共查询到20条相似文献,搜索用时 15 毫秒
1.
After sodium arsenite (100 microM) 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 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/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 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees 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 degrees C with little resistance at 45.5 degrees 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 degrees C caused by heating at 43 degrees 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. 相似文献
2.
Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C. 相似文献
3.
When CHO cells were treated either for 10 min at 45-45.5 degrees C or for 1 hr with 100 microM sodium arsenite (ARS) or for 2 hr with 20 micrograms/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5 degrees 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 micrograms/ml) or PUR (100 micrograms/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5 degrees C in all cases. However, for a challenge at 43 degrees C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43 degrees C, which was the same as heat protection observed when CHM was added before and during heating at 43 degrees C without the initial heat treatment. These differences between the initial treatments and between 43 and 45 degrees 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 degrees C with or without the presence of CHM. Heating cells at 43 degrees C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45 degrees C immediately after heating at 43 degrees 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 micrograms/ml) or PUR (100 micrograms/ml) to inhibit protein synthesis during heating at 43 degrees C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43 degrees C, protein synthesis was required during 43 degrees C for the development of additional thermotolerance to 45 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
Mechanism of the decrease in the major cell surface protein of chick embryo fibroblasts after transformation. 总被引:33,自引:0,他引:33
The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with 14C-leucine, is decreased 3–6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4–5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation.Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16–26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5–6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts. 相似文献
5.
Heat shock induced proteins in plant cells 总被引:1,自引:0,他引:1
Thomas Barnett Mitchell Altschuler Carl N. McDaniel Joseph P. Mascarenhas 《Genesis (New York, N.Y. : 2000)》1979,1(4):331-340
Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells. 相似文献
6.
Effects of photoperiod and temperature on the cholinesterase activity in the ganglia of Schistocerca gregaria 总被引:1,自引:0,他引:1
H.C. Bauer 《Journal of insect physiology》1976,22(5):683-688
The presence of acetyl cholinesterases, cholinesterases, and non-specific esterases in four different ganglia of Schistocerca gregaria was demonstrated using various substrates, as well as by means of inhibition with physostigmine salicylate and BW 284 C51. The contribution of these enzymes to the total esterase activity in each ganglion was also determined quantitatively.In animals kept under a L:D = 12:12 regime with concurrent changes of temperature (about 30°C during the light period and 20°C during the dark period), the activity of the AChE displays a maximum at about the time of light change or 1 to 2 hr later. The activity peak lasts for about 2 hr. If average enzyme activities are calculated, no statistically significant differences between ‘day’ and ‘night’ animals are apparent.An elevation of the environmental temperature from 20°C to 30°C during the light period led to a significant rise in enzyme activity in all ganglia after 1 to 3 hr. Lowering the environmental temperature from 30°C to 20°C during the light period led to a significant decrease in enzyme activity only in the suboesophageal ganglion after 2 hr. Artificial stimulation of the animals in a wind tunnel decreases the acetyl cholinesterase activity in the suboesophageal ganglion, whereas in the other ganglia no significant change in enzyme activity was observed. Further, no correlation was found between the enzyme activity and O2 consumption of the experimental animals. 相似文献
7.
Over 90% of the competent cells in a population of Bacillus subtilis lost their competence after being heated to 50 C for 5 min. There was only a slight loss in the number of transformants if the culture was heated for 5 min after the termination of transformation, but 90% of the transformants were lost after 1 hr at 50 C. The population as a whole grew at a slightly faster rate at 50 C than at 32 C. We postulate that a heat-labile factor is required for the uptake or retention (or both) of deoxyribonucleic acid (DNA) in the cell, since uptake of 32P-DNA into a deoxyribonuclease-resistant form was inversely proportional to the time of exposure to heat. Cells that had lost competence after being heated did not regain their competence for at least several hours, although other cells in the population became competent. These data suggest that the heat-labile factor required for competence is synthesized only once during the period that a cell remains competent. 相似文献
8.
Roeland van Wijk Angela M. Otto Luis Jimenez De Asua 《Journal of cellular physiology》1984,119(2):155-162
Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46°C for 20 min by changing their flat, well-extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37°C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F2α (PGF2α) markedly increased the heat resistance to a 46°C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2–3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF2α in these cells) was added alone or in combination with either EGF or PGF2α, it had no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2–4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46°C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells. 相似文献
9.
R E Ricklefs D D Roby 《Comparative biochemistry and physiology. A, Comparative physiology》1983,75(2):307-311
Body temperatures of South Georgia diving petrel (P. georgicus) chicks increased from about 37.5 degrees C at hatching to between 38.5 and 39 degrees C within two weeks. Temperatures of common diving petrel P. u. exsul chicks averaged 38.8 degrees C after two weeks of age. Burrow temperatures varied between 5 and 10 degrees C. Measurements of oxygen consumption and body temperature indicated that chicks achieve effective endothermy at 5 degrees C after 9 days in P. u. exsul, 5-6 days in P. georgicus, and 0 days in the Antarctic prion (Pachyptila desolata). The maximum mass-specific, cold-induced oxygen consumption of small chicks that we could measure with our apparatus (ca. 5-6 cc O2/g per hr) was achieved at 5-6 days in P. u. exsul, 3 days in P. georgicus, and 0 days in P. desolata. Mass-specific thermal conductance decreased with age and body size in all 3 species, but was highest in P. u. exsul and lowest in P. desolata. Conductance was similar at the age of effective endothermy in all 3 species (ca. 3 J/g per hr per degrees C). The period required for the development of endothermy is related to age-specific changes in both conductance and capacity for heat production and it closely parallels the length of the brooding period. It is suggested that the length of the period of thermal dependence of the chick is related to the distance between feeding areas and the nesting site. 相似文献
10.
M Edjlali D Laval-Martin R Calvayrac 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,97(1):113-117
1. Various heat treatments were applied to the wild strain Z. Klebs. of Euglena gracilis. 2. Samples of cells were taken at day 1 of the culture at 26 degrees C in a 33 mM lactate medium, when the catalatic capacities of the catalase were highest. 3. They were either submitted to heat treatments (36 and 38 degrees C), or heat-shocks (40, 42 degrees C) or non-permissive heat stress (45 degrees C) for 15 min, 1 and 2 hr. 4. After a 2-hr 45 degrees C treatment the cells were unable to recover normal physiological functions. 5. Heat treatments between 36 and 38 degrees C decreased the catalatic capacities of cells, while heat-shocks at 40 and 42 degrees C strongly reinforced these capacities of hydrogen peroxide dismutation. 6. Having been heat-shocked at 42 degrees C for 2 hr, the cells became different from control cells: (a) after several months of culture, they displayed catalatic capacities increased by 65%; (b) they were able from now on to survive a 2 hr heat shock at 45 degrees C. 相似文献
11.
P. Antaki J. Langlais P. Ross P. Gurette K. D. Roberts 《Molecular reproduction and development》1988,19(3):305-314
The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature. 相似文献
12.
Thermotolerance decay rates were determined under rapidly proliferating exponential growth conditions and under high-cell-density plateau-phase growth conditions. Thermotolerance was induced by a marginally toxic initial heat treatment of 10 min at 44 degrees C in Chinese hamster ovary cells. Second dose-response curves were obtained at 24-hr intervals for up to 96 hr after the initial heat treatment. Resistance to second heat treatment had disappeared by 72 hr after the initial heat treatment in proliferating cells, but was evident in plateau-phase cells at 96 hr. Regardless of the proliferative status, thermotolerance decayed in an exponential manner. The half-times of tolerance decay were approximately 13 hr in proliferating cells and 30 hr in plateau-phase cells. 相似文献
13.
Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin 总被引:1,自引:0,他引:1
During 4 hr after puromycin (PUR: 20 micrograms/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 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/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 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/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 degrees 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 degrees 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 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR. 相似文献
14.
Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells 总被引:2,自引:0,他引:2
Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus. 相似文献
15.
Hyperthermic enhancement of antibody-complement cytotoxicity against normal mouse B lymphocytes and its relation to capping 总被引:1,自引:0,他引:1
Normal mouse B lymphocytes were exposed to water-bath hyperthermia in vitro and examined for susceptibility to antibody-complement (Ab-C) cytotoxicity. Enhancement of Ab-C cytotoxicity was observed during heat treatment at 42 or 43 degrees C. Sensitivity to Ab-C cytotoxicity returned to normal levels by 2-3 hr post exposure to 42 degrees C. No such recovery was observed when cells were preheated at 43 degrees C for 40 min. The mechanism responsible for heat-induced enhancement of Ab-C cytotoxicity may be related to the way heat affects the redistribution of membrane-bound antigen-antibody (Ag-Ab) complexes. To investigate this possibility, cells were preheated at 37, 42, or 43 degrees C. The Ab-C assay was then performed at 37 degrees C immediately or 2.5 hr after hyperthermia. The distribution of Ag-Ab complexes was evaluated by immunofluorescence. A direct correlation was found between the hyperthermic enhancement of Ab-C cytotoxicity and the hyperthermic inhibition of capping, a process where membrane-bound Ag-Ab complexes coalesce into a polar cap on the cell surface. Sensitivity to Ab-C cytotoxicity returned to normal levels when cells restored the ability to cap Ag-Ab complexes following 42 degrees C hyperthermia. Cells heated at 43 degrees C were still sensitive to Ab-C cytotoxicity and did not recover the capping ability even 2.5 hr after heat treatment. 相似文献
16.
Y Mugiya K Sugano 《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,99(4):899-902
1. Effects of ammonium-induced acidosis on white muscle RNA, DNA and protein concentrations were examined in the rainbow trout, Oncorhynchus mykiss. Serum protein concentrations were also determined. 2. A single administration of ammonium chloride at a dose of sublethal level caused no changes in these parameters after 3 and 6 hr. 3. Multiple administrations at a lesser dose caused significant decreases in RNA and serum protein concentrations 48 and 72 hr after the first administration. 4. DNA and muscle protein concentrations remained unchanged throughout the experimental period. 5. Significant decreases in RNA-DNA ratios and RNA-muscle protein ratios were primarily attributed to decreases in RNA concentrations. 相似文献
17.
CHO cells subline HA-1 were made thermotolerant by a priming heat treatment (43 degrees C, 30 min). Later, 4, 16, or 24 hr, they were either irradiated or heated (43 degrees C, 30 min) and irradiated. Thermotolerance had no effect on the radiation sensitivity of the cells as measured by the D0 value of the clonogenic survival curve. However, the N value of the curve (width of shoulder) showed a significant increase at 24 hr, indicating an increased capacity to accumulate sublethal damage. This indicates that the fractionation schedule 43 degrees C, 30 min + 37 degrees C, 24 hr + 43 degrees C, 30 min + X ray required approximately 100 rad more radiation than 43 degrees C, 30 min + X ray to reduce survival to the same level. The same priming treatment was given to RIF-1 tumors growing in C3H mice. Later, 24 hr, when the tumors were either irradiated or heated (43 degrees C, 30 min) and irradiated, it was found that thermotolerance had no effect on the radiosensitivity of the cells as measured by in vitro assay. However, thermal radiosensitization was not apparent 24 hr after the priming treatment. 相似文献
18.
We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a heat treatment at 45.5 degrees C administered 12 hr later. The thermotolerance ratio at 10(-3) isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2-2 micrograms/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 micrograms/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 micrograms/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance development by treatment with a combination of 2 micrograms/ml tunicamycin and 10 micrograms/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis-independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein. 相似文献
19.
Songul Gurel Ekrem Gurel Rajvinder Kaur Joshua Wong Ling Meng Han-Qi Tan Peggy G. Lemaux 《Plant cell reports》2009,28(3):429-444
A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for
example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various
temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal
43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at
43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased
frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs
were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2
to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing
calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar
treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable
transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is
several-fold higher than published frequencies. 相似文献
20.
Mouse fibroblast LM cells have been heated at 44 degrees C for different periods. Potassium content of the cells was measured at certain intervals during the postheating period at 37 degrees C for up to 24 hr. The level of K+ decreased gradually in time starting within some hours after the heat treatment. The rate of K+ loss as well as the ultimate level reached was heat-dose dependent. When the potassium content of the cell population was determined 16 hr after the heat treatment, a correlation was observed between the concentration of potassium and the level of cell survival. When X irradiation was applied immediately after hyperthermia, radiosensitization on the level of cell survival was obtained as expected, the extent being dependent on the severity of heat treatments. No added K+ loss was observed, however, when hyperthermia was combined with radiation. It is suggested that plasma membrane related functions are disturbed by the heat treatment. This points to membranes as possible candidates for primary targets in the case of cell inactivation by heat alone, and not with respect to the radiosensitization by hyperthermia. 相似文献