共查询到20条相似文献,搜索用时 15 毫秒
1.
Soumya Ivaturi Catherine J. Wooten Maikhanh D. Nguyen Gene C. Ness Dayami Lopez 《Biochemical and biophysical research communications》2014
Several findings suggest that the low-density lipoprotein (LDL) receptor may internalize different lipoprotein particles via diverse pathways. Using a combination of discontinuous sucrose gradients and Triton solubilization studies, we demonstrated that the LDL receptor could be located simultaneously in clathrin-coated pits and caveolae in rat and human liver and in human hepatocyte-like C3A cells. Treatment with the cholesterol biosynthesis inhibitor, zaragozic acid A, shifted the distribution of the LDL receptor to clathrin containing fractions, whereas treatment with cholesterol or LDL shifted the receptor distribution towards caveolin-1 containing fractions. The LDL-dependent shift of the LDL receptor to caveolae coincided with a reduction in internalization of Bodipy-LDL. Redistribution within plasma membrane microdomains in response to specific treatments resulting in changes in LDL receptor function represents a novel paradigm that could be exploited in the development of a new class of therapeutic drugs. 相似文献
2.
To evaluate the peroxisomal requirement for beta-oxidation of hydroxyeicosatetraenoic acids (HETES), we tested 5-, 12- and 15-HETE oxidation in wild-type and mutant Chinese hamster ovary (CHO) cells. Mutant CHO cells contain peroxisomal ghosts, have random cytosolic localization of catalase and lack two of the enzymes necessary for peroxisomal beta-oxidation. Reverse-phase HPLC indicated that 33% of 12-HETE radioactivity was converted by wild-type CHO cells during a 2 h incubation to one major and several minor polar metabolites. Wild-type CHO cells also converted 15-HETE to one major and several minor polar metabolites. Neither 12- nor 15-HETE were converted to any metabolites by the mutant CHO cell lines, despite appreciable cellular uptake of these hydroxyeicosanoids. 5-HETE was not converted to any metabolic products by either the wild-type or the mutant CHO cells. Docosahexaenoic acid beta-oxidation was substantially reduced in the mutants as compared to the wild-type cells, palmitic acid beta-oxidation was reduced to an intermediate extent in the mutants, but octanoate beta-oxidation and citrate synthase activity were not impaired. Protein immunoblotting for mitochondrial manganese superoxide dismutase indicated a single band of identity at 20 kDa in both wild-type and mutant CHO cells. Since mutant CHO cells fail to convert 12- and 15-HETE to oxidative metabolites but contain normal mitochondrial enzymatic activities, intact peroxisomes appear to be the organelle responsible for HETE oxidation. 相似文献
3.
Enhanced internalization of ricin in nigericin-pretreated Chinese hamster ovary cells. 总被引:5,自引:4,他引:1
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Biochemical and electron microscopic autoradiographic studies with [125I] ricin have revealed that nigericin-pretreated Chinese hamster ovary cells are more efficient than untreated cells in the internalization of the toxin into the cells. These results suggest that the enhanced rate of internalization of ricin in nigericin-pretreated cells may account for the enhancement of cytotoxicity of ricin in Chinese hamster ovary cells by nigericin. 相似文献
4.
Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. 总被引:10,自引:0,他引:10
We examined the intracellular signal transduction of two endothelin receptor subtypes (ETA and ETB) by transfection and stable expression of individual receptor cDNAs in Chinese hamster ovary cells. Both receptors showed a rapid and marked stimulation of phosphatidylinositol hydrolysis and arachidonic acid release in response to agonist interaction. The two receptors, however, exhibited different responses in the cyclic AMP transduction cascades. ETA mediated the accumulation of cyclic AMP formation, whereas ETB displayed an inhibitory action on the forskolin-stimulated cyclic AMP accumulation. In both receptors, the responses of phosphatidylinositol hydrolysis, arachidonic acid release, and cyclic AMP formation were induced in complete agreement with the endothelin-binding selectivity of each receptor subtype. Endothelin, added together with GTP, activated the adenylate cyclase activity in membrane preparations of ETA-expressing cells, indicating the direct linkage of ETA to the adenylate cyclase system. Pertussis toxin treatment of ETA-expressing cells resulted in partial inhibition of the endothelin-induced cyclic AMP accumulation, whereas the same treatment of ETB-expressing cells completely abolished the endothelin-induced inhibition of cyclic AMP formation. Thus, the two endothelin receptor subtypes are coupled to multiple but distinct signal transduction cascades through different G proteins. 相似文献
5.
Chinese hamster ovary cell mutants defective in the internalization of ricin. 总被引:6,自引:2,他引:4
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Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin. 相似文献
6.
We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-3H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A) Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[3H]fucitol quantitatively from [3H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [3H]fucose-labelledglycoproteins from Lec1 cells were separated by SDSPAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [3H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDSPAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[3H]fucitol. Trypsin treatment of [3H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked 相似文献
7.
Gregan B Jürgensen J Papsdorf G Furkert J Schaefer M Beyermann M Rosenthal W Oksche A 《The Journal of biological chemistry》2004,279(26):27679-27687
Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation. 相似文献
8.
L. D. Recht Vic Raso Roger Davis Rebecca Salmonsen 《Cancer immunology, immunotherapy : CII》1996,42(6):357-361
Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization
and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the
underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization
rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin,
7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfRneg) and two lines transfected with either wild-type TfR (Tfrwt) or an internalization-deficient (TfRδ7 – 58del) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfRwt cells (ID50<1 nM) while both TfRneg cells and TfRδ7 – 58del were only minimally affected (ID50>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfRwt and TfRδ7 – 58del cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by
co-treatment of TfRwt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfRδ7 – 58del cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin
efficacy than receptor number.
Received: 15 March 1996 / Accepted: 28 May 1996 相似文献
9.
We describe a method of collecting large quantities of mitotic cells from a population of Chinese hamster ovary cells which were exponentially growing on positively charged dextran microcarriers in suspension culture. These cells were treated for 2.5 h with colcemid, and mitotic cells were harvested from the oicrocarriers by increasing spinner velocity. A yield of 2–3% of the total population was obtained using this method; of the cells collected, 85–95% were in metaphase as determined by microscopic inspection. Both synchrony and cell viability were excellent in the selected population. 相似文献
10.
A method for the purification of centrosomes from cultured Chinese hamster ovary cells is described. The centrosomes produced by application of this method show good retention of their intracellular morphology: the centrioles are surrounded by an “osmiophilic halo” containing numerous pericentriolar or satellite bodies. The latter spherical structures are approx. 55 nm in diameter and possess a densely staining central core surrounded by an envelope of lighter material. The number of satellite bodies associated with the centrioles seems variable, as does their spatial disposition within the osmiophilic halo. 相似文献
11.
12.
We used a conventional procedure involving treatment with 5-bromodeoxyuridine and visible light to isolate a stable, temperature-sensitive, auxotrophic variant-TsNd-6-from its parental Chinese hamster ovary cell clone K1. At the nonpermissive temperature of 39.5 °C, TsNd-6 requires thymidine, hypoxanthine, and glycine for growth. Folinic acid can substitute for hypoxanthine and glycine at the elevated temperature. 相似文献
13.
Genetic and biochemical analysis of mutation(s) affecting ricin internalization in Chinese hamster ovary cells 总被引:1,自引:0,他引:1
We have previously reported the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in the internalization of ricin (Ray, B., and Wu, H.C. (1982) Mol. Cell. Biol. 2, 535-544). These mutants also do not exhibit the enhancement of ricin internalization by nigericin pretreatment at a low concentration, which is observed in the wild-type CHO cells. An analysis of somatic cell hybrids between the mutant and the toxin-sensitive wild-type CHO cell line shows that all of the phenotypes associated with the toxin resistance mutation are dominant in the hybrid cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]palmitic acid-labeled cell extracts from the mutant and toxin-resistant hybrid cell lines has revealed an increased incorporation of [3H] palmitic acid into two proteins with apparent molecular weights near 30,000 in the mutant and hybrid cells as compared to that in the wild-type cell line. Our studies indicate that these two fatty acyl proteins might be related to a dominant mutation(s) which results in a decreased uptake of ricin. 相似文献
14.
A "hybrid" discontinuous gradient consisting of 6% Percoll overlaid on metrizamide separated mitochondria from other organelles in a Chinese hamster ovary cell postnuclear supernatant in a single 15-min centrifugation. The mitochondrial preparation contained about 25% of the mitochondrial marker, cytochrome-c oxidase, in a form that was about 90% latent. Based on the postnuclear supernatant, cytochrome-c oxidase activity was enriched approximately 45-fold. Trace amounts of lysosomal, rough endoplasmic reticular, Golgi, peroxisomal, plasma membrane, and cytosolic markers were found in the preparation. Electron microscopy revealed that the preparation consisted almost exclusively of mitochondria with only minor amounts of contaminating organelles. Analysis of the mitochondrial preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the mitochondrial preparation had a unique protein profile compared to the postnuclear supernatant and other gradient interfaces. Separation of the mitochondria into membrane and lumenal (matrix) fractions by treatment with 100 mM Na2CO3, pH 11.5, also indicated that the mitochondria were intact; they were rich in lumenal proteins. The data indicate that the mitochondria represent maximally about 2.2% of Chinese hamster ovary cell postnuclear supernatant protein. These isolated mitochondria should prove useful for problems in molecular cell biology. 相似文献
15.
Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with [35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts. 相似文献
16.
After exposure to compactin, the competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 22% of CHO-K1 cells contained abnormally high numbers of chromosomes. In two populations of cells selected for compactin resistance 31 and 33% of the cells contain more than 22 chromosomes. Some cell lines isolated from these populations have the wild type chromosome number of 20-21, while others have a broad distribution of chromosome number, often with a mean around 36-40. Finally, Chinese hamster ovary cells that are mutant for 3-hydroxy-3-methylglutaryl-CoA reductase and therefore auxotrophic for mevalonate were starved for that compound. This treatment also increased the number of cells containing extra chromosomes. These results indicate that interruption of the cellular supply of mevalonate results in abnormal chromosome number. 相似文献
17.
18.
Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells 总被引:15,自引:0,他引:15
M C Willingham I H Pastan G G Sahagian 《The journal of histochemistry and cytochemistry》1983,31(1):1-11
Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system. 相似文献
19.
Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor 总被引:36,自引:15,他引:21
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Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors. 相似文献
20.
The process of clathrin-mediated endocytosis tightly regulates signaling of the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A fundamental question in the cell biology of membrane receptor endocytosis is whether activated receptors can initiate the formation of clathrin-coated pits (CPs) or whether they are simply mobilized to pre-existing CPs. Here, using various approaches, including a dynamic assay to monitor the distribution of CPs and GPCR-beta-arrestin complexes in live HeLa cells, we demonstrate for the first time that activated GPCRs do not initiate the de novo formation of CPs but instead are targeted to pre-existing CPs. 相似文献