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Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM. 相似文献
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S K Cunningham S J Sequeira J Chambers A Heffernan T J McKenna 《Journal of steroid biochemistry》1988,31(1):125-130
We have demonstrated that conventional methods for measuring total urinary aldosterone (TUA) may markedly and inconsistently underestimate aldosterone output, since under the conditions usually employed (pH 1.0), the hydrolysis of aldosterone conjugates in urine is incomplete. The use of more acidic hydrolysis conditions (pH 0.2) overcomes this problem. However free aldosterone may be damaged at this pH. Therefore to accurately measure TUA output, it is necessary to isolate the undamaged aldosterone chromatographically and to correct for procedural losses based on the recovery of aldosterone tracer added to the urine prior to hydrolysis. We compared a number of laboratory estimates of aldosterone status (including urinary free aldosterone) with the 24-h urinary sodium output in normal subjects, since this provides a good bioassay of aldosterone. Sodium output correlated best with "optimised" 24 h TUA, i.e. hydrolysed at pH 0.2, (r = -0.589, P less than 0.001), and with plasma aldosterone (r = -0.504, P less than 0.005). Both aldosterone in random urine specimens and plasma renin activity correlated poorly with 24-h sodium output. Therefore, while the measurement of optimised TUA excretion provides the best index of aldosterone activity, assay of aldosterone in random specimens of plasma, which is more convenient for patient and laboratory, may be adequate for many clinical purposes. 相似文献
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The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS
Chemical Abstracts Service
- DMSO
dimethylsulfoxide
- ETOH
ethanol
- HGPRT
hypoxanthine-guanine phosphoribosyl transferase
- HGPRT+
HGPRT-competent
- HGPRT–
HGPRT-te]deficient
- MC
metabolic cooperation
- MC+
metabolic cooperation-competent
- MC–
metabolic cooperation-deficient
- MEM
minimum essential medium
- PDBu
phorbol dibutyrate
- PMA
phorbol myristate acetate
- 6TG
6-thioguanine
- 6TGr
6-thioguanine-resistant
- 6TGs
6-thioguanine-sensitive
- V79/MC assay
Chinese hamster V79 lung fibroblast assay for metabolic cooperation 相似文献
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Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate 总被引:1,自引:0,他引:1
T J FitzGerald M McKenna L Rothstein C Daugherty K Kase J S Greenberger 《Radiation research》1986,107(2):205-215
Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells. 相似文献
10.
Photoaffinity labeling of the purified skeletal muscle calcium antagonist receptor by a novel benzothiazepine, [3H]azidobutyryl diltiazem 总被引:1,自引:0,他引:1
K Naito E McKenna A Schwartz P L Vaghy 《The Journal of biological chemistry》1989,264(35):21211-21214
Previous photoaffinity-labeling studies with [3H]azidopine, (+) [3H]PN200-110, and [3H]LU 49888 have demonstrated that 1,4-dihydropyridines (nifedipine-like drugs) and phenylalkylamines (verapamil-like drugs) bind exclusively to the 165-kDa alpha 1 subunit of skeletal muscle calcium channels. However, it has not been conclusively determined whether benzothiazepines (diltiazem-like drugs), which represent the third group of calcium antagonists, also bind to the alpha 1 subunit. Here we report data obtained with a newly developed benzothiazepine photoaffinity probe, [3H]azidobutyryl diltiazem. This drug competes with diltiazem for the benzothiazepine-binding site and, in purified calcium channel preparations, specifically labels the 165-kDa polypeptide which does not change its electrophoretic mobility upon disulfide reduction. These data show that benzothiazepines, just like 1,4-dihydropyridines and phenylalkylamines, bind to the alpha 1 subunit of the skeletal muscle calcium channels. 相似文献