Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Determination of 8-oxoguanine and 8-hydroxy-2'-deoxyguanosine in the rat cerebral cortex using microdialysis sampling and capillary electrophoresis with electrochemical detection

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values 相似文献   

Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase

DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

The Relation between Erythrocyte Trans Fat and Triglyceride,VLDL- and HDL-Cholesterol Concentrations Depends on Polyunsaturated Fat

Background

Trans fatty acids (TFA) lower HDL and increase triglyceride concentrations while polyunsaturated fatty acids (PUFA) lower triglycerides and may decrease HDL concentrations. The effect of the interaction between trans fat and PUFA on lipids is uncertain.

Methods

Men and women (n = 1032) in the Genetics of Lipid-Lowering Drugs and Diet Network (GOLDN) study were included. Fatty acids in erythrocyte membranes were measured with gas chromatography while data on potential confounders were obtained from questionnaires. To test the interaction between total erythrocyte PUFA (ePUFA) and TFA (eTFA) on lipid concentrations we distributed eTFA into tertiles and dichotomized ePUFA at the median concentration.

Results

For the 1^st, 2^nd and 3^rd tertiles of eTFA, multivariate-adjusted means±s.e.m for HDL were 46.2±1.1, 46.3±1.1 and 45.5±1.0 mg/dL among those with low ePUFA, respectively, while they were 50.0±1.1, 46.9±1.1 and 44.7±1.1 mg/dL among those with high ePUFA, respectively (P for interaction = 0.01). For the 1^st, 2^nd and 3^rd tertiles of eTFA, multivariate-adjusted means±s.e.m for triglycerides were 178.6±11.3, 144.7±10.9 and 140.8±10.6, respectively, among those with low ePUFA, while they were 133.8±11.3, 145.7±10.9 and 149.3±11.5, respectively, among those with high ePUFA (P for interaction = 0.005). Results for VLDL were similar to those for triglycerides. No significant interactions were observed for LDL or total cholesterol.

Conclusions

The relation between trans fat and HDL, VLDL and triglycerides may depend on PUFA. The benefit of avoiding trans fat may be greater among individuals with higher PUFA intake. Supplementation with PUFA among individuals with relatively high trans fat intake may have limited benefits on lipid profiles.     

Assessment of ecological risks in weed biocontrol: Input from retrospective ecological analyses

Prediction of the outcomes of natural enemy introductions remains the most fundamental challenge in biological control. Quantitative retrospective analyses of ongoing biocontrol projects provide a systematic strategy to evaluate and further develop ecological risk assessment. In this review, we highlight a crucial assumption underlying a continued reliance on the host specificity paradigm as a quantitative prediction of ecological risk, summarize the status of our retrospective analyses of nontarget effects of two weevils used against exotic thistles in North America, and discuss our prospective assessment of risk to a federally listed, threatened species (Cirsium pitcheri) based on those studies. Our analyses quantify the fact that host range and preference from host specificity tests are not sufficient to predict ecological impact if the introduced natural enemy is not strictly monophagous. The implicit assumption when such use is made of the host specificity data in risk assessment is that population impacts are proportional to relative preference and performance, the key components of host specificity. However, in concert with shifting awareness in the field, our studies demonstrate that the environment influences and can alter host use and population growth, leading to higher than expected direct impacts on the less preferred native host species at several spatial scales. Further, we have found that straightforward, easily anticipated indirect effects, on intraguild foragers as well as on the less preferred native host plant species, can be both widespread and significant. We conclude that intensive retrospective ecological studies provide some guidance for the quantitative prospective studies needed to assess candidate biological control agent dynamics and impacts and, so, contribute to improved rigor in the evaluation of total ecological risk to native species.     

Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

Rat liver histidyl-tRNA synthetase. Purification and inhibition by the myositis-specific anti-Jo-1 autoantibody

Myositis is an autoimmune inflammatory muscle disease of unknown etiology. We demonstrate directly that the antigen to the myositis-specific anti-Jo-1 antibody is histidyl-tRNA synthetase. The anti-Jo-1 antibody inhibits human HeLa and rat liver histidyl-tRNA synthetase. Using conventional and immunoaffinity chromatography with immobilized anti-Jo-1 antibody, we have purified rat liver histidyl-tRNA synthetase which has a subunit M_r 64,000 and an estimated native M_r suggesting an α₂ structure. The evidence indicates that the Jo-1 antigen is histidyl-tRNA synthetase, and that some of the histidyl-tRNA synthetase structure are conserved across species.