Assessment of the total effective xenoestrogen burden in extracts of human placentas

We have standardized a method to assess the total effective xenoestrogen burden (TEXB) in human placentas by the extraction and separation by high-performance liquid chromatography of two fractions containing lipophilic xenoestrogens (alpha) and endogenous hormones (beta), followed by assessing their estrogenicity in MCF-7 breast cancer cell-based E-Screen and Yeast Estrogen Screen (YES) bioassays. The means of TEXB alpha concentrations (in estradiol equivalent (Eeq) units) were 1.32 and 0.77 Eeq pM g^?1 placenta in the E-Screen and YES, respectively; TEXB beta concentrations were 6.97 and 11.56 Eeq pM g^?1 placenta, respectively. The interclass correlation coefficient was low and a fair level of agreement was observed after kappa test correction. According to the E-Screen and YES, TEXB alpha was ≥LOD in 70.0 and 55.0% of the placentas and 92.5 and 82.5% in beta, respectively. Although both bioassays can be recommended for assessing TEXB, there is greater experience with the use of the E-Screen for estrogenic assessment after extensive extraction of complex human matrices.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Development of Mice with Osteoblast-Specific Connexin43 Gene Deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43^fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43^±; Colα1(I)-Cre or Cx43^±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43^fl/?; Colα1(I)-Cre or Cx43^fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.     

Modifications in Connexin Expression in Liver Development and Cancer

The establishment of an elaborate gap junctional intercellular communication network, especially between hepatocytes, is important for normal liver development. In fact, the production of the gap junction building blocks, the connexins, undergoes several well-defined changes throughout the hepatic differentiation process. This ultimately results in the acquisition of an adult connexin expression pattern which is critical for maintaining the fully differentiated hepatocyte-specific phenotype. Abnormalities of connexin production are observed in a number of pathological conditions, such as during liver cancer. This article provides an overview of these processes with emphasis on the underlying molecular mechanisms.     

Bisphosphonates and Connexin 43: A Critical Review of Evidence

Abstract

Bisphosphonates (BPs) are drugs commonly used in the treatment of various disease arising or affecting bone tissue. There is a standard use in bone neoplasia and metastasis, hormonal and developmental disorders as well as for compensation of adverse effects in several medical therapies. Many in-vivo and in-vitro studies have assessed the efficacy of this drug and its function in cellular scale. In this concern, BPs are described to inhibit the resorptive function of osteoclasts and to prevent apoptosis of osteoblasts and osteocytes. They can preserve the osteocytic network, reduce fracture rate, and increase the bone mineral content, which is therapeutically used. Connexin 43 (Cx43) is a crucial molecule for basal regulation of bone homeostasis, development, and differentiation. It is described for signal transduction in many physiological and pathological stimuli and recently to be involved in BP action.     

Increased Co-localization of Connexin43 and ZO-1 in Dissociated Adult Myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium-a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immuno-precipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Comparison of Connexin 43, 40 and 45 Expression Patterns in the Developing Human and Mouse Hearts

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.