Cytochrome P-450 in human liver microsomes: high-performance liquid chromatographic isolation of three forms and their characterization

Three forms of cytochrome P-450, designated as P-450-HM1, P-450-HM2, and P-450-HM3, were isolated from human liver microsomes using high-performance liquid chromatography (HPLC) techniques. Each purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of SDS-PAGE, the molecular weights of P-450-HM1, P-450-HM2, and P-450-HM3 were estimated to be 51,000, 54,000, and 52,000, respectively. The oxidized absolute spectra of these three forms of cytochrome P-450 showed Soret absorption peaks at around 417 nm, indicating that these forms were in the low spin state. In a reconstituted system, P-450-HM1 showed the highest catalytic activities of nifedipine and (S)- or (R)-nilvadipine oxidases. The same form showed higher activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. P-450-HM2 showed high N-demethylase activities for benz-phetamine and aminopyrine, and also showed the highest activity of testosterone 16 beta-hydroxylase among the three forms, while it did not show detectable activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. Anti-P-450-HM1 immunoglobulin G (IgG), but not anti-P-450-HM2 IgG, inhibited the activities of testosterone 6 beta-hydroxylase and nifedipine and nilvadipine oxidases in human liver microsomes. Anti-P-450-HM1 IgG was also inhibitory against progesterone 6 beta- and 16 alpha-hydroxylases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated mitochondrial biogenesis in skeletal muscle is associated with testosterone-induced body weight loss in male mice

Androgen reduces fat mass, although the underlying mechanisms are unknown. Here, we examined the effect of testosterone on heat production and mitochondrial biogenesis. Testosterone-treated mice exhibited elevated heat production. Treatment with testosterone increased the expression level of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), ATP5B and Cox4 in skeletal muscle, but not that in brown adipose tissue and liver. mRNA levels of genes involved in mitochondrial biogenesis were elevated in skeletal muscle isolated from testosterone-treated male mice, but were down-regulated in androgen receptor deficient mice. These results demonstrated that the testosterone-induced increase in energy expenditure is derived from elevated mitochondrial biogenesis in skeletal muscle.     

Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

Activation regimens for full‐term development of rabbit oocytes injected with round spermatids

The present study was designed to investigate the effect of activation regimens on full‐term development of rabbit oocytes after round spermatid injection (ROSI). In the first series, rabbit oocytes were treated with 5 µM ionomycin before ROSI, after ROSI, or before and after ROSI. In addition, non‐treated oocytes were subjected to intracytoplasmic sperm injection (ICSI) using ejaculated spermatozoa. Cleavage rate of ROSI oocytes activated before and after ROSI (55%) was comparable with that of ICSI oocytes (60%), and significantly higher than those of ROSI oocytes activated either before or after ROSI (29–39%; P < 0.05). No offspring were produced by transfer of the cleaving ROSI oocytes, while 8% of the cleaving ICSI oocytes transferred gave birth to offspring. In the second series, oocytes were exposed to 5, 10, or 20 µM ionomycin, followed by ROSI, 5 µM ionomycin treatment, and incubation with 5 µg/ml cycloheximide (CHX) + 2 mM 6‐dimethylaminopurine (DMAP). Significantly higher cleavage rates were derived from oocytes activated with 10 and 20 µM ionomycin before ROSI (91% and 82%, respectively; P < 0.05) compared to those activated with 5 µM ionomycin before ROSI (53%). Live offspring were obtained when the cleaving ROSI oocytes with the initial ionomycin treatment at 5 and 10 µM were transferred (offspring rate 2% and 4%, respectively). These activation regimens, however, were not valid for the ROSI using cryopreserved round spermatids. In conclusion, rabbit ROSI oocytes were capable of developing into full‐term when the oocytes were activated with a combined treatment of ionomycin and CHX/DMAP. Mol. Reprod. Dev. 76: 573–579, 2009. © 2008 Wiley‐Liss, Inc.     

The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.     

Repression of nitrate uptake by replacement of Asp105 by asparagine in AtNRT3.1 in Arabidopsis thaliana L

An Arabidopsis mutant (rnc1) with a mutation at the 313th nucleotide from the translational start site of AtNRT3.1 was isolated. The mutation resulted in the replacement of aspartate by asparagine at the 105th amino acid in a region conserved among higher plants. In the rnc1 mutant, both the nitrate concentrations in plants and the nitrate uptake from the medium were <13% compared with those of the wild type, while AtNRT3.1 mRNA was accumulated similarly and both AtNRT1.1 and AtNRT2.1 mRNA were decreased. These results suggest that the replacement of Asp105 in AtNRT3.1 markedly reduces nitrate uptake and accumulation.     

Membrane Stretching Triggers Mechanosensitive Ca<Superscript>2+</Superscript> Channel Activation in <Emphasis Type="Italic">Chara</Emphasis>

In order to confirm that mechanosensitive Ca²⁺ channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca²⁺ (Δ[Ca²⁺]_c). However, the observed Δ[Ca²⁺]_c decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (ΔE _m) and stretching or compression of the plasma membrane. Significant ΔE _m values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). ΔE _m appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large ΔE _m values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.     

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice

Recessively inherited loss-of-function mutations in the parkin , DJ-1 , or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila .     

γH2A is a component of yeast heterochromatin required for telomere elongation

Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a Δrif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.Key words: γH2A, H2AS129 phosphorylation, heterochromatin, telomere, Sir complex, Tel1/Mec1, Rif1/2, Cdc13, yKu proteins