Biomedical subjects

Masahiko Ito

Publications and source records attributed to Masahiko Ito.

At least 19 recordsLinked to original sources

PubMed2006

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor.

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Active Transport, Cell Nucleus↗

PubMed2006

Involvement of the STAT5 signaling pathway in the regulation of mouse preimplantation development.

The signal transducer and activator of transcription 5 (STAT5) is an essential factor in the signal transduction pathways for a number of cytokines that regulate the growth and differentiation of mammalian cells. In this study, we investigated the STAT5 signaling pathway in mouse embryos, to elucidate the mechanism of cytokine signal transduction that regulates preimplantation development. The results of the RT-PCR analysis showed that both STAT5A and B were expressed throughout preimplantation development. Immunocytochemistry revealed that the STAT5A/B proteins were located in the nucleus from the early 1-cell stage to the blastocyst stage. STAT5 activation appeared to be regulated by Janus kinases (JAKs) and SRC family kinases (SFKs), since inhibitors of these kinases inhibited the localization of STAT5 proteins to the nucleus. The JAK inhibitor Ag490 reduced both the developmental rate of the embryos and the expression levels of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5 proteins function in preimplantation development by mediating the signals from cytokines.

Active Transport, Cell Nucleus↗

PubMed2006

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe.

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.

Animals↗

PubMed2006

Calcium signals for egg activation in mammals.

A dramatic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) occurs in eggs at fertilization common to all animal species examined to date, and this serves as a pivotal signal for egg activation characterized by resumption of meiotic cell division and formation of the pronuclei. In mammalian eggs, repetitive [Ca(2+)](i) rises (Ca(2+) oscillations) each of which accompanies a propagating wave across the egg occur due to release of Ca(2+) from the endoplasmic reticulum mainly through type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor. Ca(2+) oscillations are induced by a cytosolic sperm factor driven into the egg cytoplasm upon sperm-egg fusion. A current strong candidate of the sperm factor is a novel sperm-specific isozyme of phospholipase C (IP(3)-producing enzyme), PLCzeta. Recent extensive research has reveled characteristics of PLCzeta such as the Ca(2+) oscillation-inducing activity after injection of PLCzeta-encoding RNA or recombinant PLCzeta into mouse eggs, extremely high Ca(2+)-sensitivity of the enzymatic activity in vitro, and nuclear translocation ability possibly related to cell-cycle-dependent regulation of Ca(2+) oscillations. [Ca(2+)](i) rises cause successive activation of calmodulin-dependent kinase II and E3 ubiquitin ligase, lead to proteolysis of ubiquitinated cyclin B1 and inactivation of metaphase-promoting factor (Cdk1/cyclin B1 complex), and result in the release of eggs from meiotic arrest.

Animals↗

PubMed2005

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development.

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.

Active Transport, Cell Nucleus↗

PubMed2005

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia.

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.

Base Sequence↗

PubMed2005

HLA-B polymorphism in Japanese HIV-1-infected long-term surviving hemophiliacs.

Approximately 30% of patients with hemophilia in Japan were infected with human immunodeficiency virus (HIV) in early 1980s through contaminated blood products. In 1995, a cohort of HIV-infected, asymptomatic patients with hemophilia was set up for follow-up study. Although the patients met the criteria for long-term non-progressor (LTNP) at the entry to the cohort, some of them later developed lymphopenia during five more years of observation. We collected blood samples from 80 long-term survivors; 42 of them did not require antiviral therapy, but the rest were under treatment. Analysis of HLA-B genotype revealed that carriers of known HIV-resistant alleles such as HLA-B*5701, B*5801, and alleles of B27 antigenic group were not increased in frequency, but that HLA-B*1507 was increased in the cohort (6.25% vs. 1.03%, OR = 6.40, p = 0.039). We also observed the decrease in carriers of HLA-B*5401 (3.75% vs. 14.95%, OR = 0.22, p = 0.016). HLAB* 5401 is a relatively common allele in East Asian populations and belongs to the same B22 antigenic group as B55 and B56 which were reported to associate with rapid progression. Our data indicated that HLA class I is one of the host factors involved in the retardation of HIV disease progression as also reported in the previous studies; however, the alleles associated with this resistance were not the same because of divergent host genetic background.

Alleles↗

PubMed2005

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells.

We report the effects of hemicellulase-treated Agaricus blazei (ABH) on the maturation of bone-marrow-derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up-regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T-cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin-12 (IL-12) p40, tumour necrosis factor-alpha, or IL-1beta. In addition, ABH suppressed LPS-induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS-induced cytokine secretion, while only slightly decreasing up-regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan-induced or CpG oligodeoxynucleotide-induced IL-12p40 production in DCs. The inhibition of LPS-induced responses was not associated with a cytotoxic effect of ABH nor with an anti-inflammatory effect of IL-10. However, ABH decreased NF-kappaB-induced reporter gene expression in LPS-stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS-stimulated but non-ABH-pretreated DCs. These observations suggest that ABH regulates DC-mediated responses.

Agaricus↗

PubMed2005

Effects of the cell wall of Kluyveromyces marxianus YIT 8292 on the plasma cholesterol and fecal sterol excretion in rats fed on a high-cholesterol diet.

The cellular components involved in the hypocholesterolemic activity of Kluyveromyces marxianus YIT 8292 were examined in rats fed on a high-cholesterol diet. Whole cells (KM) were heated at 115 degrees C for 10 minutes and fractionated into water-soluble extract 1 and the insoluble residue (KM-CW). After mechanical disruption by glass beads, KM-CW was separated into the cell wall (KM-W) and water-soluble extract 2. Plasma total cholesterol was decreased by feeding KM-CW or KM-W, but was not changed by feeding extract 1 or extract 2. Feeding KM-CW and KM-W increased the fecal sterol excretion and concentration of short-chain fatty acids (SCFA) in the cecum. The hypocholesterolemic activity of KM-CW was completely abolished by the enzymatic degradation of alpha-mannan and beta-glucan. These results suggest that alpha-mannan and beta-glucan were the major active components of KM, and that its hypocholesterolemic activity may be attributable to the increasing fecal sterol excretion and/or production of SCFA.

Animals↗

PubMed2004

Detection of white spot syndrome virus (WSSV) from hemolymph of Penaeid shrimps Penaeus japonicus by reverse passive latex agglutination assay using high-density latex particles.

A simple reverse passive latex agglutination (RPLA) method for detecting white spot syndrome virus (WSSV) in the hemolymph of infected Kuruma shrimp (Penaeus japonicus) was developed. It was confirmed that WSSV could be detected from the shrimp hemolymph when the latex particles blocked with a casein protein were used as detection reagent. It became clear from the result of the infection trial that viruses are detectable by RPLA before the appearance of overt symptoms of this disease. In addition, an amplification product of 982 bp (s) derived from WSSV by PCR was detected in all the samples in which WSSV was detected by RPLA. This newly developed RPLA assay can examine many samples in a simple manner since hemolymph can be extracted more easily than any other organs. This assay can be used conveniently for virus detection in the culture pond of shrimps or in the field.

Animals↗

PubMed2004

Localization of janus kinase 2 to the nuclei of mature oocytes and early cleavage stage mouse embryos.

Jak2, which is a member of the Janus tyrosine kinase family, plays essential roles in cytokine signal transduction and in the regulation of cell growth and gene expression. To investigate the involvement of Jak2 in the regulation of early preimplantation development, we examined the expression of Jak2 in mouse embryos. Reverse transcription-polymerase chain reaction assays revealed that the relative amount of Jak2 mRNA was highest in unfertilized oocytes, gradually decreased until the four-cell stage, and remained at low levels until the blastocyst stage. Immunocytochemistry showed that Jak2 was localized predominantly to the female pronucleus in one-cell embryos. The immunofluorescence signal was very weak or undetectable in the male pronucleus. In unfertilized oocytes and one-cell embryos at M phase, Jak2 was localized to the chromosomes. After cleavage to the two-cell stage, the intensity of the immunofluorescence signal decreased in the nucleus while the embryos were in late G2. This decrease was independent of DNA synthesis because it was not affected by inhibition of DNA replication. However, inhibition of protein synthesis repressed the disappearance of Jak2 from the nucleus. These results suggest a novel function for Jak2 in the regulation of early preimplantation development.

Animals↗

PubMed2004

Potent hypocholesterolemic activity of the yeast Kluyveromyces marxianus YIT 8292 in rats fed a high cholesterol diet.

The hypocholesterolemic activities of 81 yeast strains were examined in rats fed a high cholesterol diet (HCD). Male Wistar rats were fed an HCD or an HCD supplemented with 10% yeast for 7 d. It was found that the hypocholesterolemic activities of the yeasts varied remarkably between strains. Kluyveromyces marxianus YIT 8292 exhibited the most potent hypocholesterolemic activity among the yeasts that were tested. K. marxianus YIT 8292 significantly decreased not only plasma total cholesterol but also liver total cholesterol when administered as a dietary admixture at a concentration of 3%. In contrast, brewer's yeast and baker's yeast, which have been predominantly used for food, did not exhibit hypocholesterolemic activity even when administered at a concentration of 10%. These results suggest that K. marxianus YIT 8292 may be utilized as a novel food material with the ability to contribute to the prevention of hypercholesterolemia.

Animals↗

PubMed2003

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase.

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

Amino Acid Sequence↗

PubMed2003

Antioxidative effects of lactic acid bacteria on the colonic mucosa of iron-overloaded mice.

The antioxidative effects of lactic acid bacteria on lipid peroxidation in the colonic mucosa were investigated. Among 49 strains of lactic acid bacteria, Streptococcus thermophilus YIT 2001 showed the highest inhibitory activity against lipid peroxidation in liposomes induced by ferrous iron. Feeding a diet containing 0.4% St. thermophilus YIT 2001 (2 x 10(8) colony-forming units per mouse per day) for 2 weeks caused a significant decrease of lipid peroxide (thiobarbituric acid reactive substance) in the colonic mucosa of iron-overloaded mice (0.07% Fe in the diet). The mucosal lipid peroxide level did not correlate with the soluble iron concentration of the cecal contents. Therefore, it is suggested that the antioxidative effect of St. thermophilus YIT 2001 in the colonic mucosa was not due to the removal of ferrous iron from the reaction system of lipid peroxidation.

Animals↗

PubMed2003

Characterization of MCH-mediated obesity in mice.

Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus. Recently, we demonstrated that chronic intracerebroventricular infusion of MCH induced obesity accompanied by sustained hyperphagia in mice. Here, we analyzed the mechanism of MCH-induced obesity by comparing animals fed ad libitum with pair-fed and control animals. Chronic infusion of MCH significantly increased food intake, body weight, white adipose tissue (WAT) mass, and liver mass in ad libitum-fed mice on a moderately high-fat diet. In addition, a significant increase in lipogenic activity was observed in the WAT of the ad libitum-fed group. Although body weight gain was marginal in the pair-fed group, MCH infusion clearly enhanced the lipogenic activity in liver and WAT. Plasma leptin levels were also increased in the pair-fed group. Furthermore, MCH infusion significantly reduced rectal temperatures in the pair-fed group. In support of these findings, mRNA expression of uncoupling protein-1, acyl-CoA oxidase, and carnitine palmitoyltransferase I, which are key molecules involved in thermogenesis and fatty acid oxidation, were reduced in the brown adipose tissue (BAT) of the pair-fed group, suggesting that MCH infusion might reduce BAT functions. We conclude that the activation of MCH neuronal pathways stimulated adiposity, in part resulting from increased lipogenesis in liver and WAT and reduced energy expenditure in BAT. These findings confirm that modulation of energy homeostasis by MCH may play a critical role in the development of obesity.

Adipose Tissue↗

PubMed2003

Prevalence of drug-resistance-associated mutations in antiretroviral drug-naive Zambians infected with subtype C HIV-1.

The ability of HIV-1 to evolve resistance to antiretroviral drugs leads to treatment failure. By nucleotide sequencing of HIV-1 subtype B isolates, amino acids responsible for drug resistance have been identified. Less information is available, however, on the extent and distribution of these amino acids in HIV-1 nonsubtype B viruses circulating mainly in developing countries. More HIV-infected patients in the developing world are now using antiretroviral drugs, and hence there is a need to monitor drug resistance mutations in HIV-1 non-subtype B viruses. This study examines the prevalence of drug resistance mutations in 28 antiretroviral drug-naive HIV-1-infected Zambians. HIV-1 proviral DNA was extracted from peripheral blood mononuclear cells. The region encompassing gag p17 to env C2-V3-C3 was amplified by the polymerase chain reaction followed by direct sequencing. Sequence analyses for drug resistance-associated mutations in th e protease and reverse transcriptase genes, and HIV-1 subtyping, were done. Overall, 92.8% of the generated sequences were HIV-1 subtype C. The generated sequences revealed only secondary associated, but no primary, drug-resistance mutations The most frequent secondary mutations in the protease and RT genes were, respectively, I93L(91.7%), L89M (79.2%), M3611V (79%, 4.2%), and R211K (70.8%), S48T (62.5%). The atypical residues M41N (3.6%) and D67A (3.6%) were detected in the RT gene. This study reveals many naturally occurring polymorphisms in HIV-1 subtype C isolates from antiretroviral drug-naive individuals. Such polymorphisms could lead to rapid treatment failure and development of drug-resistant HIV-1 mutants in individuals undergoing antiretroviral therapy.

Adolescent↗

PubMed2002

Chronic intracerebroventricular infusion of MCH causes obesity in mice. Melanin-concentrating hormone.

Melanin-concentrating hormone (MCH) is a cyclic amino acid neuropeptide localized in the lateral hypothalamus. Although MCH is thought to be an important regulator of feeding behavior, the involvement of this peptide in body weight control has been unclear. To examine the role of MCH in the development of obesity, we assessed the effect of chronic intracerebroventricular infusion of MCH in C57BL/6J mice that were fed with regular or moderately high-fat (MHF) diets. Intracerebroventricular infusion of MCH (10 microg/day for 14 days) caused a slight but significant increase in body weight in mice maintained on the regular diet. In the MHF diet-fed mice, MCH more clearly increased the body weight accompanied by a sustained hyperphagia and significant increase in fat and liver weights. Plasma glucose, insulin, and leptin levels were also increased in the MCH-treated mice fed the MHF diet. These results suggest that chronic stimulation of the brain MCH system causes obesity in mice and imply that MCH may have a major role in energy homeostasis.

Adipose Tissue↗

PubMed2002

Melanin-concentrating hormone receptor subtypes 1 and 2: species-specific gene expression.

To assess the contribution of potential central nervous system pathways implicated in the control of appetite regulation and energy metabolism, it is essential to first identify appropriate animal models. Melanin-concentrating hormone (MCH), a conserved cyclic neuropeptide implicated in the modulation of food intake, has been shown to bind and activate two G-protein-coupled receptors, called GPR24 and MCHR2, expressed in human brain and other tissues. Here we show that several non-human species (rat, mouse, hamster, guinea pig, and rabbit) do not have functional MCHR2 receptors, or encode a nonfunctional MCHR2 pseudogene while retaining GPR24 expression. We identified three species for further evaluation that express both MCH receptor subtypes. We cloned and functionally characterized dog, ferret, and rhesus GPR24 and MCHR2 in mammalian cells and studied their brain distribution patterns by in situ hybridization. The homology, expression profile, and functional similarity of the receptors in the dog, ferret, and rhesus to that of human support the potential use of these species as preclinical animal models in the development of therapeutic agents for obesity or other MCH-mediated disorders.

Amino Acid Sequence↗