Biomedical subjects

Tadashi Mabuchi

Publications and source records attributed to Tadashi Mabuchi.

13 recordsLinked to original sources

PubMed2007

Genetic analysis of human complement factor H polymorphisms.

Human complement factor H (factor H) is polymorphic, with five previously reported FH alleles and three previously reported HF alleles (HF*A, HF*B, and HF*Q0). The relationship between the FH and HF alleles is not clear, and the genetic basis of factor H phenotypes has not yet been identified. In this study, nucleotide sequence analysis of complementary DNA (cDNA) from individuals with each HF phenotype identified seven mutated sites in the factor H gene. However, in four cases, the same cDNA sequence was observed in individuals with two different HF phenotypes. Western blotting and 2-DE also showed that a 160 kDa protein corresponding to factor H was expressed in individuals with HF phenotypes. In addition, factor H cross-reacting 45 and 42 kDa polypeptides were detected in individuals with HF A, HF B, or HF AB phenotypes, but not in individuals with the HF Q0 (a null allele) phenotype. Thus, HF phenotype did not correlate well with factor H gene or protein structural variation. Evidence is provided to support the hypothesis that the HF phenotypes do not correspond to polymorphism in factor H, but instead correspond to polymorphism in factor H-related protein 1. A novel PCR-RFLP method was developed and used to detect four polymorphisms (G257A, G1492A, A2089G, and G2881T) in the factor H gene in 54 unrelated Japanese individuals. This method could be useful for studies on genetic disease associated with these mutations.

Alleles↗

PubMed2006

Suppression of cell proliferation by interferon-alpha through interleukin-1 production in adult rat dentate gyrus.

The therapeutic use of interferon-alpha (IFN-alpha), a proinflammatory cytokine, is known to cause various neuropsychiatric adverse effects. In particular, depression occurs in 30-45% of patients, frequently interrupting treatment. IFN-alpha-treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by IFN-alpha remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Therefore, we investigated the effect of subchronic IFN-alpha treatment on neurogenesis in the adult rat dentate gyrus (DG). Immediately after the administration of IFN-alpha for 1 week, a decrease in the number of 5-bromo-deoxyuridine-labeled proliferating cells was observed in the DG; however, no effect was detected on the expression of mature neuronal phenotype in the newly formed cells 3 weeks later. Also, an increase in the level of interleukin-1beta (IL-1beta), a major proinflammatory cytokine, was observed in the hippocampus following the administration of IFN-alpha. Furthermore, coadministration of an IL-1 receptor antagonist completely blocked the IFN-alpha-induced suppression of the cell-proliferative activity in the DG. Our results indicate that IFN-alpha suppresses neurogenesis in the DG, and that IL-1beta plays an essential role in the suppression. The decreased cell proliferation caused by IFN-alpha-induced IL-1beta may be responsible, at least in part, for IFN-alpha-induced depression.

Animals↗

PubMed2006

Expression of syndecans, a heparan sulfate proteoglycan, in malignant gliomas: participation of nuclear factor-kappaB in upregulation of syndecan-1 expression.

Invasion of tumor cells into the surrounding normal brain tissues is a prominent feature of malignant gliomas. Malignant glioma cells secrete thrombospondin-1 which participates in the motility of glioma cells and binds cell surface heparan sulfate proteoglycan. To clarify the invasion mechanism of tumor cells, expression of the syndecans (syndecan-1, -2, -3, and -4), a major cell surface heparan sulfate proteoglycan family, was analyzed in malignant gliomas. Involvement of nuclear factor-kappaB (NF-kappaB) on syndecan-1 expression was also investigated. Using reverse transcription-PCR, the authors analyzed the expression of syndecan-1, -2, -3, and -4 in 10 malignant glioma cell lines, 2 glioblastoma specimens, and 2 normal brain specimens. All malignant glioma cell lines and glioblastoma specimens expressed all types of syndecan mRNA, except in one glioma cell line that lacked syndecan-3 expression. On the other hand, normal brain specimens expressed syndecan-2, -3, and -4 mRNA, but did not syndecan-1 mRNA. Syndecan-1 protein was localized in the cell surface of all malignant glioma cell lines by flow cytometry. Various levels of active nuclear factor-kappa B (NF-kappaB) was detected in all malignant glioma cell lines using immunoblotting. The expression of active NF-kappaB and syndecan-1 increased in U251 glioma cells after tumor necrosis factor-alpha or interleukin-1beta treatment, which can activate NF-kappaB. The amplification of active NF-kappaB and syndecan-1 by tumor necrosis factor-alpha or interleukin-1beta was suppressed by an inhibitor of NF-kappaB activation (emodin). Emodin also downregulated the expression of syndecan-1 mRNA in U251 cells. These results indicate that malignant glioma cells express all types of syndecans and suggest that NF-kappaB participates in the upregulation of the syndecan-1 expression at the transcriptional level, and increased expression of syndecan-1 could associate with extracellular matrices including thrombospondin-1.

Brain Neoplasms↗

PubMed2006

Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence.

Selection of good quality oocytes is important for improvement of assisted reproductive technology. Here, we studied the relationship of the mitochondrial distribution in metaphase II stage (MII) oocytes with fertility, since mitochondria in ooplasm are essential for energy production required for fertilization and embryo development. To observe mitochondria non-invasively, we used oocytes from a transgenic mouse, in which enhanced green fluorescent protein is targeted to the mitochondrial matrix and thus fluorescence is observed exclusively in the mitochondria. Control oocytes with mitochondria distributed around the nucleus showed normal embryo developmental competence, whereas oocytes with abnormal diffuse and fragmented mitochondria showed a significantly lower rate of embryo development after activation by intracytoplasmic sperm injection or strontium, which is a very effective agent for activation of mouse oocytes. Also, we showed that the reduced developmental competence of oocytes with diffuse and fragmented mitochondria caused by vitrification and thawing is similar to that of oocytes with abnormal mitochondrial foci obtained naturally. These findings suggest that abnormal mitochondrial distribution in oocytes at MII is a cause of developmental retardation and therefore normal mitochondrial distribution could be used as a criterion for selection of good oocytes.

Animals↗

PubMed2006

Reduced expression of the transporter associated with antigen processing 1 molecule in malignant glioma cells, and its restoration by interferon-gamma and -beta.

OBJECT: It remains unclear whether malignant glioma cells can deliver tumor antigens efficiently to major histocompatibility complex (MHC) Class I molecules. To elucidate the mechanism of antigen presentation in malignant gliomas, the authors examined the expression of the transporter associated with antigen processing 1 (TAP1), which transports antigens to MHC Class I molecules, and low-molecular-mass polypeptide 2 (LMP2), which is a subunit of a proteasome. They also analyzed the effects of interferon (IFN)-gamma and IFN-beta on the expression of these molecules. METHODS: Five glioma cells expressed undetectable or very low levels of TAP1 protein and did not express TAP1 messenger (m)RNA. Normal brain tissue and glioma tissue specimens also showed undetectable levels of TAP1 protein and the same levels of LMP2 protein as lymphoblastoid B cells. Treatments of the tumor cells with IFN-gamma, or -beta enhanced the expression of both TAP1 protein and mRNA as well as the expression of MHC Class I molecules. The expression of LMP2 protein was not altered by the IFN treatments. The authors analyzed structural alterations in the TAP1 promoter region in eight malignant glioma cell lines. Single nucleotide polymorphism was found in 446 bp up-stream of the translation start site of the TAP1 gene, and a point mutation was found in 34 bp upstream of the TAP1 gene. CONCLUSIONS: Malignant glioma cells may be less immunogenic due to low levels of TAP1 expression. Upregulated expression of TAP1 and MHC Class I molecules by IFN-gamma and -beta may enhance antigen presentation in malignant glioma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Three copies of the ATP2 gene are arranged in tandem on chromosome X in the yeast Saccharomyces cerevisiae.

We previously reported that there were three copies of ATP1 coding for F1-alpha and two copies of ATP3 coding for F1-gamma on the left and right arm of chromosome II, respectively. In this study, we present evidence that there are three closely linked copies of ATP2 encoding the beta subunit of the F1F0-ATPase complex on the right arm of chromosome X in several laboratory strains, including Saccharomyces cerevisiae strain S288C, although it was reported by the yeast genome project that ATP2 is a single-copy gene. Chromosome X fragmentation, long-PCR, chromosome-walking and ATP2-disruption analysis using haploid wild-type strains and prime clone 70645 showed that the three copies of ATP2 are present on the right arm of chromosome X, like those of ATP1 on chromosome II. Each was estimated to be approximately 4 kb apart. We designated the ATP2 proximal to the centromere as ATP2a, the middle one as ATP2b and the distal one as ATP2c. The region containing the three ATP2s is composed of two repeated units of approximately 7 kb; that is, both ends (ATP2a, ATP2c) accompanying the ATP2-neighboring ORFs are the same. A part of YJR119c, YJR120w, YJR122w (CAF17) and YJR123w (RP55), which were reported by the yeast genome project, are contained in the ATP2 repeated units; and the middle ATP2 of the three ATP2s, ATP2b, is located between the two repeated units. Expression of all three copies of ATP2 (ATP2a, ATP2b, ATP2c) was confirmed because a single or double ATP2-disruptant could grow on glycerol, but a triple ATP2-disruptant could not. In addition, of the three copies of ATP1 and ATP2, even if only one copy of the ATP1 and ATP2 genes remained, the cells grew on glycerol.

Blotting, Southern↗

PubMed2005

Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells.

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.

Animals↗

PubMed2004

Oocyte mitochondria: strategies to improve embryogenesis.

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.

Animals↗

PubMed2004

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.

Acrosome↗

PubMed2004

Overexpression of heparin-binding growth-associated molecule in malignant glioma cells.

The highly invasive and angiogenic characteristics of malignant gliomas depend on the production of growth factors and angiogenic factors. Heparin-binding growth-associated molecule (HB-GAM) is a secreted growth factor that is mitogenic for endothelial cells. To examine the expression profile of HB-GAM in malignant glioma cells, messenger ribonucleic acid (mRNA) expression was analyzed in 10 malignant glioma cell lines, two glioblastoma tissue specimens, and two normal brain tissue specimens by the reverse transcription-polymerase chain reaction. HB-GAM mRNA was expressed in all specimens including normal brain tissue specimens. Western blot analysis revealed that HB-GAM protein contents in glioma cell lines and glioblastoma tissues were 1.8 to 6.3 times higher than those in normal brain tissues. The effect of neutralizing anti-platelet-derived growth factor (PDGF) antibody was also examined on the production of HB-GAM in malignant glioma cells, since malignant glioma cells secrete PDGF that upregulates HB-GAM expression. Treatment of U251 and T98G glioblastoma cells with the anti-PDGF antibody did not affect the HB-GAM production. These results suggest that HB-GAM is overexpressed in malignant glioma cells and is involved in tumor growth.

Antibodies↗

PubMed2003

Spatiotemporal changes of levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase, in subcellular compartments during spermatogenesis in the rat testis.

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.

Animals↗

PubMed2003

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies, ATP3a and ATP3b, on the right arm of chromosome II.

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.

Base Sequence↗

PubMed2003

Localization of a mitochondrial type of NADP-dependent isocitrate dehydrogenase in kidney and heart of rat: an immunocytochemical and biochemical study.

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.

Animals↗