Functional gap junctions in thymic epithelial cells are formed by connexin 43

A multiparametric study was carried out to investigate the presence and possible role of communicating junctions in the thymus, particularly in the thymic epithelium, the major component of the thymic microenvironment. The presence of direct cell-cell communication mediated by gap junctions was demonstrated in human and murine thymic epithelial cells (TEC) by means of in situ and in vitro immunohistochemical labeling as well as in vitro fluorochrome injection and double whole-cell patch clamp experiments. Moreover, both immuno- and Northern blot studies revealed that the gap junction protein connexin 43 and its mRNA were present in TEC. Importantly, we showed that thymic endocrine activity, as ascertained by thymulin production, could be specifically downmodulated in vitro by a gap junction inhibitor, octanol. We also investigated the existence of gap junctions between TEC and thymocytes. In thymic nurse cells we were able to detect cell-cell communication, although only a minor percentage of epithelial/thymocyte pairs were coupled in a given moment. In contrast, intercellular communication was not detected between cultured phagocytic cells of the thymic reticulum and the respective rosetting thymocytes. We suggest that gap junctions formed by connexin 43 may represent a novel (and rather cell type-specific) pathway for intrathymic cellular communication, including TEC/TEC as well as possible TEC/thymocyte interactions.     

Classical swine fever virus is genetically stable in vitro and in vivo

Summary.  Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene’93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5′ NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies. Received January 15, 1999 Accepted May 2, 1999     

A sequence database allowing automated genotyping of Classical swine fever virus isolates

Classical swine fever (CSF) is a highly contagious viral disease of pigs. According to the OIE classification of diseases it is classified as a notifiable (previously List A) disease, thus having the potential for causing severe socio-economic problems and affecting severely the international trade of pigs and pig products. Effective control measures are compulsory, and to expose weaknesses a reliable tracing of the spread of the virus is necessary. Genetic typing has proved to be the method of choice. However, genotyping involves the use of multiple software applications, which is laborious and complex. The implementation of a sequence database, which is accessible by the World Wide Web with the option to type automatically new CSF virus isolates once the sequence is available is described. The sequence to be typed is tested for correct orientation and, if necessary, adjusted to the right length. The alignment and the neighbor-joining phylogenetic analysis with a standard set of sequences can then be calculated. The results are displayed as a graph. As an example, the determination is shown of the genetic subgroup of the isolate obtained from the outbreaks registered in Russia, in 2005. After registration (Irene.greiser-wilke@tiho-hannover.de) the database including the module for genotyping are accessible under http://viro08.tiho-hannover.de/eg/eurl_virus_db.htm.     

Connexin43 gap junctions exhibit asymmetrical gating properties

A communication-deficient cell line (RIN cells, derived from a rat islet tumour), stably transfected with cDNA coding for rat connexin43 (Cx43), was chosen to further assess the mechanism of voltage gating of Cx43 gap junction channels. The experiments were carried out on preformed cell pairs using a dual whole-cell, voltage-clamp method. The junctional current, I _j, revealed a time- and voltage-dependent inactivation at transjunctional voltages V _j > ± 40mV. When an asymmetrical pulse protocol was used (in cell 1 the holding potential was maintained, in cell 2 it was altered to establish a variable V _j), the channels exhibited an asymmetrical gating behaviour: V _j,0 = −73.7 mV and 65.1 mV for negative and positive V _j, respectively (V _j at which I _j is half-maximally inactivated); g _j(min) = 0.34 and 0.29 (normalized minimal conductance); τ = 350 ms and 80 ms at V _j = 100 mV (time constant of I _j inactivation). Hence, these parameters were more sensitive to positive V _j values. When a symmetrical pulse protocol was used (the holding potentials in cell 1 and cell 2 were altered simultaneously in steps of equal amplitude but of opposite polarity), the V _j -dependent asymmetries were absent: V _j,0 = −60.5 and 59.5; g _j (min) = 0.27 and 0.29; τ = 64 ms and 47 ms at 100 mV. Putative explanations for these obervations are discussed. A possibility is that the number of channels alters with the polarity of V _j. Received: 1 September 1995/Received after revision and accepted: 9 November 1995     

Pericyte endothelial gap junctions in human cerebral capillaries

Summary Human cerebral tissue has been ultrastructurally studied and gap junctions have been visualized between endothelial cells and pericytes that permit ion exchange. We propose that the functional interrelationship between endothelium and pericytes may play a role in the alteration of capillary diameter for the control of local cerebral blood flow.     

Classical swine fever virus NS5B-GFP fusion protein possesses an RNA-dependent RNA polymerase activity

Summary.  RNA-dependent RNA polymerase (RdRp) is the replicase of positive-strand RNA viruses. Expression and characterization of the replicase are the first steps in the elucidation of the virus replication mechanism. We expressed nonstructural protein 5B (NS5B) of classical swine fever virus (CSFV) as a fusion protein with green fluorescent protein (GFP) in porcine kidney cells (PK-15 cells), natural host cells of CSFV. The expressed CSFV NS5B-GFP fusion protein possessed RdRp activity. By fluorescence microscope it was observed that the density of the fusion protein near cytoplasmic membranes was higher than that in other parts of cells. This was in contrast to the distribution of the GFP alone which was uniformly distributed throughout the cytoplasm. The GFP is a signal for the location of NS5B in a host cell that allows in vitro and in vivo investigation of the distribution of plus-strand RNA virus RdRp. Received September 24, 2001; accepted March 23, 2002 Published online July 19, 2002     

Spontaneous lung dysfunction and fibrosis in mice lacking connexin 40 and endothelial cell connexin 43

Gap junction proteins (connexins) facilitate intercellular communication and serve several roles in regulation of tissue function and remodeling. To examine the physiologic effects of depleting two prominent endothelial connexins, Cx40 and Cx43, transgenic mice were generated by breeding Cx40-deficient mice (Cx40(-/-)) with a vascular endothelial cell (VEC)-specific Cx43-deficient mouse strain (VEC Cx43(-/-)) to produce double-connexin knockout mice (VEC Cx43(-/-)/Cx40(-/-)). The life span in VEC Cx43(-/-)/Cx40(-/-) mice was dramatically shortened, which correlated with severe spontaneous lung abnormalities as the mice aged including increased fibrosis, aberrant alveolar remodeling, and increased lung fibroblast content. Moreover, VEC Cx43(-/-)/Cx40(-/-) mice exhibited cardiac hypertrophy and hypertension. Because VEC Cx43(-/-)/Cx40(-/-) mice demonstrated phenotypic hallmarks that were remarkably similar to those in mice deficient in caveolin-1, pulmonary caveolin expression was examined. Lungs from VEC Cx43(-/-)/Cx40(-/-) mice demonstrated significantly decreased expression of caveolin-1 and caveolin-2. This suggests that expression of caveolin-1 may be linked to expression of Cx40 and endothelial Cx43. Moreover, the phenotype of caveolin-1(-/-) mice and VEC Cx43(-/-)/Cx40(-/-) mice may arise via a common mechanism.     

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.     

Plaque assay for African swine fever virus on swine macrophages

Summary.  A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses. Received December 28, 2001; accepted February 20, 2002 Published online April 26, 2002     

Phylogenetic analysis of swine fever virus strains

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.     

Proteins specified by African swine fever virus

Infection of MS cells with African swine fever virus (ASFV) produces inhibition of protein synthesis which is detectable from 4.5 hours after infection. At least 34 viral polypeptides have been indentified with molecular weights ranging between 9500 and 243,000 daltons. Three of these proteins show affinity for the cell nucleus and nine are in both the nuclear and cytoplasmic fractions. Ten early proteins were found, and most of the structural proteins were late proteins. Most of the proteins are synthesized within the first 8 hours after infection. At least nine proteins induced antibodies in the natural infection. Six of these proteins are structural proteins. The antigenic determinants of VP172, VP162, VP146, VP73, VP34, and IP23.5 are in the primary structure of the proteins.     

The role of myoendothelial gap junctions in the formation of arterial aneurysms: the hypothesis of "connexin 43:40 stoichiometry"

Heterocellular myoendothelial gap junctions (MEGJs) are essential in coordinating and regulating vasomotion. Little is known about their potential role in disease states. We discuss how alteration in the Cx 43:40 expression ratio at the level of MEGJs may begin a chain of reactions in the arterial wall resulting in an aneurysm formation. In this model, we assumed that aneurysm is a chronic arterial disease associated with medial degeneration and intimal hyperplasia. It also was assumed that MEGJs are composed of Cx43 and Cx40 in different stoichiometry and that the characteristic of a given junction is in the favor of its most abundantly expressed constituent. The hypothesis of Cx 43:40 stoichiometry indicates that impaired MEGJs may play a role in the pathogenesis of arterial aneurysms. Cx43 upregulation and Cx40 downregulation (increased Cx 43:40 stoichiometry) may induce a cascade of inflammatory, electrical, metabolic and proliferative derangements in the arterial wall, which finally lead to the matrix degradation, intimal hyperplasia, endothelial-medial dissociation and loss of endothelium-dependent hyperpolarizing currents, irregular vasomotion, impaired growth factor activation, and arterial sympathetic deprivation. The final consequence of these alterations is aneurysm formation.     

Proteins specified by African swine fever virus

Summary At least 28 polypeptides have been identified in intracellular virus, with molecular weights ranging from 11,500 to 243,000 daltons. By treatment with Nonidet P-40 and 2-mercaptoethanol it is possible to obtain subviral particles that have lost some proteins and have a density in CsCl of 1.31 g/cm³ which is higher than that of the complete virus (1.23 g/cm³). After addition of NaCl the virus loses its major protein VP73 which indicates that it is localized in the viral envelope. Cores obtained after this treatment are made up of at least 14 proteins. Incorporation of³H-fucose and³H-glucosamine in intracellular virus occurs in three minor components. The protein VP42 is possibly the cell actin and appears to be strongly associated with the virus. It is not possible to eliminate it under conditions where the viral envelopes desappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.With 5 Figures     

Epitopic diversity of African swine fever virus

African swine fever (ASF) is caused by an icosahedral cytoplasmic, double stranded DNA virus. In the acute form of the disease, pigs die from disseminated intravascular coagulation (DIC) with extensive damage of the free and fixed macrophage systems and the reticular epithelial cells of the thymus; mortality is virtually 100%. In recent years, subacute and chronic forms of ASF have become more prevalent in the field, especially in outbreaks occurring outside the continent of Africa, and virus isolated from these outbreaks have often been of lesser virulence. In pigs experimentally infected with such isolates, a number of immunopathological manifestations have been encountered, e.g. hypergammaglobulinemia associated with necrotizing pneumonia, persistent infection in the presence of ASF-specific antibodies, and lack of demonstrable virus neutralizing antibodies. Nevertheless, the immune systems of pigs that have clinically recovered have not been impaired by the infection. We suggest that the heterogeneous composition of the virus population in a given isolate may be one of the causes of the anomalous immune responses. When a number of biological markers, i.e., hemadsorption characteristics, plaque size, infectivity, virulence, antigenic determinants, and genomic structure, were used to characterize the virus clones derived from various ASF virus (ASFV) isolates, considerable heterogeneity was apparent. In the present investigation, 20 monoclonal antibodies (MAb), which specifically identified the 14 kDa viral protein within the cytoplasmic membrane of the infected cells, were used to determine epitopic differences among a number of virus clones derived from various isolates. All of the non-African isolates examined contained two epitopically different groups of virus clones, and the reaction profiles obtained were distinctly different from those obtained with the clones of an African isolate (Tengani). It was concluded that an ASFV isolate is composed of a biologically diverse virus population with distinctly different members which are only identified after cloning.     

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down-regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special-type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up-regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up-regulation of Cx26 and/or Cx43 in the carcinoma cells of over two-thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems. © 1998 John Wiley & Sons, Ltd.