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PubMed1996

Changes in the expression and distribution of connexin 43 in isolated cultured adult guinea pig cardiomyocytes.

In the present study, we have investigated the changes in the expression and distribution of the principal gap-junction channel protein in ventricular muscle, connexin 43 (Cx43), during the first 2 weeks of culturing adult guinea pig cardiomyocytes at low density to prevent formation of cellular contacts. In freshly isolated cardiomyocytes, immunoreactive Cx43 occupied 6.5 +/- 0.4% of the pixel area of the cell, with 85% being localized to dense particles at the step-like end projections of the myocytes (intercalated disk regions) and 15% being within the sarcoplasm or along the lateral surface of the myocytes ("nondisk" distribution). During the myocytes' first 48 h in culture, immunoreactive Cx43 decreased by 27.5% from control values, to 4.7 +/- 0.5% of the cells' pixel area (P < 0.01). Cx43 particles also redistributed: after 48 h in culture approximately 90% of the immunoreactive Cx43 was localized in the sarcoplasm and nondisk regions of the myocyte. After 7 days, immunoreactive Cx43 only occupied 50% of the cells' control pixel area (P < 0.01) and was nearly uniform in its punctate pattern throughout the sarcoplasm. This distribution remained the same during the 2nd week in culture. Changes in myosin light chain staining during 8 days in culture largely paralleled those in Cx43 staining. Laser confocal microscopic analysis of double-immunolabeled myocytes that had been in culture for 24-48 h showed colocalization of Cx43 with clathrin in approximately 30% of the sarcoplasmic Cx43 particles. Thus it is demonstrated that the expression of Cx43 decreases significantly during the first 48 h in culture after myocyte isolation and that Cx43 also undergoes substantial redistribution but for the next 2 weeks remains more or less unchanged and at relatively high levels (approximately 50%). These data indicate that cardiomyocytes in isolation maintain their ability to reconnect with each other for up to at least 2 weeks. This is the first time that this property has been investigated in cultured adult ventricular cardiomyocytes.

Analysis of Variance↗
PubMed2000

Colocalization of doublecortin with the microtubules: an ex vivo colocalization study of mutant doublecortin.

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.

Animals↗
PubMed1997

The cut-homeodomain transcriptional activator HNF-6 is coexpressed with its target gene HNF-3 beta in the developing murine liver and pancreas.

Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells. We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions. We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6, but not HNF-3 beta, expression continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

Amino Acid Sequence↗
PubMed1998

In situ hybridization with 33P-labeled RNA probes for determination of cellular expression patterns of liver transcription factors in mouse embryos.

Murine hepatocyte nuclear factor-3beta (HNF-3beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelium, and pancreatic acinar cells. We have previously identified a hepatocyte and pancreatic cut-homeodomain transcription factor, HNF-6, which is required for HNF-3beta promoter activity. In this study, we used in situ hybridization studies of stage-specific embryos to demonstrate that HNF-6 and its target gene, HNF-3beta, are coexpressed in the foregut endoderm and in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelium, and pancreatic ductal epithelium and exocrine acinar cells. In support of the role of HNF-6 in regulating HNF-3beta expression in developing hepatocytes, their liver expression levels are both transiently reduced between 14 and 15 days of gestation. At day 18 of gestation and in adult pancreas, HNF-6 and HNF-3beta transcripts remain colocalized in the exocrine acinar cells, but their expression patterns diverge in endocrine cells. HNF-3beta expression is restricted to the endocrine cells of the islets of Langerhans, whereas the ductal epithelium expresses HNF-6. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

Animals↗
PubMed1998

The intracellular expression pattern of the human papillomavirus type 11 E1--E4 protein correlates with its ability to self associate.

The function of the human papillomavirus type 11 (HPV 11) E1--E4 spliced protein is not known. E1--E4 protein in HPV-infected tissue is detected in the cytoplasm of differentiated epithelial cells and as immunoreactive bands corresponding to potential monomers, dimers and trimers in immunoblots. The yeast two-hybrid system was employed to test for self association of the HPV 11 E1--E4 protein. To confirm the results of the yeast two-hybrid experiments, coimmunofluorescence studies of a green fluorescent fusion protein (GFP-E1--E4) and a T7 epitope-tagged E1--E4 protein were performed in C33a keratinocytes. E1--E4 protein was shown to self associate in the yeast two-hybrid system, and this result was confirmed by colocalization of GFP-E1--E4 and T7-E1(wedge)E4 proteins in keratinocytes. Analysis of E1--E4 mutants established that the C-terminus was required for self association and that sequences in the N-terminus influenced the intracellular localization of E1--E4 protein. The intracellular expression patterns of GFP-E1--E4 and GFP-E1--E4 mutants were correlated with E1--E4 binding in the yeast two-hybrid system. Those E1--E4 mutants that did not self associate in the yeast two-hybrid system were detected as diffuse cellular fluorescence when expressed as GFP fusions. In contrast, GFP-E1--E4 was detected as a perinuclear aggregate. All E1--E4 mutants capable of associating with E1--E4 in the yeast two-hybrid system were detected as aggregates when expressed as GFP fusion proteins in keratinocytes.

Animals↗
PubMed1994

Structural organization and neuropeptide distributions in the equine enteric nervous system: an immunohistochemical study using whole-mount preparations from the small intestine.

The architecture and neurochemistry of the enteric nervous system was studied by use of whole-mount preparations obtained by microdissection of the horse jejunum. A myenteric plexus and two plexuses within the submucosa were identified. The external submucosal plexus lying in the outermost region of the submucosa had both neural and vascular connections with the inner submucosal plexus situated closer to the mucosa. Counts of neurones stained for NADH-diaphorase demonstrated the wide variation in size, shape and neurone content of individual ganglia in both the external and internal submucosal plexuses. The average number of cells/ganglion was similar in each plexus (about 25 cells). Immunoreactivities for galanin, vasoactive intestinal peptide and neuropeptide Y were observed in nerve cell bodies and fibres of each of the plexuses. Immunoreactivity for substance P was extensive and strong in nerve fibres of all plexuses but was weaker in cell bodies of the submucosal neurones and absent in the cell bodies of the myenteric plexus. Comparative quantitative analysis of immunoreactive cell populations with total cell numbers (enzyme staining) was indicative of neuropeptide colocalization in the external submucosal plexus.

Animals↗
PubMed1999

A furostanol glycoside from rhizomes of Costus spicatus.

A new furostanol glycoside was isolated from the rhizomes of Costus spicatus. Its structure was established as (3 beta,22 alpha,25R)-26-(beta -D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[6-deoxy-alpha-L-mannopyranosy l-(1-->4)]- beta-D-glucopyranoside. The structural identification was performed using detailed analysis of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR and COLOC) and chemical conversions.

Carbohydrate Conformation↗
PubMed1998

Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody.

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

Animals↗
PubMed1998

Voltage-dependent anion channel proteins in synaptosomes of the torpedo electric organ: immunolocalization, purification, and characterization.

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca2+ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate-cytochrome-c oxidoreductase and antimycin-insensitive NADH-cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [14C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30-35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDACI (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.

Amino Acid Sequence↗
PubMed2001

The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

Acute Disease↗
PubMed1999

Syntaxin 1 interacts with the L(D) subtype of voltage-gated Ca(2+) channels in pancreatic beta cells.

Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.

Animals↗
PubMed1999

Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization.

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.

Amino Acid Sequence↗
PubMed1999

Association of two nuclear proteins, Npw38 and NpwBP, via the interaction between the WW domain and a novel proline-rich motif containing glycine and arginine.

We have previously reported a nuclear protein possessing a WW domain, Npw38 (Komuro, A., Saeki, M., and Kato, S. (1999) Nucleic Acids Res. 27, 1957-1965). Here we report a Npw38-binding protein, NpwBP, isolated from HeLa cell nuclear extracts and its characterization using a cloned cDNA. NpwBP contains two proline-rich regions that are capable of binding to the WW domain of Npw38. The binding analysis using an oligopeptide-immobilized membrane revealed that the WW domain of Npw38 preferentially recognizes a short proline-rich sequence, PPGPPP, surrounded by an arginine residue, so we named it a PGR motif. Localization analysis using green fluorescent protein fusion protein and immunostaining showed that Npw38 and NpwBP are colocalized in the same subnuclear region. Coimmunoprecipitation experiments confirmed the association between Npw38 and NpwBP, which were expressed as epitope-tagged forms in COS7 cells. Furthermore, the N-terminal region of NpwBP has binding ability for poly(rG) and G-rich single-stranded DNA. These results suggest that NpwBP is a physiological ligand of Npw38 and that the Npw38-NpwBP complex may function as a component of an mRNA factory in the nucleus.

Amino Acid Sequence↗
PubMed2000

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation.

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.

Apoptosis↗
PubMed2000

Peroxiredoxin IV is a secretable protein with heparin-binding properties under reduced conditions.

Peroxiredoxins (PRxs) play a role in protecting protein free thiol groups against oxidative damage and thioredoxin-dependent peroxidase activity. This report describes the characteristics of the fourth member of the mammalian PRxs, PRx IV. Rat PRx IV produced in Sf21 cells by a baculovirus expression system has two bands with different electrophoretic mobilities, 31 and 27 kDa [Matsumoto et al. (1999) FEBS Lett. 443, 246-250]. The 27-kDa PRx IV lacks the NH(2)-terminal 36 amino acids which correspond to a predicted leader peptide, which is required for secretion from cells. Thus, the 31-kDa form is probably a precursor form, and the 27-kDa form, a secretable form which is enzymatically active. Pulse-chase experiments of PRx IV-transfected COS-1 cells showed that PRx IV is processed within 10 min and released from cells. The secretable form contains both reduced and oxidized forms. The reduced form binds to both a heparin affinity column and human umbilical vein endothelial cells, while the oxidized form does not. The equilibrium dissociation constants, K(D), for heparin and heparan sulfate as judged by surface plasmon resonance experiments were 19 and 870 nM, respectively. The secretable form corresponds to the major bands found in most tissues, as evidenced by immunoblot analysis. Within cells, secretable form was largely localized on the endoplasmic reticulum, as judged by colocalization with calreticulin. Moreover, PRx IV has glutathione-dependent peroxidase activity in addition to thioredoxin-dependent activity. These data indicate that PRx IV is a secretable protein and may exert its protective function against oxidative damage by scavenging reactive oxygen species in the extracellular space.

Animals↗
PubMed1999

Transforming growth factor-betas and their signaling receptors are coexpressed in Crohn's disease.

OBJECTIVE: To evaluate mechanisms that contribute to tissue repair and tissue remodeling in Crohn's disease (CD). SUMMARY BACKGROUND DATA: Transforming growth factor-betas (TGF-betas) are involved in different chronic inflammatory disorders. They function by binding to two receptors, type I (TbetaR-I) subtype ALK5 and type II (TbetaR-II), which are concomitantly required for signal transduction. METHODS: Tissues were obtained from 18 patients with CD (10 female patients, 8 male patients, median age 38.7 years [range 16 to 58 years]) undergoing surgery because of CD-related complications. Tissue samples of 18 healthy organ donors (10 female subjects, 8 male subjects, median age 50.3 years [range 15 to 65 years]) served as controls. The expression and localization of TGF-beta1, TGF-beta2, TGF-beta3, TbetaR-IALK5, TbetaR-II, and TbetaR-III were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: On Northern blot analysis, 94% of the CD samples exhibited enhanced TGF-beta1, TGF-beta3, and TbetaR-II mRNA expression compared with controls. TGF-beta2 was increased in 72%, TbetaR-IALK5 in 72%, and TbetaR-III in 82% of the patients with CD. On in situ hybridization and immunohistochemical analysis, TGF-beta1, TbetaR-IALK5, and TbetaR-II were seen to be colocalized in the lamina propria cells and in the lymphocytes closest to the luminal surface, but also in the remaining epithelial cells, and in fibroblasts of CD tissue samples. CONCLUSIONS: The concomitant overexpression of TGF-betas and their signaling receptors in CD points to a potential role of these regulatory molecules in the pathophysiology of CD. Activation of TGF-beta-mediated pathways might promote the repair of mucosal injury by enhancing the process of reepithelization, but might also contribute to extracellular matrix generation and subsequently to intramural fibrosis and intestinal obstruction.

Adolescent↗
PubMed1991

Identification and characterization of a novel cytoskeleton-associated pp60src substrate.

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.

Amino Acid Sequence↗
PubMed1996

Immunolocalization of the mNav2.3 Na+ channel in mouse heart: upregulation in myometrium during pregnancy.

mNav2.3 is a putative voltage-dependent sodium channel (NaC) gene expressed in both mouse heart and uterus that shares only 45% amino acid identity with NaCs from gene subfamily 1. Immunofluorescence studies using polyclonal antibodies against two distinct epitopes revealed that mNav2.3 protein in heart colocalized with nerve-specific antibody binding. Similar mNav2.3-specific antibody staining was observed in virgin uterus. However, mNav2.3 expression in uterine nerve disappeared during late pregnancy, concurrent with an appearance in both the longitudinal and circular uterine smooth muscle, which reached a maximum at term and quickly declined within 2 days postpartum. mNav2.3 expression in term uterus often colocalized on the myocyte surface with connexin 43. The immunofluorescence results are supported by Western analysis in which the 217-kDa NaC increased during late pregnancy and declined 2 days postpartum. These data provide perhaps the most dramatic example of NaC regulation. The acute and transient upregulation in myometrium during gestation suggests the Nav2.3 channel plays a role in uterine function at term.

Animals↗