Effect of tunicamycin and cycloheximide on the secretion of acid hydrolases from I-cell cultured fibroblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Miller AL
PROVIDER: S-EPMC1161736 | biostudies-other | 1980 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Cultured rat hepatocytes were used to measure hepatic synthesis of rat plasma glycoproteins. [3H]Glucosamine was progressively incorporated into the protein of hepatocyte culture media very-low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and the p greater than 1.21 g/ml fraction after 3.5 and 6.5 h incubation. Apolipoproteins B, E and C, as well as transferrin, were identified as glycoproteins. The association of radioactivity with apolipoprotein C of hepatocyte very-low-density and high-density lipoproteins suggests that apolipoprotein C-III-3, the only C apoglycoprotein in the rat, is synthesized de novo by the hepatocytes. Treatment of hepatocytes with tunicamycin, a specific inhibitor of protein glycosylation, resulted in a substantial decrease in [3H]glucosamine incorporation into hepatocyte very-low-density, low-density and high-density lipoproteins and p greater than 1.21 g/ml protein, but had little or no effect on secretion. In the rat, hepatic secretion of lipoproteins and transferrin does not appear to be dependent on prior protein glycosylation.
| S-EPMC1163550 | biostudies-other
Project description:1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.
| S-EPMC1265220 | biostudies-other
Project description:The effect of inhibitors on the glycosylation, activity and secretion of lipoprotein lipase was studied in brown adipocytes cultured from newborn mice. Such cells synthesized and secreted active lipoprotein lipase. It is generally accepted that active lipoprotein lipase is a homodimer. Glycosylation of lipoprotein lipase was analysed by PAGE of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase immunoprecipitated from cells incubated for 1-2 h with [35S]methionine. The most prevalent 35S-labelled lipase subunit (Mr 57,000-58,000) in these cells contained endo H-resistant oligosaccharide chains, the next most prevalent contained totally endo H-sensitive chains, and the least prevalent subunit contained partially endo H-sensitive chains. Complete blocking of the glycosylation of lipoprotein lipase with tunicamycin (1 microgram/ml) for 24 h resulted in synthesis of an inactive non-secretable form of lipase with a smaller subunit (Mr 51,000-52,000). Immunofluorescent studies showed that unglycosylated lipase in tunicamycin-treated cells was retained in the endoplasmic reticulum. Cells treated with 1 microM-monensin, an intra-Golgi transport inhibitor, synthesized an active form of lipase which was not secreted, but was retained in the Golgi. The lipase in monensin-treated cells contained only partially or totally endo H-sensitive chains. Blocking either Golgi mannosidase I with 4 mM-1-deoxymannojirimycin or Golgi mannosidase II with 10 microM-swainsonine resulted in production of a form of lipoprotein lipase which was active and secreted, and which contained only endo H-sensitive chains. Our findings demonstrate that core glycosylation of lipoprotein lipase in the endoplasmic reticulum is required for lipase activity and transport from the reticulum, whereas processing of the oligosaccharide chains to endo H-resistant (complex) type chains in the Golgi is not required for either the activity or the secretion of lipoprotein lipase.
| S-EPMC1151315 | biostudies-other
Project description:When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase-sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms.
| S-EPMC1151545 | biostudies-other
Project description:The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.
| S-EPMC1179284 | biostudies-other
Project description:The combination of ascorbic acid and rutin, commonly used in oral preparations for their antioxidant and anti-inflammatory properties, can also be used to protect skin cells from the effects of UV radiation in sunlight. Here, we tested the potential protective effect of ascorbic acid and rutin used together in UVB-irradiated human skin fibroblasts, and assessed the proteomic profile of these cells, grown in a three-dimensional (3D) system. Proteomic findings revealed a combined effect of ascorbic acid and rutin in UV-irradiated fibroblasts against overexpression of pro-inflammatory signaling proteins and DNA reorganization/expression. These effects were not observed when cells were treated with either compounds alone. The antioxidant effects of ascorbic acid and rutin also prevented protein modifications by lipid peroxidation products. Further, ascorbic acid stimulated rutin-protein adduct formation, which supports intra/extracellular signaling and the Nrf2/ARE antioxidant pathway, contributing to the protective effects against UV-induced oxidative stress. The combined effect of ascorbic acid and rutin suggests that this combination of compounds is potentially effective against skin damage caused by UV radiation.
| S-EPMC7230807 | biostudies-literature
Project description:1. Chinese-hamster ovary fibroblasts were cultured to provide a homogeneous cell population. Homogenates obtained from these cells were fractionated by centrifugation techniques and the resulting fractions were analysed for protein and for enzymes representative of certain subcellular particles. 2. Unlike those in rat liver homogenates, the mitochondrial and lysosomal populations proved impossible to separate by differential centrifugation owing to the similarity of their sedimentation properties. Their resolution was possible by using isopycnic centrifugation in a continuous sucrose density gradient. 3. The mitochondrial population equilibrated at a density of 1.17g.cm(-3) as in rat liver homogenates. However, the lysosomal population equilibrated at a lower rather than a higher density position than the mitochondria and the probable reasons for this are discussed. 4. The lysosomal population subdivided into two groups characterized by differences in acid hydrolase content and equilibrium densities. The fraction with a density of 1.15g.cm(-3) contained the majority of arylsulphatases A and B, of cathepsin and of beta-acetylglucosaminidase activities, whereas that with a density of 1.09g.cm(-3) contained the majority of the acid phosphatase and acid ribonuclease activities. The probable division of the lysosomal population of a single cell into a number of distinguishable subgroups is suggested.
| S-EPMC1177613 | biostudies-other
Project description:A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.
| S-EPMC1149042 | biostudies-other
Project description:Purpose:Inflammation, hyaluronan production, and adipogenesis are the main pathological events leading to Graves' orbitopathy (GO). Guggulsterone (GS), a phytosterol found in the resin of the guggul plant, is a well-known treatment for several inflammatory disorders, such as arthritis, obesity, and hyperlipidemia. Here we investigated the effects of GS treatment on GO pathology. Methods:Using primary cultures of orbital fibroblasts from GO patients and non-GO controls, we examined the effects of GS on hyaluronan production and the production of proinflammatory cytokines induced by interleukin (IL)-1?, using real-time reverse transcription-polymerase chain reaction analysis, western blots, and enzyme-linked immunosorbent assays. Further, adipogenic differentiation was evaluated by quantification of Oil Red O staining and assessment of protein levels of peroxisome proliferator activator gamma (PPAR?), CCAAT-enhancer-binding proteins (C/EBP) ? and ?, and sterol regulatory element-binding protein-1 (SREBP-1). Results:Treatment with noncytotoxic concentrations of GS resulted in the dose-dependent inhibition of IL-1?-induced inflammatory cytokines, including IL-6, IL-8, MCP-1, and COX-2, at both mRNA and protein levels. The hyaluronan level was also significantly suppressed by GS. Moreover, GS significantly decreased the formation of lipid droplets and expression of PPAR?, C/EBP ?/?, and SREBP-1 in a dose-dependent manner. GS pretreatment attenuated the phosphorylation of nuclear factor-kappa B induced by IL-1?. Conclusions:Our data show significant inhibitory effects of GS on inflammation, production of hyaluronan, and adipogenesis in orbital fibroblasts. To our knowledge, this is the first in vitro preclinical evidence of the therapeutic effect of GS in GO.
| S-EPMC7401490 | biostudies-literature
Project description:Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution ((13)C(3)-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2-4 h. Reference values in AFS were 0-8.2 ?mol/L for 15-25 weeks of gestation and 3.2-12.0 ?mol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 ?mol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20-154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS.
| S-EPMC3173643 | biostudies-literature