Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle

CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.     

Fas has a role in cerebral malaria, but not in proliferation or exclusion of the murine parasite in mice

We examined the susceptibility of murine Fas-deficient mutants to malaria infection in order to investigate the role of Fas in an experimental murine model of cerebral malaria (CM). We infected mice of B6 and CBA wild-type and mutant backgrounds with Plasmodium berghei ANKA. The incidence of CM in the mutant mice (B6-lpr, CBA-lpr^cg) was decreased by about 50% compared with wild-type control strains at 2 weeks after infection. We did not observe significant differences of parasitemia during a murine malaria infection with nonlethal Plasmodium yoelii 17XNL between wild-type and lymphoproliferative (lpr) mutant mice of C3H and MRL genetic backgrounds, although B6-lpr mice exhibited significantly higher parasitemia than did B6 mice 12 to 18 days after infection. These results suggest Fas has a possible role in CM but may not play a major role in the proliferation or exclusion of a murine malaria parasite in a nonlethal infection.     

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.     

Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.     

Measuring gluconeogenesis using a low dose of 2H2O: advantage of isotope fractionation during gas chromatography

The contribution of gluconeogenesis to glucose production can be measured by enriching body water with (2)H(2)O to approximately 0.5% (2)H and determining the ratio of (2)H that is bound to carbon-5 vs. carbon-2 of blood glucose. This labeling ratio can be measured using gas chromatography-mass spectrometry after the corresponding glucose carbons are converted to formaldehyde and then to hexamethylenetetramine (HMT). We present a technique for integrating ion chromatograms that allows one to use only 0.05% (2)H in body water (i.e., 10 times less than the current dose). This technique takes advantage of the difference in gas chromatographic retention times of naturally labeled HMT and [(2)H]HMT. We discuss the advantage(s) of using a low dose of (2)H(2)O to quantify the contribution of gluconeogenesis.     

Comparative determination of polymorphs of indomethacin in powders and tablets by chemometrical near-infrared spectroscopy and X-ray powder diffractometry

The purpose of this research was to develop a rapid chemometrical method based on near-infrared (NIR) spectroscopy to determine indomethacin (IMC) polymorphic content in mixed pharmaceutical powder and tablets. Mixed powder samples with known polymorphic contents of forms α and γ were obtained from physical mixing of 50% of IMC standard polymorphic sample and 50% of excipient mixed powder sample consisting of lactose, corn starch, and hydroxypropyl-cellulose. The tablets were obtained by compressing the mixed powder at 245 MPa. X-ray powder diffraction profiles and NIR spectra were recorded for 6 kinds of standard materials with various polymorphic contents. The principal component regression analysis was performed based on normalized NIR spectra sets of mixed powder standard samples and tablets. The relationships between the actual and predicted polymorphic contents of form g in the mixed powder measured using x-ray powder diffraction and NIR spectroscopy show a straight line with a slope of 0.960 and 0.995, and correlation coefficient constants of 0.970 and 0.993, respectively. The predicted content values of unknown samples by x-ray powder diffraction and NIR spectroscopy were reproducible and in close agreement, but those by NIR spectroscopy had smaller SDs than those by x-ray powder diffraction. The results suggest that NIR spectroscopy provides a more accurate quantitative analysis of polymorphic content in pharmaceutical mixed powder and tablets than does conventional x-ray powder diffractometry.     

CYP1A1-mediated mechanism for atherosclerosis induced by polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) have been known to induce atherosclerosis. It has been reported that the metabolic activation of PAHs by cytochrome P450 (CYP) is an important step for PAH-induced atherosclerosis. We recently reported that PAHs down-regulated the liver X receptor (LXR) alpha-regulated genes via aryl hydrocarbon receptor (AHR) as one of the causes responsible for atherosclerosis induced by PAHs. Thus, the aim of this study was to clarify the role of CYP1A1 in the suppression of LXR-mediated signal transductions by 3-methlychoranthrene (MC), one of the PAHs. We found that LXR-mediated transactivation was inhibited by the PAH, but not by halogenated aromatic hydrocarbon, which is scarcely metabolized by CYP1A1. The repression of LXR-mediated signal transductions by MC was restored by co-treatment of HepG2 cells with a CYP1A1 inhibitor, alpha-naphthoflavone, and by the transfection of short interference RNA for CYP1A1. Based on these lines of evidence, we propose that the metabolic activation of PAHs by CYP1A1, but not the activation of AHR by PAHs, is a direct mechanism for atherosclerosis via the suppression of LXR-mediated signal transductions.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

Conservative sorting in a primitive plastid. The cyanelle of Cyanophora paradoxa

Higher plant chloroplasts possess at least four different pathways for protein translocation across and protein integration into the thylakoid membranes. It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor ('conservative sorting'), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e. are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic precursor proteins. In the absence of data on cyanobacterial protein translocation, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a peptidoglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria. We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein.