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Burt V. Bronk Joe D. Patton David N. Mellard 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,697(3):278-285
Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C. 相似文献
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Joe T. Ritchie 《Plant and Soil》1981,58(1-3):81-96
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Xu Zhang Wei Zhang Santosh L. Saraf Mehdi Nouraie Jin Han Michel Gowhari Johara Hassan Galina Miasnikova Adelina Sergueeva Sergei Nekhai Rick Kittles Roberto F. Machado Joe G. N. Garcia Mark T. Gladwin Martin H. Steinberg Paola Sebastiani Donald A. McClain Victor R. Gordeuk 《Human genetics》2015,134(8):895-904
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Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha 总被引:1,自引:0,他引:1
Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol. 相似文献
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Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187 总被引:4,自引:0,他引:4
P J Bennet J P Moatti A Mansat H Ribbes J C Cayrac F Pontonnier H Chap L Douste-Blazy 《Biochimica et biophysica acta》1987,919(3):255-265
Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction. 相似文献
10.
Phospholipase C from human sperm specific for phosphoinositides 总被引:3,自引:0,他引:3
H Ribbes M Plantavid P J Bennet H Chap L Douste-Blazy 《Biochimica et biophysica acta》1987,919(3):245-254
Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization. 相似文献