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At least 37 records · Page 2Linked to original sources
PubMed1992

WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Identification of HCV-associated antigen(s) in hepatocytes.

HCV-associated antigens (HCAg) were localized morphologically using immunofluorescence methods. Fluorescence was found in the cytoplasm of individual liver cells or in groups of cells. Nuclear fluorescence was not observed. Blocking and absorption studies suggest that HCAg is related to nucleocapsid or envelope proteins.

Animals↗
PubMed1991

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal.

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.

Blotting, Western↗
PubMed1992

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes.

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa Gs alpha, and 39 and 41 KDa Gi alpha subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [alpha-32P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160, 100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21-28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.

Amylases↗
PubMed1978

Stereological model system for free cells and base-line data for human peripheral blood-derived small T-lymphocytes.

T-lymphocytes derived from human peripheral blood and passed through a nylon-wool column, were employed to develop and test a new stereological model system for free spherical cells, allowing a quantitative characterization of the cell and its components at the ultrastructural level. Electron micrographs were recorded in a hierarchical manner at three different levels of magnification and subjected to point counting procedures. The resulting parameters were expressed in relation to various reference compartments, both absolute and relative. Results indicated that the average volume of a small, non-activated T-lymphocyte was 103.8 micron3, the nuclear volume 47.5 micron3 and the cytoplasmic volume 55.9 micron3. On the average, the cytoplasm contained 30 mitochondria, 0.7 micron3 RER-cisternae, 0.2 micron3 cisternae and vesicles of the Golgi apparatus and about 231,000 free ribosomes (most of them single). The ratio of eu- to heterochromatin volume was 0.5. The design and application of the stereological model system are discussed with regard to dynamic studies of a variety of free cells, such as macrophages, neutrophilic granulocytes and various lymphocytes.

Adult↗
PubMed1992

Arachidonic acid incorporation in cardiomyocytes, endothelial cells and fibroblast-like cells isolated from adult rat heart.

The incorporation of radiolabeled arachidonic acid (3[H]-AA) in normoxic cardiomyocytes (MC), cardiac endothelial cells (EC) and fibroblast-like cells (FL) isolated from adult rat heart was studied. Deposition of 3[H]-AA in the cellular lipid pool was assessed with biochemical and autoradiographic techniques. Extraction and subsequent analysis of lipids from the three different cell types revealed that MC contained significantly more triacylglycerols than EC and FL. The proportion of (unlabeled) AA was also higher in MC triacylglycerols than in EC and FL. The quantity of phospholipids did not differ among the three cell types studied. However, the content of (unlabeled) AA in the MC phospholipid pool was twice as high as in EC and FL. The amount of 3[H]-AA incorporated in the cellular lipid pool of MC, EC and FL depended on the concentration of AA in the incubation medium and the incubation time. In EC and FL incorporation of 3[H]-AA was highest in the cellular phospholipid pool (0.01 microM AA, 30 min incubation). With increased concentration of AA and longer incubation times, the cellular triacylglycerol pool became more important as site of 3[H]-AA incorporation. In MC, comparable amounts of 3[H]-AA were incorporated in the cellular triacylglycerol and phospholipid pools (0.01 and 1 microM AA). At higher AA concentrations (10 microM) the triacylglycerol pool was the preferred site of 3[H]-AA deposition. Autoradiographic analysis at the light microscopic level revealed that the extra-nuclear space was readily stained when the three cell types were incubated with 3[H]-AA. These findings indicate that all cellular lipid pools and membranes are most likely site of deposition of radiolabeled arachidonic acid.

Animals↗
PubMed1992

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA.

Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.

Cell Compartmentation↗
PubMed1979

Water permeability of yeast cells at sub-zero temperatures.

A combined cryomicroscopic-multiple nonlinear regression analysis technique has been used to determine the water permeability of the yeast cell Saccharomyces cerevisiae during freezing. The time rate of change in volume of "supercooled" yeast cells was photographically monitored using a "cryomicroscope" which is capable of controlling in a programmable manner both the temperature and the time rate of change in temperature of the cell suspension being studied. Multiple nonlinear regression analysis together with a thermodynamic model of cell water transport during freezing was then used to statistically deduce the subzero temperature dependence of the cell water permeability. The water permeability process for S. cerevisiae being cooled at subzero temperatures was found to be rate-limited by the passage of water through either the plasmalemma, the cell wall, or a combination of these two permeability barriers. The hydraulic water permeability coefficient for yeast at 20 degrees C is approximately 1--2 x 10(-13) cm3/dyne sec, if extrapolation from subzero temperatures to room temperature is permissible, while the apparent activation energy governing the permeability process at subzero temperatures is approximately 45--68 kJ/mol (11--16 kcal/mol).

Cell Compartmentation↗
PubMed1992

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Animals↗
PubMed1992

Transport of methotrexate in basolateral membrane vesicles from rat liver.

Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na+ gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: Km = 0.15 +/- 0.05 microM and Vmax = 11.4 +/- 1.1 pmol 10 s-1 mg-1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by anion-exchange inhibitor 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl-, SO4(2-), and oxalate2-. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO4(2-), oxalate2-) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K+ diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.

Carcinoma↗
PubMed1992

Differentiation of HL-60 cells by phorbol ester is correlated with up-regulation of protein kinase C-alpha.

To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.

Acid Phosphatase↗
PubMed1992

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.

Animals↗
PubMed1991

Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin-like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphosphoinositides.

Using a polyclonal antibody raised against a synthetic peptide of the catalytic region of protein kinase C, we have carried out a combined immunocytochemical and immunochemical analysis to follow the subcellular localisation of this enzyme in response to mitogenic stimulation with insulin-like growth factor I and bombesin. These investigations show a time dependent translocation of protein kinase C from the cytoplasm to the nucleus since 5 min stimulation reaching a maximal effect after 45 min. These results show clearly that mitogen induced translocation of protein kinase C to the nucleus follows temporally the earlier changes in nuclear polyphosphoinositide metabolism previously demonstrated.

Amino Acid Sequence↗
PubMed1992

Interleukin-3 induces translocation and down-regulation of protein kinase C in human platelets.

Protein kinase C (PKC) is a family of phospholipid-dependent kinases that is involved, along with calcium mobilization, in the activation of human platelets. Since interleukin-3 (IL-3) has been shown to act, in part, by activating PKC, we investigated the effect of IL-3 on PKC activity and content in human platelets. Exposure of platelets to 10 ng/ml of IL-3 was associated with a rapid (i.e., within 3 minutes) translocation of PKC activity and content from the cytosol to the membrane fraction. In addition, treatment with IL-3 effected a time-dependent down-regulation of PKC activity and content. We speculate that IL-3 may act as a modulator of PKC-dependent pathways in the human platelet.

Blood Platelets↗
PubMed1992

The effect of changes in thiol subcompartments on T-cell colony formation and cell cycle progression: relevance to AIDS.

Recently, it has been shown that intra- and extracellular thiol levels are significantly lower than normal even in the relatively early stages of human immunodeficiency virus (HIV) infection. It is plausible that this deficiency could contribute both to the loss of T-cell function and the ability to replenish T cells associated with HIV infection. We had previously reported that the T-cell colony-forming cell (T-CFC) is impaired in HIV infection and that it can be enhanced with the thiol compounds 2-mercaptoethanol (2-ME) and N-acetylcysteine (NAC). In this study, the effect of the thiol-depleting reagents buthionine sulfoximine, cyclohexene-1-one, and copper phenanthroline on T-CFC formation and cell cycle progression was determined in HIV+ subject and/or controls. All three reagents inhibited T-CFC formation and cell cycle progression with a suggestion that colony formation by cells from HIV+ subjects was more sensitive to the effects of thiol depletion. 2-ME and NAC enhanced effect of NAC did not appear to involve increased protein kinase C translocation. Our results suggest that oxidation of membrane thiols, as well as depletion of intracellular glutathione, inhibits T-CFC formation as well as cell cycle progression for mitogen-stimulated cells in bulk culture.

Acetylcysteine↗