Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Histochemistry and Cell Biology - We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme...     

Cytochemical localization of alkaline phosphatase in the cell membrane of Dictyostelium discoideum amebae

We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).     

Lipid membrane domains in cell surface and vacuolar systems

Detergent insoluble sphingolipid-cholesterol enriched 'raft'-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed.     

Lipid membrane domains in cell surface and vacuolar systems

Detergent insoluble sphingolipid-cholesterol enriched raft-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed.     

Cytochemical detection of polysaccharides on the surface of the cell membrane complex in fungi

Cytochemical staining in toto (periodic acid, thiosemicarbazide, O_SO₄) revealed the presence of polysaccharide lamellae on the surface of the cell membrane complex of fungi. The membraneous clusters in the vacuolar bodies of Claviceps purpurea were covered with these lamellae at both surfaces, as it was also the case with the endoplasmic reticulum membranes, the tonoplast and the cytoplasmic membrane. In Saccharomyces cerevisiae, the polysaccharide lamellae were visible on the surface of the endoplasmic reticulum membranes and the plasmalemma; the strain revealed polysaccharide deposits also on the tonoplasts of small vacuoles and in glucanase vesicles. We assume that these observations give precision to the localization of the enzymes synthetizing the glycoprotein components of the fungal cell wall.     

Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

Ceramide-enriched membrane domains

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.     

Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Summary We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).     

Protein-promoted membrane domains

The current notion of biological membranes encompasses a very complex structure, made of dynamically changing compartments or domains where different membrane components partition. These domains have been related to important cellular functions such as membrane sorting, signal transduction, membrane fusion, neuronal maturation, and protein activation. Many reviews have dealt with membrane domains where lipid-lipid interactions direct their formation, especially in the case of raft domains, so in this review we considered domains induced by integral membrane proteins. The nature of the interactions involved and the different mechanisms through which membrane proteins segregate lipid domains are presented, in particular with regard to those induced by the nAChR. It may be concluded that coupling of favourable lipid-lipid and lipid-protein interactions is a general condition for this phenomenon to occur.     

Thermodynamics of membrane domains

The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid-lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase.     

Cytochemical properties of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) have been obtained by polyethylene glycol (PEG) induced fusion in suspension of the Chinese hamster metaphase cultured cells with those in interphase. As alternative approach the PEG-fusion of the Chinese hamster asynchronous culture cells in monolayer with subsequent incubation in free medium was used. A comparative cytofluorimetric investigation of PCC and chromatin of the interphase nuclei of corresponding ploidy has shown some increase (up to 10%) of acridine orange and olivomycin binding with PCC chromatin. A similar slight increase in low molecular weight ligands binding with chromatin was also found in mitotic chromosomes. The data obtained confirm the opinion about the similarity of events taking place in chromatin during physiological mitosis and premature chromosome condensation. The cytochemical study of chromatin availability to low molecular weight ligands can be used as a criterion for judging on the properties of the artificially condensed chromatin.     

Cytochemical properties of euchromatin and heterochromatin

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).     

Regulatory recruitment of signalling molecules to the cell membrane by pleckstrinhomology domains

Pleckstrin-homology (PH) domains are small protein modules found in more than 100 proteins, most of which require association with the cell membrane to mediate their biological functions. Recent studies have demonstrated that some PH domains bind specifically to phosphoinositides, and that PH-domain-mediated recruitment of certain proteins to the cell membrane is important in regulation of their activities or functions. This provides the cell with a simple and efficient mechanism for linking growth-factor-induced changes in the levels of specific membrane phosphoinositides with other signalling pathways that control diverse processes such as protein synthesis, DNA synthesis and cell adhesion.