has been shown to increase because of a reduced elastic modulus that corresponds to a reduction of wall material stiffness. 14,15 These biomechanical changes are not observed in another rat model in which a degree of hypertension similar to that observed in the 2-kidney, 1 clip (2K-1C) and the DOCA-salt models can be induced...