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  • Articles  (8)
  • Cell & Developmental Biology  (8)
  • 1985-1989  (6)
  • 1980-1984  (2)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 141 (1989), S. 510-516 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: An overall increase of 40% in nuclear-associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear-associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43°C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1-0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6-1.8. The normal levels of these two proteins were restored when cells were incubated at 37°C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector-treated cells, suggesting that “repair” of heat-induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new protein synthesis.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 37 (1988), S. 21-36 
    ISSN: 0730-2312
    Keywords: leukemia ; hematopoiesis ; lectins ; western transfer ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A high-resolution technique has been used to study differentiation-related and leukemia-associated glycoproteins. Cells are labeled with the membrane-impermeable probe sulfo-N-hydroxysuccinimidyl-biotin. Nonionic detergent extracts are subjected to affinity chromatography on a number of immobilized lectins and after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) and western transfer, the biotin-labeled glycoproteins are visualized by using avidin-horseradish peroxidase and 4-chloronaphthol. With the aid of the lectins concanavalin A, Dolichos biflours agglutinin, Lens culinaris hemagglutinin, peanut agglutinin, pokeweed mitogen, Ricinus communus agglutinin I, soybean agglutinin, Ulex europeus agglutinin I (UEA), and wheat germ agglutinin, each purifies different glycoprotein subsets from the same cell type. Mature cells of distinct hematopoietic lineages differ considerably in their cell surface glycoprotein patterns.This technique was used to analyze the glycoproteins of human leukemia cells before and after the induction of differentiation. K562 cells differentiated along different lineages after treatment with phorbol 12-myristate 13-acetate, sodium butyrate, dimethyl sulfoxide, or hemin. Limited specific alterations were observed with a number of lectins when K562 erythroleukemia cells were induced to differentiate. Among these, a number of bands were identified that were either lost or appeared after induction of differentiation with all four agents. In contrast, the glycoproteins bound by UEA were drastically diminished after induction of differentiation, and the remaining UEA-bound glycoproteins bore little resemblance to those of the cells before treatment. This high-resolution technique may be useful as a general method for the examination of cell surface glycoprotein differences. Once specific glycoprotein alterations are detected, lectin affinity chromatography and SDS-PAGE allow purification of antigens for the production of monoclonal antibodies.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 115 (1983), S. 255-259 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The objective of this study was to determine whether N6, O2-dibutyryl 3′,5′-adenosine monophosphate (db-cAMP)-induced reverse transformation in a chemically transformed mouse cell line, AKR-MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR-MCA cells were grown to confluency in the presence or absence of db-cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+ 1- +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)-treated cells exnibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db-cAMP-induced reverse transformation in AKR-MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 119 (1984), S. 77-81 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The objective of this study was to test the concept that the G1 period lacks any specific function in the life cycle of mammalian cells and hence could be drastically reduced without any effect on the generation time. HeLa cells were grown in medium containing an optimum dose (60 μM) of hydroxyurea at which the duration of S period was prolonged with little or no increase in generation time. At this concentration of hydroxyurea, we observed a maximum of 3 h (or 28.5%) reduction in the G1 period. We also studied the effects of synchronization in S phase by single and double thymidine blocks on cell size and its relationship to the duration of G1 in the subsequent cycle. By these treatments, we could reduce the G1 period by not more than 2 to 3 h. The reduction in G1 period was not directly proportional to the size (volume) of the G1 cells. These results suggest that G1 period has certain specific functions and cannot be eliminated by alterations in culture conditions.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 133 (1987), S. 553-559 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Treatment of hamster fibroblasts with the sulfhydryl-reducing agent β-mercaptoethanol (β-ME) results in increased synthesis of the glucose-regulated proteins (GRPs). The most abundant protein species being induced in the GRP78, with a minor increase also observed for GRP94. The enhanced synthesis of the GRP94 and GRP78 is primarily due to an increase in the steady state levels of the two GRP transcripts. Although β-ME has a general inhibitive affect on amino acid uptake and protein synthesis, compared to other protein synthesis inhibitors such as cycloheximide, puromycin, and amino acid analogue canavanine, β-ME is a more potent inducer of GRP gene expression. In addition, the induction by low dosage of β-ME requires de novo protein synthesis and is preceded by a drop in the rate of protein glycosylation. Our results support the hypothesis that denatured proteins can induce the GRP genes; however, a blockage ofsome post-translocational processing step in the endoplasmic reticulum, as a result of β-ME or other stress treatments, may provide the additional stimulation which transcriptionally activates the GRP genes to high levels.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 135 (1988), S. 397-406 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When CHO cells were treated either for 10 min at 45-45.5°C or for 1 hr with 100 μM sodium arsenite (ARS) or for 2 hr with 20 μg/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5°C administered 4-14 hr later, with thermotolerance ratios at 10-3 isosurvival of 4-6, 2-3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR-20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP-70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 μg/ml) or PUR (100 μg/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5°C in all cases. However, for a challenge at 43°C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43°C, which was the same as heat protection observed when CHM was added before and during heating at 43°C without the initial heat treatment. These differences between the initial treatments and between 43 and 45°C may possibly be explained by reports that show that heat causes more redistribution of HSP-70 to the nucleus and nucleolus than ARS and that redistribution of HSP-70 can occur during heating at 42°C with or without the presence of CHM. Heating cells at 43°C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45°C immediately after heating at 43°C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 μg/ml) or PUR (100 μg/ml) to inhibit protein synthesis during heating at 43°C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43°C, protein synthesis was required during 43°C for the development of additional thermotolerance to 45°C. These data suggest that if a considerable amount of synthesis of HSP families occurred after the initial treatment before heating at 43°C, redistribution during 43°C of the previously synthesized HSP families could lead to the additional thermotolerance to 45°C.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 132 (1987), S. 1-11 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: During 4 hr after puromycin (PUR: 20 μg/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10-3 isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5°C or 43°C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 μg/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 μg/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 μg/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 μg/ml) treatment, the addition of cycloheximide (CHM: 10 μg/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families.This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43°C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43°C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5°C caused by heating at 43°C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 132 (1987), S. 41-48 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: After sodium arsenite (100 μM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10-6 to 10-1 after 4 hr at 43°C, and as a thermotolerance ratio (TTR) of 2-3 at 10-3 isosurvival for heating at 45.5°C. Cycloheximide (CHM: 10 μg/ml) or puromycin (PUR: 100 μg/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43°C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43°C after CHM treatment were much different than those manifesting resistance to 43°C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45°C after 5 hr of heating at 43°C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45°C after 5 hr of heating at 43°C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43°C or 45.5°C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43°C with little resistance at 45.5°C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45°C caused by heating at 43°C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.
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