Mouse L cell mutants defective in receptor mediated endocytosis were isolated using selection with modeccin followed by screening for loss of mannose 6-phosphate (Man 6-P) dependent uptake or for resistance to Pseudomonas exotoxin. Mutants L146 and L15D were resistant to modeccin, lacked 215 kD Man 6-P receptor, were defective in Man 6-P dependent uptake and binding, and oversecreted several lysosomal enzymes. Mutant LEFIC was resistant to modeccin, Pseudomonas exotoxin and ricin. The mutant exhibited delayed (1) oligosaccharide processing of VSV G protein, (2) transport of VSV G to the cell surface and (3) release of radiolabeled VSV G into virions. LEFIC was blocked in proteolytic processing of the Sindbis glycoprotein pE2 and showed intracellular accumulation of Sindbis nucleocapsid. The mutant showed a delay in secretion of fibronectin. An intersection of the secretory and endocytic pathways is suggested by the isolation of this cross-toxin resistant mutant defective in intracellular transport of membrane and secretory proteins.

No description available.

No description available.

As the plasma membrane of the cardiac muscle cell (sarcolemma) plays a major role in the cardiac physiology and homeostasis, the aim of this study is to identify using mass spectrometry (MS) and data base mining the integral and peripheral proteins that are making its composition and to uncover putative novel proteins and to ultimately reveal the differences in protein composition between the sarcolemma of control rat hearts and hearts submitted to ischemia and ischemia-reperfusion to find disease-specific markers. Toward this goal we have developed a new method to purify cardiac sarcolemma using an approach that avoids denaturing conditions and combines subcellular fractionation and immunoadsorption: An initial cardiac sarcolemma preparation obtained by sucrose gradient centrifugation was enriched 27-fold in 5-nucleotidase activity and 5-12-fold in sarcolemmal specific markers. Subsequently, this enriched preparation was incubated with antibodies directed against intercalated disc proteins, N-cadherin and Connexin43, and then immunoadsorbed to superparamagnetic beads (Dynabeads Pan Mouse IgG). Samples from the enriched sarcolemma and immunoabsorbed fractions were separated by 1-D SDS-PAGE and in-gel digested with trypsin. The tryptic fragments were identified, analyzed and assigned to proteins using tandem MS-MS spectrometry in combination with Mascot search software. Out of the 518 identified proteins, 40% were attributed to the sarcolemma and associated cytoskeleton, of which 65% were peripheral proteins and the remainders were either membrane spanning or lipid anchored proteins. Further functional classification of the sarcolemma proteins indicates that the majority (65%) are involved in signaling, trafficking and cell adhesion. The MS analysis also reveals the presence of 71 novel proteins, which are currently being evaluated as relevant candidates for further study. Comparing the sarcolemma and immunoselected intercalated disc preparations; we noticed that the distribution of protein abundance in all of the localization and functional categories follows almost the same trend. This led us to conclude that in order to have an easy, fast and a reliable technique to compare different heart states in terms of sarcolemma protein composition the sarcolemma preparation would serve just as well as an immunoselected preparation with less time, effort and materials. In order to find out differences in protein composition between normal sarcolemma and sarcolemma submitted to global ischemia/ischemia-reperfusion; sarcolemma fractions (SF) obtained from Langendorff rat heart models were analyzed by mass spectrometry. The results of this study revealed that global ischemia induced an increase in calcium binding proteins (i.e. annexins) concomitantly with a decrease in transport proteins (i.e. Na/K ATPase). After ischemia, the signaling proteins category showed a decrease followed by a return to the control level after reperfusion. The amount of G alpha inhibiting 2 protein, caveolin, flotilin and myosin varied significantly during the ischemia/reperfusion protocol.

No description available.

No description available.

The main objective of this study was to determine the distribution of these perforatorial polypeptides in the head of the rat spermatozoon. / Immunoblotting demonstrated that seven perforatorial polypeptides tested in this study, had epitopes in common. Immunogold labeling of spermatozoa showed that the distribution of the 34, 43, 57 and 63 kDa polypeptides corresponded exactly to the regions of the perinuclear theca immunolabeled with the whole perforatorium serum. However, antibodies affinity purified against the 13, 13.4 and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner layer of the ventral spur. / These results suggest (1) the perforatorium is biochemically distinct from the postacrosomal sheath with the exception of a restricted region referred to as the ventral spur; (2) there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer and of the postacrosomal sheath; and (3) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, by may also compose portions of the outer periacrosomal layer, and postacrosomal sheath; and (4) Actin is not part of the perforatorium in mature rat spermatozoa.

The lectins Ulex europaeus 1 (UEA 1), Ricinus communis 1 (RCA 1), Wheat Germ Agglutinin (WGA), and Lens culinaris agglutinin (LCA) which are specific for L-fucose, D-galactose, N-acetylglucosamine, and D-mannose respectively, have been used to localize cytochemically the presence of these sugar residues in certain human, mammalian, and amphibian cell types. Thin sections of Lowicryl K4M-embedded human distal colon, rat duodenum, and frog dorsal root ganglion were incubated with UEA 1-gold, RCA 1-gold, WGA/ovomucoid-gold, or LCA-gold. UEA-1, RCA-1, and LCA binding sites were present in the nuclei of human colonic cells, rat duodenal goblet and columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells. Most of the nuclear binding sites were localized in the euchromatin compartment, while extranuclear labelling was largely restricted to sites known to contain glycoproteins. WGA binding sites were present in the nuclei of rat duodenum columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells, but no labelling of human colonic cell nuclei was observed. Thus, we report the presence of these sugars in the nuceloplasm of human colonic cells (with the exception of WGA), rat duodenal villous columnar cells, and frog dorsal root ganglion cells examined.

Cres is a member of the cystatin superfamily of cysteine protease inhibitors; however, it differs from typical cystatins in that it is a serine protease inhibitor. In the testis, Cres exhibits expression solely in spermatids; while in the epididymis, Cres is secreted by principal cells of the initial segment to associate with sperm in the lumen. In the cauda region Cres is endocytosed by clear cells. The presence of Cres mRNA in the efferent ducts by RT-PCR analysis is suggestive of its synthesis in the region. In Cres-/- mice, statistically significant reductions were noted in tubule, epithelial, and luminal profile areas in the testis and epididymis, as compared to wild-type mice. Cres -/- mice revealed degenerating germ cells in the testis. Immature germ cells were present in the epididymal lumen and the epithelial principal cells contained large irregularly shaped lysosomes, unlike the small spherical ones of wild type mice, suggesting of lysosomal storage diseases. Taken together the data indicate the importance of Cres for sperm production and maintenance of epithelial integrity of the testis and epididymis. / Cres est un membre de la superfamille des cystatines. Les cystatines sont reconnues pour être des inhibiteurs de protéases de cysteine. Cres diffère des autres membres car il s'agit d'un inhibiteur de protéases de serines. Dans le testicule, Cres est exprimé seulement par les spermatides. Dans l'épididyme, Cres est secrété par les cellules principales du segment initial pour ensuite s'associer aux spermatozoïdes présents dans la lumière. Dans la région de la queue, Cres est incorporé par endocytose dans les cellules claires. La présence de l'ARN messager de Cres dans les canaux efférents, qui a été démontrée par RT-PCR, suggère qu'il est produit dans cette région. Dans les souris Cres -/-, une réduction significative de la taille des tubules, de la lumière et de l'épaisseur de l'épithélium, en comparaison avec les souris contrôles (sauvages), a été observée dans le testicule et l'épididyme. Une dégénération des cellules germinales dans le testicule a aussi été observée chez les souris Cres -/- et des cellules immatures étaient présentes dans la lumière de l'épididyme. De plus, les cellules principales possédaient de larges lysosomes de forme irrégulière, très différents des petits lysosomes sphériques qui sont généralement observés dans l'épididyme. Ces informations indiquent l'importance de Cres dans le maintien de l'intégrité des épithéliums du testicule et de l'épididyme, et donc dans la spermatogenèse et la maturation des spermatozoïdes.

A morphological study was done of Tomes' process, the organic matrix and the hydroxyapatite crystals of enamel. The membrane of Tomes' process where secretion of rod and interrod material occurs is extensively infolded. The interrod secretion site extends circumferentially around the ameloblast and granules fuse with deep membrane infoldings. The infoldings associated with secretion sites can be abolished by injecting vinblastine. Newly secreted enamel proteins do not accumulate extracellularly. So-called "stippled material" is an artifact of fixation and results from degradation of previously calcified enamel. Simultaneous visualization of the organic matrix and hydroxyapatite crystals is not possible because the aqueous stains needed to reveal the organic matrix dissolve the young enamel crystals. Osmium tetroxide, however, protects against beam damage and dissolution by water. When visualized separately the organic matrix seems to be contained within the space occupied by the crystal. Using stereo electron microscopy, crystals appear as thin flat ribbons. Images previously described as hexagonal cross-cut crystals are due to visualizing a three-dimensional structure in two dimensions.