Biosynthesis of glycosaminoglycans by cultured mastocytoma cells.
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ABSTRACT:
SUBMITTER: Lewis RG
PROVIDER: S-EPMC1177831 | biostudies-other | 1973 Jun
REPOSITORIES: biostudies-other
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Project description:Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized (14)C-labelled glycosaminoglycan when incubated with UDP-[(14)C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of (14)C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized (14)C-labelled glycosaminoglycan when incubated with UDP-[(14)C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The (14)C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The (14)C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3'-phosphate 5'-sulphatophosphate, as well as UDP-[(14)C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.
| S-EPMC1177832 | biostudies-other
Project description:Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.
| S-EPMC1148290 | biostudies-other
Project description:IntroductionThe endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx.MethodsGAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis.Results and conclusionsHyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.
| S-EPMC9280546 | biostudies-literature
Project description:Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
| S-EPMC3919470 | biostudies-literature
Project description:Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.
| S-EPMC1162095 | biostudies-other
Project description:Cobalt manganese ferrite nanoparticles have application potential in the biomedical field, however there is limited information concerning the biological response. The aim of this work was to investigate the cytotoxic potential of cobalt-manganese ferrite nanoparticles in canine mastocytoma tumor cells (C2) and adipose-derived mesenchymal stromal stem cells (ASCs) cultured under a static magnetic field (MF). In this study, we investigated the viability and proliferation rate of ASC and C2 cells cultured with Co0.2Mn0.8Fe2O4 nanoparticles under 0.5T MF. We observed cells morphology and measured intracellular ROS generation. Thermal observations were used to characterize the thermotrophic cell behavior in different condition and RNA level of heat shock proteins and apoptotic genes was measured. Nanoparticles reduced cell viability, caused cell damage, i.e., through the formation of reactive oxygen species (ROS) and increased transcriptional level of apoptotic genes (Bcl-2, Bax, p53, p21). In addition, we have found that C2 mastocytoma cells cultured with metal oxide nanoparticles under MF exhibited unexpected biological responses, including thermotolerance and apoptotic response induced by the expression of heat shock proteins and ROS produced under a MF. Our results suggest that stimulation using MF and Co0.2Mn0.8Fe2O4 nanoparticles is involved in mechanisms associated with controlling cell proliferative potential signaling events. We can state that significant differences between normal and cancer cells in response to nanoparticles and MF are apparent. Our results show that nanoparticles and MF elevate the temperature in vitro in tumor cells, thereby increasing the expression of ROS as well as heat shock proteins.
| S-EPMC5434168 | biostudies-literature
Project description:Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.
| S-EPMC9884262 | biostudies-literature
Project description:PurposeTo describe the ophthalmic symptoms and histopathological findings in a rare case of an eyelid mastocytoma in an adult.ObservationsA man in his early 60s developed a painless, non-tender, non-pruritic, mobile nodule on the right lower eyelid beneath the inferior orbital rim. The lesion grew to 15 × 9 mm over eleven months. Biopsy revealed a diffuse infiltrate of histiocytoid and spindle-shaped mast cells forming cords and small nests between collagen fibers in the superficial and deep dermis. Mast cell lineage was confirmed by immunohistochemistry. Physical examination revealed no other cutaneous lesions and no evidence of systemic disease. Serum tryptase level was normal. Annual full-body examination by a dermatologist for 4.5 years has revealed neither recurrence in the eyelid nor cutaneous involvement at other sites.Conclusions and importanceMast cell tumors limited to the human eyelid are extremely uncommon with only four previously reported cases, including one in an adult. This case highlights the rare possibility of a solitary mastocytoma presenting in the eyelid of an adult.
| S-EPMC5787878 | biostudies-literature
Project description:Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.
| S-EPMC1137663 | biostudies-other
Project description:The mechanism of inhibition of phosphatidylcholine (PC) biosynthesis by the isoprenoid farnesol was investigated in the human leukaemic CEM-C1 cell line. Cells were preincubated with 20 microM farnesol for up to 2 h and pulsed with [3H]choline. PC biosynthesis was inhibited to one-quarter at the step catalysed by cholinephosphotransferase (CPT). CPT activity in cellular homogenates from farnesol-treated cells was significantly decreased, but no changes in cytidylyltransferase activity or diacylglycerol concentration were observed. Measurements of CPT activity in the experiments in which farnesol was added directly to the homogenates or microsomal fractions demonstrated that farnesol did not affect CPT activity. However, cytosol from farnesol-treated samples decreased microsomal CPT activity almost twice as much as did cytosol from controls. This effect was found to be heat-stable, and disappeared after dialysis, but could not be attributed to farnesol present in the cytosol. The effect of farnesol was specific when compared with other structurally similar isoprenoids. We conclude that farnesol brings about changes in cultured cells, leading to decreased CPT activity, and thus to the inhibition of PC biosynthesis.
| S-EPMC1134625 | biostudies-other