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1.
The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se. 相似文献
2.
It has been proved that 3H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of 3H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process. 相似文献
3.
ts Cl mouse L cells are temperature-sensitive (ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function. 相似文献
4.
Mara Rossini Susan Baserga C. H. Huang C. James Ingles Renato Baserga 《Journal of cellular physiology》1980,103(1):97-103
tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [3H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ~ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage. 相似文献
5.
The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of 32P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and
degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine
(PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol
mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids
at the two temperatures have been discussed. 相似文献
6.
P M Silverman 《Archives of biochemistry and biophysics》1973,157(1):222-233
The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg2+ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-32P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg2+ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis. 相似文献
7.
A temperature-sensitive mutant of BHK, designated is BN-2, shows a rapid drop in 3H-thymidine incorporation along with accumulation of the cells in the G1 phase of the cycle when asynchronous cultures are shifted from 33.5°C to the nonpermissive temperature of 39.5°C. Synchronized cultures of ts BN-2 cells did not enter DNA synthesis when shifted up in G1. Shift-up of cultures at the beginning of the S phase resulted in an approximately normal rate of DNA synthesis for about 2 hr. The rate of DNA synthesis then quickly declined, and the cells became arrested in mid-S after completion of approximately 0.5 rounds of DNA replication. At the same time, the majority of the cells were observed to lose the nuclear membrane and displayed premature chromosome condensation. These events were followed by the appearance of cells containing several micronuclei and eventual cell disruption and death. The nonpermissive temperature appeared to have no effect on either the elongation of short fragments of DNA or the execution of mitosis after the completion of the S phase under permissive conditions. The ts defect in this mutant may directly limit the initiation of DNA synthesis or alter the regulation of chromatin condensation. 相似文献
8.
Density dependent stimulation of thymidine incorporation in BHK21 cells by active material released from the same cells 总被引:4,自引:0,他引:4
Cultures of BHK2l cells continuously release into serum-free medium, active materials which stimulate the incorporation of thymidine in non-confluent, but not in confluent cultures of homotypic cells. The activity is not removed by centrifugation at 30,000 g for four hours, but is non-dialysable and retained by membranes with a nominal limitation for MW 25,000. Activity is stable at 4° for several weeks, but destroyed in 30 minutes at 56°. BHK2l cells also release active material capable of enhancing the effect of added serum. This is also heat labile and its action is density-dependent. 相似文献
9.
10.
The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis. 相似文献
11.
12.
Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37°C is preceded by storage
at 4°C for 2–24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14
d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37°C are optimal in retinas from 8 to 12 d
embryos.
This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates
that both protein and RNA synthesis are required for the 4°C storage enhancement of GS activity. The necessary RNA synthesis
occurs within the first 48 h following transfer to 37°C and does not require concomitant protein synthesis. Uridine uptake,
but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4°C storage when compared with
whole retina controls incubated at 37°C for the total time. In contrast, both uptake and incorporation of amino acids are
increased in 4°C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37°C. This suggests that enhancement
of GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following
4°C storage. 相似文献
13.
A particulate enzyme preparation capable of catalyzing the transfer of d-[U-14C]apiose and d-[U-14C]xylose from uridine 5′-(α-d-[U-14C]apio-d-furanosyl pyrophosphate) (UDP[U-14C]Api) and uridine 5′-(α-d-[U-14C]xylopyranosyl pyrophosphate) (UDP[U-14C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-14C]apiose or d-[U-14C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a Km for UDP[U-14C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-14C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min. 相似文献
14.
The inhibitory effects of substituted nitro- and sulphobenzofurazans on DNA, RNA and protein synthesis were compared in a new malignant fibrosarcoma cell line at 37°C and 41°C. The effects of these drugs with and without mild hyperthermia were evaluated by determining the % inhibition of incorporation of 3H-precursors into DNA, RNA and protein. None of the sulphobenzofurazan derivatives (Sbf) were effective inhibitors of nucleic acid and protein synthesis at 37°C nor did they enhance the inhibitory effect of hyperthermia alone. The nitrobenzofurazan derivatives (Nbf) at concentrations 10% that used for the Sbf derivatives strongly inhibited biopolymer synthesis in a dose related manner; 4-chloro-7-nitrobenzofurazan (Nbf-Cl) being the most potent inhibitor. Hyperthermia amplified the effect of all the Nbf compounds tested on RNA and protein synthesis but did not further affect DNA synthesis. This selective synergistic effect was most pronounced when the lowest concentrations of Nbf compounds were studied. The synergism however, did not follow a uniform pattern. 6-Mercaptopurine and 6-(1-methyl-4-nitro-5-imidazoyl)thiopurine (Azathioprine) (100 μM) had marginal effects on nucleic acid and protein synthesis when the cells were exposed to these two thiopurines for 1 h at both 37°C and 41°C and they had only a moderate inhibitory effect after exposure for 15 h. 相似文献
15.
Root exudate components change litter decomposition in a simulated rhizosphere depending on temperature 总被引:3,自引:0,他引:3
The release of root exudates into the rhizosphere is known to enhance soil biological activity and alter microbial community structure. To assess whether root exudates also stimulated litter decomposition, in a rhizosphere model system we continuously injected solutions of glucose, malate or glutamate through porous Rhizon® soil solution samplers into the soil at rhizosphere concentrations. The effect of these substances on the decomposition of 14C-labelled Lolium perenne shoot residues present in the soil was evaluated by monitoring 14CO2 evolution at either 15°C or 25°C. The incorporation of the 14C into the microbial biomass and appearance in the dissolved organic matter (DOM) pool was estimated after 32 d incubation. The presence of malate and glutamate increased the mineralization of L. perenne residues by approximately 20% relative to the soil without their addition at 15°C, however, no significant effects on residue decomposition were observed at 25°C. The incorporation of the 14C-label into the microbial biomass and DOM pool was not affected by the addition of either glucose, malate or glutamate. Although nearly the same amount of L. perenne residues were mineralized at either temperature after 32 d, less 14C was recovered in the microbial biomass and DOM pools at 25°C compared to 15°C. Alongside other results, this suggests that the rate of microbial turnover is greater at 25°C compared to 15°C. We conclude that the addition of labile root exudate components to the rhizosphere induced a small but significant increase on litter decomposition but that the magnitude of this effect was regulated by temperature. 相似文献
16.
Escherichia coli JM83 {F? ara Δ(lac-proAB) rpsL [?80dΔ(lacZ)M15]} in midlog growth phase at 30 °C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 μV/cm using an inductor coil. Exposed and unexposed control cells were maintained at 30.8 ± 0.1 °C and 30.5 ± 0.1 °C, respectively. Quadruplicate samples of exposed and unexposed E. coli cells were simultaneously radiolabeled with 35S-L-methionine at 10 min intervals over 2 hr. Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis. The results showed that E. coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells. Thus small prokaryotic cells (less than 2 μm × 0.5 μm) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT. © 1994 Wiley-Liss, Inc. 相似文献
17.
Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus 总被引:1,自引:0,他引:1
Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers. 相似文献
18.
19.
The effect of cell crowding on DNA synthesis (incorporation of 3HTdR and 32PO4) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after 3HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells. 相似文献