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PubMed1994

Isolation and characterization of gap junctions in the osteoblastic MC3T3-E1 cell line.

Gap junctions are channels connecting cells that function in cell-to-cell communication. Gap junctions are abundant in osteoblastic cells. Membranes enriched for gap junction plaques were obtained by differential centrifugation, followed by treatment of the membranes with potassium iodide and sarkosyl before sucrose density gradient centrifugation. Electron microscopy showed that the preparation was enriched for electron-dense membranes consistent with gap junctions. Coomassie Blue staining of SDS-PAGE preparations revealed a prominent band at approximately 41 kD. Western analysis with a site-directed antibody, CT-360 (D. Laird, California Institute of Technology, Pasadena, CA), to the C-terminal portion of the rat heart connexin 43 molecule was positive in the MC3T3-E1 cell line, a phenotypic osteoblastic cell line derived from normal neonatal mouse calvariae. Western analysis using a monoclonal antibody, R5.21C, to rat liver connexin 32 was negative. Additionally, a prominent band at 59 kD was detected by CT-360 in both gap junction-enriched preparations and cell lysates. Treatment of diluted samples of gap junction-enriched preparations with sulfhydryl reducing agents in combination with detergents resulted in the enhancement and diminution of the 41 and 59 kD bands, respectively. Immunoprecipitation following [35S]methionine/[35S]cysteine labeling revealed a significant band detected at 122 kD in addition to the 41 kD band. To demonstrate functional gap junctions, transfer of lucifer yellow dye to surrounding cells was monitored after microinjection of a target cell. Between passages 10 and 25 in culture, functional cell coupling was found in approximately 70% of injected cells. Coupling was detected within 1-2 minutes after injection. Simultaneous microinjection of the CT-360 antibody with lucifer yellow resulted in the decoupling of cells. In conclusion, (1) MC3T3-E1 cells possess a 41 kD protein that is recognized by connexin 43 antibody to rat heart gap junction; (2) multimers of the MC3T3-E1 gap junctions occur in the preparation; and (3) functional coupling demonstrated by dye transfer may be regulated by region(s) in the C terminus of the connexin molecule.

3T3 Cells↗
PubMed2006

Differential activities of the core planar polarity proteins during Drosophila wing patterning.

During planar polarity patterning of the Drosophila wing, a "core" group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell-cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell-cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells.

Oxidative stress plays a key role in aging diseases of the posterior pole of the eye such as age-related macular degeneration. The oxidative stress response of in vitro RPE cells has been studied for a small number of genes. However, a comprehensive transcriptional response has yet to be elucidated. The purpose of this study was to determine if the transcription of a common set of genes is altered by exposure of ARPE-19 cells to three major generators of oxidative stress, hydrogen peroxide (H2O2), 4-hydroxynonenal (HNE), and tert-butylhydroperoxide (tBH). As expected, a common response was observed that included 35 genes differentially regulated by all three treatments. Of these, only one gene was upregulated, and only by one oxidant, while all other responses were downregulation. The majority of these genes fell into five functional categories: apoptosis, cell cycle regulation, cell-cell communication, signal transduction, and transcriptional regulation. Additionally, a large number of genes were differentially regulated by one oxidant only, including the majority of the conventional oxidative stress response genes present on the Clontech Human 1.2 microarray. This study raises questions regarding the generality of results that involve the use of a single oxidant and a single cell culture condition.

Apoptosis↗
PubMed1996

Consequences of transcellular biosynthesis of leukotriene C4 on organ function.

Formation of eicosanoids is a special mode of cell communication whereby production of eicosanoids by mixed cell populations differs from that expected from each individual cell. Transcellular biosynthesis of leukotriene C4 occurs via transfer of the reactive intermediate leukotriene A4 from neutrophils to vicinal acceptor cells devoid of 5-lipoxygenase activity such as platelets or vascular cells. Evidence for the in vivo relevance of transcellular eicosanoid metabolism results from experiments using the isolated beating rabbit heart perfused with activated neutrophils. The resultant leukotriene C4 synthesis is timely related to the pressor response of the coronary arteries and inflammatory damage of the heart by edema formation and neutrophil infiltration into the organ. Blockade of leukotriene C4 synthesis by 5-lipoxygenase inhibitors or leukotriene C4 actions by respective receptor antagonists facilitated significant protective effects. Further confirmation of the potential role of LTC4 in myocardial ischemia comes from in vivo studies in the rabbit.

Animals↗
PubMed1993

Dye and electric coupling between osteoblast-like cells in culture.

Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.

1-Octanol↗
PubMed1987

[The microenvironment and hematopoiesis].

Successive steps in hematopoietic differentiation occur in the bone marrow in close contact with non hematopoietic components of the microenvironment. The crucial importance of these interactions in the regulation of early hematopoietic events has been convincingly demonstrated in vivo in the mutated steel mouse, and confirmed more recently in vitro in long-term cultures. Parts of the signal mediating these interactions are represented by locally secreted CSF-like growth factors or more recently characterized inhibitors of proliferation. more importantly however appears to be direct cell-cell communication, especially in the regulation of erythropoiesis and recent data suggest that specific adhesion processes might be required initially. As neoplastic clonal dominance is usually not maintained in vitro, experimental evidence for the involvement of the microenvironment in the regulation of leukemic hemopoiesis is still pending. However, it has been shown recently that in some conditions stromal cells may be manipulated in vitro and alter growth properties of leukemic cells. Understanding mechanisms regulating the interactions between hematopoietic and stromal cells may be of considerable importance since it should be possible to delineate and subsequently modulate in vitro the factors affecting stem cell properties. This might lead to alternative therapeutic strategy.

Animals↗
PubMed1988

Junctional communication and cellular differentiation.

Gap junctions provide pathways of direct cell to cell communication in the tissues of metazoan animals. Cells joined by gap junctions share their small ions and molecules but can maintain distinctive activities through expression of different macromolecules which are too large to pass through the junctions. The junctional channels are made of a tissue invariant, evolutionarily conserved 16-18 k protein but the formation and maintenance of active coupling also requires one or more connexins, a family of tissue-specific proteins ranging in size from 21 k to 70 k. Junctions can be isolated as complexes containing both types of protein by mild procedures using high pH but the connexins can be removed by detergent, urea and protease treatment without destroying the characteristic junctional-morphology of hexagonally packed channels in the double membrane structures. There is also some evidence for the participation in the complex of tissue-specific proteoglycans which perhaps interact with the tissue-specific connexins and account for specificity of junction formation. Such specificity in mixed cultures leads to the production of communication compartments, groups of cells joined by junctions but separated by reduced trans-boundary coupling from cells in adjacent compartments. Compartmentation also occurs in vivo resulting in specific patterns of junctional communication which have been mapped in most detail in mouse skin. These mapping data and the changes which are associated with abnormal proliferation have lead to new ideas on intercellular control.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effects of dinitrotoluenes on morphological cell transformation and intercellular communication in Syrian hamster embryo cells.

The effects of four isomers of dinitrotoluene (DNT) and technical DNT (a mixture of DNT isomers and other compounds, with 2,4-DNT as the major constituent) were studied in two short-term in vitro assays. None of the isomers or technical DNT induced an increase in morphological transformation of Syrian hamster embryo (SHE) cells. Four DNT metabolites (2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, and 2,4-dinitobenzoic acid), representing different stages in reduction or oxidation of DNT isomers, were also negative for induction of morphological transformation. The DNT isomers were tested in an intercellular communication assay based on dye transfer. 2,4-DNT, 2,6-DNT, and technical DNT inhibited intercellular communication in the SHE cell line BPNi at toxic concentrations. This may be reminiscent of in vivo data showing promoting activity of these compound. 2,3-DNT and 3,4-DNT did not inhibit communication.

Animals↗
PubMed2004

Resistance of B16 melanoma cells to CD47-induced negative regulation of motility as a result of aberrant N-glycosylation of SHPS-1.

The adhesion receptor SHPS-1 activates the protein-tyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton. SHPS-1 also contributes to cell-cell communication through association with CD47. Although functional alteration of SHPS-1 is implicated in cellular transformation, the role of the CD47-SHPS-1 interaction in carcinogenesis has been unclear. A soluble SHPS-1 ligand (CD47-Fc) has now been shown to bind to Melan-a non-tumorigenic melanocytes but not to syngeneic B16F10 melanoma cells. Treatment of B16F10 cells with 1-deoxymannojirimycin, which prevents N-glycan processing, restored the ability of SHPS-1 derived from these cells to bind CD47-Fc in vitro, indicating that aberrant N-glycosylation of SHPS-1 impairs CD47 binding in B16F10 cells. CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells. However, a monoclonal antibody that reacts with SHPS-1 on both Melan-a and B16F10 cells inhibited the migration of both cell types similarly. CD47 binding induced proteasome-mediated degradation of SHPS-1 in a tyrosine phosphorylation-independent manner. Furthermore, overexpression of SHPS-1 reduced the level of tyrosine phosphorylation of focal adhesion kinase, and this effect was reversed by CD47 binding. These results suggest that CD47 binds to and thereby down-regulates SHPS-1 on adjacent cells, resulting in inhibition of cell motility. Resistance to this inhibitory mechanism may contribute to the highly metastatic potential of B16 melanoma.

Animals↗
PubMed1998

Expression and activity of epidermal growth factor receptor in late fetal rat lung is cell- and sex-specific.

Epidermal growth factor (EGF) augments late fetal lung maturation by advancing the ontogeny of fetal lung development and by stimulating surfactant synthesis. Previous studies have indicated that fibroblastalveolar epithelial cell communications mediate surfactant synthesis in the fetal lung and EGF acts through such a mechanism. We investigated the hypothesis that is differential activity and expression of the epidermal growth factor receptor (EGF-R) in fetal lung fibroblasts during the canalicular stage of lung development mediates EGF effects. To test this hypothesis, we examined fetal rat lung fibroblasts (FLFs) and type II cells of late gestation (canalicular and saccular stages; 17-22 days) by EGF-R binding techniques, SDS-PAGE, and Western blot analysis. Specific EGF binding increased 181% in day 18 female FLFs, with male FLFs exhibiting a similar increase on day 19. In contrast, specific EGF binding was low in type II cells, did not increase during late gestation, and there were no sex-specific differences. SDS-PAGE and Western blot analysis revealed a predominant 170-kDa EGF-R band in fibroblasts that increased with gestation (peak = 19 days), and was stronger in females. Immunoprecipitation of EGF-treated cells demonstrated the tyrosine kinase activity of the identified receptor. In contrast, type II cells showed minimal signal that did not increase until day 21 of gestation. We also examined whole fetal lung sections by immunohistochemistry to determine cell-specific expression of the EGF-R in vivo. Immunohistochemistry revealed specific EGF-R staining in columnar and cuboidal epithelia of small conducting airways and in mesenchyme of epithelial-mesenchymal borders (including subepithelial mesenchyme). In contrast, alveolar epithelia showed minimal staining, while subalveolar mesenchyme EGF-R staining peaked at day 19 of gestation. We conclude that cell-specific and sex-specific differences in EGF-R binding and EGF-R immunolocalization appears in the fetal lung at a developmental stage that is critical for alveolar epithelial cell differentiation. The results suggest a role for EGF-R activation in late fetal alveolar epithelial cell maturation, which is mediated through mesenchymal-epithelial cell communication.

Animals↗
PubMed2003

Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA) activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.

Animals↗
PubMed1995

Protein tyrosine phosphatases expressed in developing brain and retinal Müller glia.

Regulation of protein function through tyrosine phosphorylation is critical to many developmental processes involving cell-cell communication. A number of protein tyrosine phosphatases (PTPs) have been identified in the early postnatal and mature central nervous system (CNS), but the PTPs expressed during its development have not been well characterized. Using a polymerase chain reaction with degenerate primers, we analyzed PTPs expressed in fetal (E18) rat brain and Müller glia cultures from embryonic chick retina, two systems in which cell-to-cell contacts are numerous. Fetal rat brain expressed four known receptor-like PTPs (PTP delta, LAR, LAR-PTP2, LRP (PTP alpha)) and the non-receptor phosphatase PTP1B. Müller glia exhibited a distinct but overlapping pattern of expression: four known receptor PTPs (PTP alpha, PTP gamma, PTP delta, PTP zeta) and PTP1B. In addition, two novel PTPs, termed MG-PTP1 and 2 (Müller glia PTP 1 and 2) were identified in Müller glia cDNA. MG-PTP1 was related to, but distinct from PTP delta, while MG-PTP2 was related to, but distinct from the cytosolic T-cell phosphatase. These results demonstrate that a distinct but overlapping set of PTPs is expressed in the developing brain and retinal Müller glia, including two novel PTPs that may participate in neural cell communication.

Animals↗
PubMed2000

Cell-to-cell interaction prevents cell death in cultured neonatal rat ventricular myocytes.

OBJECTIVES: Loss of cardiac cells and the anatomical or functional remodeling of intercellular coupling occur under several pathological conditions. We have assessed the significance of intercellular coupling for cell death. METHODS AND RESULTS: Ventricular cells obtained from 1 day old Wistar rats were cultured. Apoptosis was detected by nick-end labeling. Cells were plated at low and high cell density (3x10(4)/ml and 12x10(4)/ml, respectively). Cultured myocytes died spontaneously by apoptosis in a time dependent manner. The increase of the apoptotic cell population in a culture with high cell density on day 4 (1+/-1.2%, n=4) was significantly lower than that in a culture with low cell density (20+/-5.5%, n=4). The progression of apoptosis in the culture of low cell density was prevented in part after application of the medium extract from the culture of high cell density; the apoptotic cell population on day 6 decreased from 57+/-8.0% (n=4) to 36+/-3.8% (n=4). Treatment of the cultured myocytes at high cell density with antisense oligonucleotide for connexin43 (Cx43) for 24 h on day 2 resulted in a significant decrease in Cx43 expression as judged by Western blot, dye transfer and immunocytochemistry using mouse monoclonal antibody for Cx43. In association with the down-regulation of Cx43, the progress of apoptosis was accelerated; the apoptotic cell population on day 5 in the antisense-treated cultures (27+/-5.7%, n=4) was significantly higher than the sense-treated cultures (5+/-1.1%, n=4). The effect of Cx43 antisense treatment to promote apoptosis was not reversed by application of high cell-density culture medium. CONCLUSIONS: These findings suggest that cell-cell communication through gap junction formation and some humoral factors play important roles in the survival of cultured myocytes.

Actinin↗
PubMed1998

Modulation of gap junction expression during transient hyperplasia of rat epidermis.

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of alpha1 (Cx43) and beta2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, alpha1 is expressed in the lower, less differentiated portion, while beta2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in alpha1 and beta2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with beta2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in alpha1 and beta2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.

Animals↗
PubMed1997

Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction.

Actin cables have been reported to act in vivo as contractile 'purse strings' capable of closing embryonic wounds through generation of circumferential tension. Furthermore, their involvement in wounds within in vitro model systems suggests that actin cable contraction may be an important mechanism involved in the process of wound closure. The aim of this study therefore, was to investigate the appearance of actin cables in a contracting fibroblast populated collagen lattice, an in vitro model of events associated with wound contraction. Utilising this in vitro model, the time-course of actin cable production was investigated and the involvement of integrin receptors analysed using immunofluorescent labelling techniques. Over a period of hours distinct cellular cable-like structures developed at the edges of collagen lattices coinciding with the onset of contraction. Cellular organisation within the cable was evident as was polymerisation of actin microfilaments into elongated stress fibres forming a continuous cell-cell 'actin cable' around the circumference of the lattice. Immunolocalisation demonstrated that integrin receptor subunits beta 1 and alpha 2 but not alpha 5 were involved in apparent intimate cell-cell contact between juxtaposed fibroblasts within this actin cable. This study demonstrates the involvement of integrin receptors in actin cable formation within collagen lattice systems undergoing reorganisation. Such integrin involvement may enable participating cells to respond to the tensional status of their surrounding environment and via cell-cell communication, to permit a co-ordinated contraction of the cable. It is concluded that integrin receptor involvement in active actin cable contraction may be involved in the process of wound contraction.

Actins↗
PubMed2005

Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.

The nematode gonad is an exemplary system for the study of organogenesis and fundamental problems in developmental and cellular biology. Nematode gonads vary dramatically across species (Chitwood, B.G., Chitwood, M.B., 1950. Introduction to Nematology." University Park Press, Baltimore; Felix, M.A., Sternberg, P.W., 1996. Symmetry breakage in the development of one-armed gonads in nematodes. Development 122, 2129-2142). As such, comparative developmental biology of gonadogenesis offers the potential to investigate changes in developmental and cellular processes that result in novel organ morphologies and thus may give insights into how these changes can affect animal bauplane. Pristionchus pacificus is a free-living nematode that diverged from the model nematode Caenorhabditis elegans around 200-300 million years ago. The morphology and development of P. pacificus is highly homologous to that of C. elegans. However, many differences in morphology and the underlying molecular signaling networks are easy to identify, making P. pacificus ideal for a comparative approach. Here, we report a detailed description of the P. pacificus hermaphrodite gonad using electron and fluorescent microscopy that will provide a basis for both phenotypic studies of genetic mutations and in vivo molecular studies of cloned genes involved in P. pacificus gonad development. We report that the morphology of the P. pacificus gonad is distinct from that of C. elegans. Among these differences are germ line patterning differences, heterochronic differences, novel gonadal arm-migrations, novel cellular composition of some somatic tissues (e.g., the number of cells that comprise the sheath and different spermathecal regions are different), the absence of a somatic tissue (e.g., the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, we report a set of cell ablations in P. pacificus that indicate extensive cell communication between the somatic gonadal tissues and the germ line. Individual ablation experiments in P. pacificus show significant differences in the effects of individual somatic tissues on germ line patterning in comparison to C. elegans.

Animals↗
PubMedDate not supplied

"In vitro" expression of the cardiogenic potentiality of chick embryo heart-forming cells.

Chick embryos between final presomitic and 4 somites stage were studied. The subcephalic fold was handly severed from the embryos and cultivated in liquid medium for 7 days. Because of the embryo age, no heart anlage was observed at the moment of dissection. After 4 hours of culture the cells began to migrate from the explants. After 20 hours a very extended migration ring was observed in all of the cultures; in the explants, one or more newformed tubular or spherical masses of cells throbbed rhythmically. Their size and shape were related to the embryos age: from presomitic embryos, irregular clusters appeared, while starting from two somites embryos tubular, vascular-like structures were formed. The cells of the throbbing areas at submicroscopic observation showed organizing myofibrillar apparatus into the cytoplasm; junctional complexes between the cells and gap junctions in course of organization were present in the vascular-like structures. This suggests that very early, in the lateral mesoderm are the presumptive cardiac cells which can develop "in vitro" as myocardic elements even in absence of the interactions that occur during the development "in vitro"; the observed vascular-like structures may be considered as an attempt to form a sort of cardiac primordium "in vitro", and a further step in the expression of the cardiogenic potentiality, involving cell-cell communications. The serial sections of the embryos enhanced that into the cultivated areas, vessels from yolk sac are always present; this suggests that the vascular structures, i.e. the endothelium may be involved in the determination of the myocardic elements.

Animals↗
PubMed2006

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein.

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.

14-3-3 Proteins↗