Archaea: an archetype for replication initiation studies?

Whereas the process of DNA replication is fundamentally conserved in the three domains of life, the archaeal system is closer to that of eukarya than bacteria. In the time since the complete genome sequences of several members of the archaeal domain became available, there has been a burst of research on archaeal DNA replication. These studies have led to both expected and surprising findings. This review summarizes the search for origins of replication in archaea, and our current knowledge of initiation, the process by which replication origins are recognized, the DNA molecule is unwound and the replicative helicase is loaded onto the DNA in preparation for DNA synthesis. The similarities and differences of the initiation process in archea, bacteria and eukarya are also summarized.     

A novel series of histone deacetylase inhibitors incorporating hetero aromatic ring systems as connection units

A series of structurally novel HDAC inhibitors, in which a hetero aromatic ring connects the spacer with the hydrophobic group, has been designed and synthesized. These new inhibitors are very potent in in vitro enzymatic assays and display antiproliferation activity against two human cancer cell lines.     

Alpha-keto amides as inhibitors of histone deacetylase

Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.     

Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity

The neuroprotective effects of lithium, a mood stabilizer, against glutamate-induced excitotoxicity in rat cortical neurons were associated with a decrease in Tyr1472 phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit and a loss of receptor activity. Since this receptor tyrosine phosphorylation is mediated by the Src-family tyrosine kinases, we investigated the effects of lithium on the Src kinase activity. Levels of phosphorylated Src kinase at Tyr416, an index of Src activation, were reduced after treatment with LiCl (1 mM) for more than 3 days. Protein levels of Src-family kinases such as Src, Fyn, and Yes were unchanged by lithium treatment. The activities of cytosolic protein tyrosine kinase and protein phosphatase were also unchanged by lithium treatment, indicating the selectivity and the modulation. Moreover, the levels of postsynaptic densities (PSD) and SynGAP, the scaffolding proteins of the NMDA receptor complex, were unaltered by lithium. A Src kinase inhibitor, SU6656, and an NR2B antagonist, ifenprodil, partially blocked glutamate excitotoxicity. Our results suggest that lithium-induced inactivation of Src kinase contributes to this drug-induced NMDA receptor inhibition and neuroprotection against excitotoxicity.     

Determination of 9-nitrocamptothecin by precolumn derivatization and its metabolite 9-aminocamptothecin in a biological fluid using reversed-phase high-performance liquid chromatography with fluorescence detection

A novel insoluble topoisomerase I inhibitor, 9-nitrocamptothecin (9-NC), is in advanced stages of clinical development and has been used to treat a diverse array of tumor types, including breast, ovarian, pancreatic and haematological malignancies. We have established a sensitive high-performance liquid chromatography method using fluorescence detection for the quantitation of 9-NC. Non-fluorescent 9-NC is converted to fluorescent 9-aminocamptothecin (9-AC) via a one-step pre-column derivative reaction. The quantitative limit of 9-NC was 1 ng/ml and the method was reproducible with the respective intra- and inter-day variability falling below 5.0 and 9.0%. The determination of both 9-NC and its metabolite 9-AC in dog plasma was also achieved using the same chromatographic and detection conditions. In dog plasma, the quantitative limits of 9-AC and 9-NC were 0.25 and 1 ng/ml, respectively. The presence of 9-AC in the samples yielded no interference with the determination of 9-NC. However, individual matrices can affect the conversion efficiency of 9-NC, thus indicating that standard samples should be run for each matrix.     

Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice

Our goal was to determine whether mice genetically altered to lack either creatine kinase (M/MtCK(-/-)) or adenylate kinase (AK(-/-)) show altered properties in the dynamic regulation of myocardial oxygen consumption (MVO(2)). We measured contractile function, oxygen consumption, and the mean response time of oxygen consumption to a step increase in heart rate [i.e., mitochondrial response time (t(mito))] in isolated Langendorff-perfused hearts from wild-type (n = 6), M/MtCK(-/-) (n = 6), and AK(-/-) (n = 4) mice. Left ventricular developed pressure was higher in M/MtCK(-/-) hearts (88.2 +/- 6.8 mmHg) and lower in AK(-/-) hearts (46.7 +/- 9.4 mmHg) compared with wild-type hearts (60.7 +/- 10.1 mmHg) at the basal pacing rate. Developed pressure fell slightly when heart rate was increased in all three groups. Basal MVO(2) at 300 beats/min was 19.1 +/- 2.4, 19.4 +/- 1.5, and 16.3 +/- 1.9 micromol x min(-1) x g dry wt(-1) for M/MtCK(-/-), AK(-/-), and wild type, respectively, which increased to 25.5 +/- 3.7, 25.4 +/- 2.6, and 22.0 +/- 2.6 micromol. min(-1) x g(-1), when heart rate was increased to 400 beats/min. The t(mito) was significantly faster in M/MtCK(-/-) hearts: 3.0 +/- 0.3 versus 7.3 +/- 0.6 and 8.0 +/- 0.4 s for M/MtCK(-/-), AK(-/-), and wild-type hearts, respectively. Our results demonstrate that MVO(2) of M/MtCK(-/-) hearts adapts more quickly to an increase in heart rate and thereby support the hypothesis that creatine kinase acts as an energy buffer in the cytosol, which delays the energy-related signal between sites of ATP hydrolysis and mitochondria.     

Partial AUC estimation and regression

Accurate diagnosis of disease is a critical part of health care. New diagnostic and screening tests must be evaluated based on their abilities to discriminate diseased from nondiseased states. The partial area under the receiver operating characteristic (ROC) curve is a measure of diagnostic test accuracy. We present an interpretation of the partial area under the curve (AUC), which gives rise to a nonparametric estimator. This estimator is more robust than existing estimators, which make parametric assumptions. We show that the robustness is gained with only a moderate loss in efficiency. We describe a regression modeling framework for making inference about covariate effects on the partial AUC. Such models can refine knowledge about test accuracy. Model parameters can be estimated using binary regression methods. We use the regression framework to compare two prostate-specific antigen biomarkers and to evaluate the dependence of biomarker accuracy on the time prior to clinical diagnosis of prostate cancer.