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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↗
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

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↗
PubMed1998

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Enzyme Induction↗
PubMed1994

Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain.

The three currently recognized T3 binding thyroid hormone receptor (TR) isoforms, TR alpha 1, TR beta 1, and TR beta 2, arise from two distinct genes (alpha and beta), whereas two closely related non-T3-binding receptor variants, collectively designated TR alpha 2, arise from alternate splicing of the alpha gene transcript. Using a panel of specific antisera to these isoforms we have assessed the presence or absence of TRs in oligodendrocytes and astrocytes of rat cerebrum and cerebellum. Inferences as to colocalization of the receptor isoforms and cell-specific marker proteins were based on immunohistochemical analysis of the differential emissions of paired immunofluorescent probes. Antisera against myelin basic protein (MBP) identified oligodendroglia, and glial fibrillary acidic protein identified astrocytes. MBP-positive oligodendrocytes displayed positive fluorescent signals with each of the three TR isoform-specific antisera and the antiserum to the receptor variants. These findings are consistent with the concept that the MBP gene is a direct target for thyroid hormone action. TR immunoreactivity appeared to localize primarily to the nuclei of these cells. In contrast, we observed no immunofluorescent signals for any of the TR isoforms in glial fibrillary acidic protein-positive astrocytes. These findings raise the possibility that any effect of thyroid hormone on astrocyte function and structure is mediated indirectly as a result of interaction of thyroid hormone with receptors situated in nonastrocyte cells or as a result of nonnuclear mechanisms.

Animals↗
PubMed1990

Plasmalemmal vesicles are involved in transendothelial transport of albumin, lysosomal enzymes and mannose 6-phosphate receptor fragments in capillary endothelium.

With the use of immunoelectron microscopy we have demonstrated the presence of lysosomal enzymes (acid alpha-glucosidase and glucocerebrosidase) and fragments of the 270 kDa receptor for mannose 6-phosphate and insulin-like growth factor II in blood plasma, plasmalemmal vesicles of endothelial cells and pericapillary spaces in human skeletal muscle tissue. At these locations, the three proteins colocalized with albumin known to be transported from the capillaries into the pericapillary spaces. Immunoblot analysis of plasma revealed the presence of relatively high molecular weight polypeptides in this material. These observations strongly suggest that high molecular weight species of lysosomal enzymes can pass the endothelial barrier in skeletal muscle tissue.

Albumins↗
PubMed1997

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog.

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.

Animals↗
PubMed1999

Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques.

BACKGROUND: Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS: Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS: This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

Arteriosclerosis↗
PubMed1995

Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles.

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.

Animals↗
PubMed1992

Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559.

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule-associated Rap1A was translocated to the plasma membrane in PMA-stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide-generating system.

Biological Transport↗
PubMed1999

NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides.

The distribution of neurons containing the enzymes NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has been studied in the gastrointestinal tract of lizard (Podarcis s. sicula) and snake (Thamnophis sirtalis). The techniques employed were the NADPH-d/nitroblue tetrazolium histochemical method, and the indirect immunofluorescence applied to cryostat sections and to whole-mount preparations. The colocalization of NADPH-d with NOS, with vasoactive intestinal polypeptide (VIP) and with galanin (Gal) was also studied, and a Western blot analysis using an antibody directed against mammalian Gal was performed on lizard stomach extracts. NADPH-d positive nerve cell bodies and fibres were found in the myenteric and submucous plexuses throughout the gastrointestinal tract of both reptiles. These nerve structures were also present in the other intramural nerve plexuses, although in smaller quantities. Both in lizard and snake, the stomach revealed a positive nerve population that was more dense than elsewhere in the gut. The population of the NADPH-d-positive neurons observed in the lizard was larger than that observed in the snake. The distribution of both populations was similar to those that have been described in the gut of several mammalian and non-mammalian vertebrates. Both in lizard and snake, a one-to-one correspondence was noted between NOS- and NADPH-d-containing nerve cell bodies, and the nitrergic neurons containing Gal appeared to be more numerous than those containing VIP. Western blot analysis recognised a single band with a molecular weight (3.4 kDa) very similar to that of porcine Gal. It is hypothesised that at least some of the nitrergic neurons of the lizard and snake gut are inhibitory motor neurons innervating the circular smooth musculature. In addition, the colocalization of NOS and VIP in neurons enhances their inhibitory action. The role of the neurons containing both NOS and Gal remains unknown.

Animals↗
PubMed1995

Cell type differences in Golgi retention signals for transmembrane proteins.

The transmembrane domain of Golgi resident proteins such as beta-galactoside alpha 2,6-sialyltransferase (ST) and N-acetylglucosaminyltransferase 1 (NT) contain a Golgi retention signal which confers Golgi retention to reporter proteins appended to them in the appropriate context. Thus, chimeras of the cell surface protein dipeptidyl peptidase IV containing the transmembrane domain of ST and NT are retained in the Golgi apparatus in MDCK and COS cells, as assessed by indirect immunofluorescence microscopy. Transfection of these chimeric constructs into CHO cells, however, results in their transport to vesicular structures which do not colocalize with that of an endogenous Golgi marker, mannosidase II. Furthermore, the staining pattern of these structures are not affected by brefeldin A. Biochemical analysis of the transgene products in pulse-chase experiments revealed that the chimeric proteins eventually become resistant to endoglycosidase H, suggesting that they are transported beyond the medial Golgi and therefore the vesicular structures are likely to be post-Golgi. The vesicular structures colocalized well with a lysosomal marker, cathepsin D, and also with internalized FITC-dextran chased into the lysosomal compartment. Monitoring the cell surface appearance of the chimeric protein suggests that the majority is transported directly to the lysosomal compartment. Golgi retention can be completely restored for ST and improved for NT by the inclusion of sequences flanking the transmembrane domain. Our results reflect cell type differences in the interpretation of the transmembrane domain Golgi retention signal, established that general Golgi retention of type II glycosyltransferases requires the hydrophilic flanking sequence as well as the transmembrane domain, and demonstrate that proteins which escape Golgi retention may be channeled to the lysosomal pathway.

Animals↗
PubMed1993

Analysis of Candida albicans adhesion to salivary mucin.

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.

Animals↗