TL;DR: The results suggested that N-linked oligosaccharides are not involved in the functional sites of this adhesion molecule.
Abstract: E-cadherin is a Ca2+-dependent cell-cell adhesion molecule identified as a glycoprotein with a molecular weight (MW) of 124,000. To study the role of the sugar moieties of this adhesion molecule, we tested the effect of tunicamycin on aggregation mediated by E-cadherin of teratocarcinoma cells. Immunoblot analysis using a monoclonal antibody to E-cadherin showed that in cells treated with tunicamycin this adhesion molecule is converted into two forms with MW of 118,000 and 131,000. The smaller one was exposed on the cell surface and showed a trypsin sensitivity characteristic to E-cadherin, suggesting that this is the peptide moiety of E-cadherin whose glycosylation with N-linked oligosaccharides was blocked by tunicamycin. The larger one was not removed by trypsin treatment of cells, suggesting an intracellular location. These tunicamycin-treated cells aggregated in a Ca2+-dependent manner, and the aggregation was inhibited by a monoclonal antibody to E-cadherin. These results suggested that N-linked oligosaccharides are not involved in the functional sites of this adhesion molecule.
TL;DR: Transcellular transport of fluorescein dextran through porcine arterial endothelial cells cultured on a type I collagen gel supported by a dacron sheet was studied and showed that FD transport differs from RB-LDL transport which is temperature- and dose-dependent.
Abstract: Transcellular transport of fluorescein dextran (FD) of various molecular weights (4K, 10K, 20K, 70K and 150K daltons) through porcine arterial endothelial cells cultured on a type I collagen gel supported by a dacron sheet was studied and compared with the transport of low density lipoprotein labeled with rhodamine B (RB-LDL) described previously (Hashida et al., Cell Struct. Funct. 11, 31-42, 1986). The rate of FD transport through the monolayer depended on the size of the FD. FD transport was not temperature-dependent and was not a saturable process. Our findings show that FD transport differs from RB-LDL transport which is temperature- and dose-dependent. The mechanism of the transport of FD is compared with that of RB-LDL.
TL;DR: The morphological findings in the rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.
Abstract: Dietary lectins of gluten origin have been suggested to play an important role in the mechanisms leading to the characteristic morphology of the intestine found in patients with celiac disease. To further explore this issue we have used Wheat Germ Agglutinin (WGA) or Concanavalin A (Con A) to challenge rat small intestine and study the ultrastructural changes of such a treatment. Both lectins affected the enterocytes at the base of the villi more than those at the top. The morphological findings included disarrange-ment of the cytoskeleton, increased endocytosis and shortening of the microvilli. The interrelationship between the observed changes, and their relevance for similar morphological alterations found in patients with celiac disease are discussed. In conclusion, the morphological findings in our rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.
TL;DR: Results suggest that the beta subunit is glycosylated cotranslationally in the ER then is converted to the mature type subunit (54 Kd) in the Golgi apparatus.
Abstract: (Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A- and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests. The endoplasmic reticulum (ER)-rich, and Golgi-rich subfractions were prepared from the rat kidney microsomal fraction by sucrose density gradient centrifugation. On the immunoblot, the molecular weight of the alpha subunit in both fractions was 95 kilodalton (Kd); whereas, that of the beta subunit was 50 Kd in the ER-rich fraction and 54 Kd in the Golgi-rich fraction. When treated with endoglucosidase H, the 50 Kd component was converted to 38 Kd, but the 54 Kd component was endoglucosidase H resistant. These results suggest that the beta subunit (38 Kd) is glycosylated cotranslationally in the ER (50 Kd) then is converted to the mature type subunit (54 Kd) in the Golgi apparatus.
TL;DR: Evidence is provided that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane, and that Con A-binding on the nuclear membrane shows no localization on thenuclear membrane.
Abstract: The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.
TL;DR: The data suggest that cell growth and differentiation in the M1 cells are accompanied byChanges in the intracellular K+ and Na+ concentrations but that the changes in the contents of these monovalent cations do not necessarily induce differentiation in this cell line.
Abstract: We compared intracellular K+ and Na+ ion concentrations during cell growth and differentiation of a mouse myeloid leukemia M1 cell line. Cells undergoing mitosis had higher K+ concentrations than quiescent cells. Treatment with a K+ channel blocker and furosemide enhanced cell growth and produced a slight increase in the intracellular K+ concentration. Treatment with reagents that reduced the intracellular K+ concentration stopped cell growth. Induction of differentiation in this cell line produced a decrease in the K+ concentration, which always was accompanied by an increase in the Na+ concentration. Treatment with ouabain, which decreased the intracellular K+ concentration, did not, however, induce differentiation in the M1 cell line. The data suggest that cell growth and differentiation in the M1 cells are accompanied by changes in the intracellular K+ and Na+ concentrations but that the changes in the contents of these monovalent cations do not necessarily induce differentiation in this cell line.
TL;DR: Observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.
Abstract: When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.
TL;DR: Modification of rectified Nomarski differential interference contrast optics and the epi-illumination system improved the detection of colloidal gold particles with analog video enhanced microscopy and non fading immuno-gold 5-40 nm markers are discussed.
Abstract: Modification of rectified Nomarski differential interference contrast optics (Nikon) and the epi-illumination system (Nikon IGS-cube) improved the detection of colloidal gold particles with analog video enhanced microscopy. Immuno-gold labelled microtubules of Haemanthus endosperm are visualized at a level of detection unmatched in conventional light micro-scopy. Single gold, or gold silver enhanced particles in suspension viewed with the modified epi-illumination after pressure injection into cells, are well distinguished from other granular cell components. Immuno-gold has also been detected on the surface of chromosomes and the nuclear envelopes in cells during the rapid experimental disassembly of microtubules. Thus, under certain conditions tubulin in a form other than microtubules may be detected. Practical applications of this "optical stain" for non fading immuno-gold 5-40 nm markers are discussed.
TL;DR: The results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.
Abstract: Polymorphonuclear leukocytes undergo a series of morpho-logical and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demon-strate that membrane depolarization is not associated with superoxide gener-ation : FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.
TL;DR: Values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation.
Abstract: Microtubule associated protein-1 of brain and its intracellular 350kd analogues were highly sensitive to purified Ca2+-dependent cysteine proteinase (calpain). After 15 second digestion, we detected intermediate degradation products of MAP-1 by immunoblotting using anti-MAP-1 antibody as 290, 260, 220, 170, 140, 112, 80, 68, and 32kd polypeptides. These values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation.
TL;DR: In this article, the authors constructed mutant cell strains from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene, and the transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU.
Abstract: To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.
TL;DR: A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs, and sequences homologous to pBR 322 were detectable in two Eco RI fragments of the pXLR101A-injected tadpole DNA.
Abstract: A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pX1r101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pX1r101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.
TL;DR: A high-voltage generating machine which could generate semi-rectangular pulses in PBS solution was constructed, and the effects of field strength and duration of the pulse on electric pulse-mediated transformation of mouse mammary carcinoma FM3A cells by a linear form of plasmid pSV2neo DNAs were examined.
Abstract: A high-voltage generating machine which could generate semi-rectangular pulses in PBS solution was constructed, and the effects of field strength and duration of the pulse on electric pulse-mediated transformation of mouse mammary carcinoma FM3A cells by a linear form of plasmid pSV2neo DNAs were examined. In parallel, cell survival and growth after pulsing were analyzed. When the field strength and duration of the pulse were increased, the transformation frequency increased, although the cell survival rate decreased. Under the best conditions, the transformation frequency was 2 X 10(-4), which was 80 times higher than that obtained by the calcium phosphate coprecipitation method.
TL;DR: It was found that cells treated with the inducers dexamethasone and 1-methyl-3-isobutylxanthine went through DNA synthesis (S phase) prior to lipid accumulation and that insulin enhanced this differentiation process.
Abstract: We previously established a serum-free hormone-supplemented medium for the induction of adipocyte differentiation of 3T3-L1 cells (Gamou, S. and N. Shimizu. in "Growth and Differentiation of Cells in Defined Environment", H. Murakami et al., ed., Kodansha/Springer-Verlag, pp. 173-178, 1985). Under those conditions the stage of the cell's commitment to adipocyte differentiation was separated from the stage of expression of the adipocyte phenotype. In the current study, the relationship between cell division of the growth-arrested 3T3-L1 cells and their entry into the differen-tiation program was examined by autoradiography at the individual cell level. It was found that cells treated with the inducers dexamethasone and 1-methyl-3-isobutylxanthine went through DNA synthesis (S phase) prior to lipid accumulation and that insulin enhanced this differentiation process. Under these serum-free hormone-supplemented conditions, the tumor promoter dihydroteleocidin B was found to be a strong inhibitor of adipocyte differ-entiation.
TL;DR: Results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb; and that it recognize nuclear granules in locations where desmin or desmine-related protein has not yet been reported.
Abstract: One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.
TL;DR: Results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.
Abstract: The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20 % of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.
TL;DR: Mouse FM3A cells propagated well in serum-free medium containing 0.5% serum albumin and 1 microgram of insulin/ml; but a more critical population-dependent B12 requirement was demonstrated in mouse Ehrlich and L cells and their hybrids, where a satisfactory growth response was obtained in medium supplemented with these substances.
Abstract: Mouse FM3A cells propagated well in serum-free medium containing 0.5% serum albumin and 1 microgram of insulin/ml. The vitamin B12 (B12) requirement of the cells depended on the population density. This requirement disappeared when a sufficiently large cell population was present. A combination of 1-100 ng of B12/ml and 4 micrograms of hypoxanthine/ml resulted in a synergistic increase in cell growth at low cell densities. A similar growth response was obtained when the B12 plus hypoxanthine was replaced by 4 micrograms of hypoxanthine/ml in combination with 100 ng of thymidine/ml, 1 microgram of folic acid/ml or 1 microgram of folinic acid/ml, even though 1 microgram of folic acid/ml already was present in the medium. Experiments on single cell inoculation showed that colony size and the yield of cells grown in B12-supplemented medium were much larger than those for cells grown in B12-free medium. A more critical population-dependent B12 requirement was demonstrated in mouse Ehrlich and L cells and their hybrids. At less than 100 cells there was no propagation in serum-free medium lacking B12, folinic acid and thymidine; whereas, a satisfactory growth response was obtained in medium supplemented with these substances.
TL;DR: An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum and Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin.
Abstract: An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.
TL;DR: It is concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietIC cells of rat bone marrow, and that anti-asialogANGlioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythroietic cells from ratBone marrow cells.
Abstract: Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (IV2 alpha Fuc-Gg4Cer) and IV3 alpha Gal-fucogangliotetraosylceramide (IV3 alpha GalIV2 alpha Fuc-Gg4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grunwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glycolipids on their cell surfaces; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulocytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.
TL;DR: A serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines was used.
Abstract: The ability to grow lymphoid and myeloid cells in serum-free culture medium allows researchers to analyze the factors and mechanisms required for hemopoietic cell growth and differentiation without the interference of undefined serum components. Therefore, we used a serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines. Mo was maintained in RITC for more than 8 months and had a mean viability of 59% and the same doubling times as in serum-containing medium (SCM). Under these conditions, Mo cells produced hemopoietic colony-stimulating activity that included production of a basophil/eosinophil differentiation factor of similar content to that produced in SCM. WEHI-3B cells grown for more than 12 months in RITC, or for more than 3 months in RITC without Tf and Ins, had a doubling time of 20 h, whereas cells maintained in protein-free RITC showed a 2-fold increase in doubling time then died within 3 months. The IL-3 production by WEHI-3B cells cultured in RITC was higher than the production by cells grown in SCM. When IL-3 was assayed in 32Dcl/H4 cells that had been maintained in RITC for more than 4 months, a lower response to IL-3 was found, an indication that components other than the BSA, Tf and Ins in fetal calf serum are required for optimal cell growth and differentiation.
TL;DR: A DNA transfection method by laser microbeam pricking has been recently reported and the approximate number of DNA molecules necessary to transform the recipient cell was estimated.
Abstract: A DNA transfection method by laser microbeam pricking has been recently reported (Kurata, S. et al. Exp. Cell Res. 162, 372 (1986]. The volume of external fluid transferred into the cell by the method was determined through the injection of diphtheria toxin fragment A (Yamaizumi, M. et al. Cell 15, 245 (1978]. Using these results and the results on laser DNA transfection efficiency (Kurata, S. et al. Exp. Cell Res. 162, 372 (1986], the approximate number of DNA molecules necessary to transform the recipient cell was estimated.
TL;DR: Vacuolar sap was separated by intracellular perfusion and the distribution of proteases was studied in the giant alga Chara australis, finding the optimal pH for carboxypeptidase activity was around 5, and about 95 % of total activity was localized in the vacuole.
Abstract: Vacuolar sap was separated by intracellular perfusion and the distribution of proteases was studied in the giant alga Chara australis. Caseinolytic and hemoglobin-digesting activities were found to be higher at low pH, and about 85 % of total activity was localized in the central vacuole. The optimal pH for carboxypeptidase activity measured using N-carbo-benzoxy-L-phenylalanyl-L-alanineas a substrate was around 5, and about 95 % of total activity was localized in the vacuole. Moreover a substantial portion (40 %) of aminopeptidase activity, measured at pH 5.5 using L-leucine-β-naphthylamide as a substrate, was found in the vacuole.
TL;DR: Localization of ganglioside GM1 in cholinergic neurons from the septal area of a primary culture newborn rat brain was studied with a double avidin-biotin complex system.
Abstract: Localization of ganglioside GM1 in cholinergic neurons from the septal area of a primary culture newborn rat brain was studied with a double avidin-biotin complex system. Cholinergic neurons were identified by double immunolabeling techniques that use choline acetyltransferase (ChAT) and neurofilament (NF) protein-antibodies. ChAT-positive neurons also were stained for ganglioside GM1 by using an avidin-biotin complex technique.
TL;DR: The research reported here was done to clarify the role of cholesterol in the interaction of the innersurface of plasma membrane with cytoplasmic proteins in relation to the regulatory mechanism of signal transduction.
Abstract: Since the plasticity of the plasma membrane is correlated with changes in its dynamic behavior and biological functions, the content of cholesterol in this membrane is thought to have an important role in the regulation of cell metabolism The research reported here was done to clarify the role of cholesterol in the interaction of the innersurface of plasma membrane with cytoplasmic proteins in relation to the regulatory mechanism of signal transduction Many cytoplasmic proteins of EATC or rat liver cells were found to associate with DPPC liposomes and they induced a transient increase in membrane permeability at the phase transition temperature of the liposomal lipid The association and the permeability increase were inhibited by the introduction of cholesterol into DPPC liposomes, and the sensitivity of individual proteins to the action of cholesterol differed F-actin, but not G-actin, associated selectively with the liposomes Also, the main endothermic peak of the unilamellar DPPC liposomes was shifted from 37 degrees C to 43 degrees C by this protein association, evidence of transformation from a unito multilamellar structure The introduction of cholesterol into the liposomal membrane caused a reduction in the energy content of the phase transition and in the inhibition of protein-membrane interaction We concluded that the cholesterol in the plasma membrane contributes to the regulation of cell surface signal transduction
TL;DR: Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs.
Abstract: RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis.RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 μg/ml, 37°C, 20 min) followed by sucrose density gradient centri-fugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80 % greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 μg/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20 % greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs.In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D. These results suggest that the RNA on the surfaces of nonleukemic ascites tumor cells is composed predominantly of 18S and 28S ribosomal RNA and that these RNAs might be responsible, in part if not completely, for the surface negative charges on nonleukemic tumor cells.
TL;DR: A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described.
Abstract: A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothiocyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities corresponded to the BrdU and cellular DNA contents. The durations of G1, S and G2 + M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G1, labeled S and nonlabeled G2 + M phases. The reliability of this method is discussed.
TL;DR: Inhibitory compounds could be extracted from the cell surface with the detergent, Triton CF-54, and could be separated by concanavalin A-Sepharose column chromatography and the mannosyl glycoprotein(s) located on the cellsurface is considered to function in conju-gation process.
Abstract: Concanavalin A at a concentration of 1.0 mg/ml completely inhibited the conjugation of haploid cells of Physarum polycephalum. This inhibitory effect was counteracted by α-methyl-D-mannoside. α-Mannosidase also inhibited conjugation. Neither wheat germ agglutinin nor lentil lectin had any apparent inhibitory effects. When Physarum cells were cultured in the presence of tunicamycin at a concentration of 1 μ/ml, a similar inhibitory effect on conjugation was observed. The plasma membrane from the amoebae partially inhibited conjugation. On the basis of these results, the mannosyl glycoprotein(s) located on the cell surface is considered to function in conju-gation process.Inhibitory compounds could be extracted from the cell surface with the detergent, Triton CF-54, and could be separated by concanavalin A-Sepharose column chromatography.
TL;DR: The results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.
Abstract: EL-4 tumor cells were assayed in vitro for their ability to aggre-gate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets.The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that hu-man platelet response to tumor cells is not necessarily deducable from ex-perimental data obtained with animal platelets.
TL;DR: Results are discussed in relation to the functional evolution of the initial DNA body of the oocyte from A. lactifloreus, the transcriptional abilities of its by-products, and the general process of the amplification of ribosomal genes.
Abstract: The nucleus of the premeiotic oocyte from the hoplonemertine Amphiporus lactifloreus contains a characteristic DNA-rich body that rapidly generates a peculiar nucleolar apparatus previously described as the nucleolus-DNA body complex. During early vitellogenesis, the nucleolar complex is centrally located in the nucleus and consists of many spherical and ribbon-shaped components-the nucleolar spherulae and lamellae-which produce hundreds of small peripheral nucleoli and several granular bodies as vitellogenesis progresses. Distributions of the nucleolar organizer regions (NORs) in various nucleolar components were studied by silver staining, and results were compared with previous findings obtained by cytochemical and autoradiographic techniques.The core of each spherula in the nucleolus-DNA body complex is strongly Ag-positive, and so, later on, is a minor part of each peripheral nucleolus. In contrast, the cortices of the nucleolar spherulae, in which slightly decondensed chromatin is distributed uniformly, and the nucleolar lamellae produced through self-peeling of the spherulae, do not react with silver. Thus, the NOR proteins identified by silver deposits correspond to numerous loci at which the most decondensed, transcriptionally active chromatin is made visible by the osmium-ammine reaction. These results are discussed in relation to the functional evolution of the initial DNA body of the oocyte from A. lactifloreus, the transcriptional abilities of its by-products, and the general process of the amplification of ribosomal genes.
TL;DR: Observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction.
Abstract: Effects of septic shock by repeated inoculations with Escherichia coli on the ultrastructure of the folliculo-stellate cells and cavities of the adenohypophysis of the chicken were investigated in order to determine the function of these cavities. The principal morphological modifications were dilation of the Golgi apparatus, endoplasmic reticulum and autophagic vacuoles, and necrosis phenomena in the stellate cells. The follicular cavities showed dilation, and there was heterogeneous dense material and granular elements in the follicular lumen. Based on results reported in the literature, the observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction.