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361.
Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER. 相似文献
362.
363.
Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins 总被引:6,自引:0,他引:6
M N Horst S M Basha G A Baumbach E H Mansfield R M Roberts 《Analytical biochemistry》1980,102(2):399-408
Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K2CO3 and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of 125I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine. 相似文献
364.
Himalayan gray goral is endemic to Himalayas and Hindukush ranges. Analysis of 15 fecal samples and field observations from different areas of Pakistan and Azad Kashmir suggest that goral consumes foliage of a minimum of 28 plant species. Trees, shrubs and grasses appear in the ratio of 1:36:63 and hence the species is a grazer, though may opt for browsing when forced. The species mainly subsists on six species of grasses (Chrysopogon aucheri=17.97%, Themeda anathera=13.03%, Poa pratensis=11.23%, Digitaria decumbens=9.30%, Apluda mutica=7.51%, Aristida cyanatha=3.15%), though leaves of shrubs (Myrsine africana=11.38%, Daphne oleoides=8.87%, Carissa opaca=5.94%, Dodonaea viscose=4.79%, Rubus ellipticus=2.93%, Gymnosporia royleana=1.29%) are also consumed. Food preference indices (consumed/availability) suggest that grasses are highly preferred (16.86 times of availability), followed by shrubs (3.3 times of availability), whereas trees and herbs are not preferred. Food plants contain water (77.9±0.56%), ash (8.6±0.38%), sugars (6.8±0.16%), proteins (5.6%±0.28%) and fats (1.3±0.08%). Food provides 4,440 kcal of energy and 5.45 L of water/day/adult goral, which is sufficient to meet the requirements of the species. Grasses need to be ensured in the protected area separated for management of goral population. Zoo Biol 27:371–380, 2008. © 2008 Wiley‐Liss, Inc. 相似文献
365.
The modification effect of the anoxic radiosensitizer TAN on the mutagenesis in various Salmonella tester strains after gamma-ray and helium ion irradiation was studied. The oxygen enhancement ratios (OER) for all 3 strains on the lethal assay after gamma-irradiation are approximately equal to 2. The induction of reversions in TA98 and TA100 does not modify under anoxia. The value of OER on the mutagenic assay in TA102 equals 1.6. The OER after helium ion irradiation on the lethal and mutagenic assays was less than after gamma-irradiation. The mutagenesis in 3 strains after irradiation under anoxia is enhanced by TAN. The value of the TAN modification effect after gamma-irradiation increases from 2.1 +/- 0.2 for TA102 to 5.2 +/- 0.4 for TA100. However, the TAN influence on mutagenesis in TA100 after helium ion irradiation decreases to 3.1 +/- 0.3. We conclude that peculiarities of mutagenesis in various tester strains under anoxia with TAN can be explained by considering the nature of premutational DNA damages. 相似文献