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X Zhu

Publications and source records attributed to X Zhu.

At least 775 records · Page 43Linked to original sources
PubMed1989

Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA.

We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system. An observed lag phase in DNA nicking suggests a multi-target model of hydroxyl free radical attack on DNA. In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate. These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate. We have investigated the initial rate of OH production with two OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively. We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for OH trapping by salicylate is 21.1 kcal/nmole. These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different. Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction. The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.

Catalase↗
PubMed1989

Relationship of ATP turnover, polyphosphoinositide metabolism, and protein phosphorylation in sciatic nerve and derived peripheral myelin subfractions from normal and streptozotocin diabetic rats.

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.

Adenosine Triphosphate↗
PubMed1989

Mutations in the RB1 gene and their effects on transcription.

Inactivation of both alleles of the RB1 gene during normal retinal development initiates the formation of a retinoblastoma (RB) tumor. To identify the mutations which inactivate RB1, 21 RB tumors isolated from 19 patients were analyzed with the polymerase chain reaction or an RNase protection assay or both. Mutations were identified in 13 of 21 RB tumors; in 8 tumors, the precise errors in nucleotide sequence were characterized. Each of four germ line mutations involved a small deletion or duplication, while three somatic mutations were point mutations leading to splice alterations and loss of an exon from the mature RB1 mRNA. We were unable to detect expression of the mutant allele in lymphoblasts of three bilaterally affected patients, although the mutation was present in the genomic DNA and transcripts containing the mutations were obvious in the RB tumors in the absence of a normal RB1 allele. The variations in the level of expression of mutant transcripts suggest deregulation of RB1 transcription in the absence of a functional RB1 gene product.

Alleles↗