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S Kato

Publications and source records attributed to S Kato.

At least 253 records · Page 14Linked to original sources
PubMed2000

A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D.

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.

3T3 Cells↗
PubMed2000

An evaluation of semen processing methods for eliminating HIV-1.

OBJECTIVE: Whether artificial insemination can provide adequate protection for discordant couples where the man is HIV-1 positive and the woman is HIV-1 negative is uncertain because of the paucity of HIV-1 elimination data assessing current sperm-washing techniques. We evaluated how effectively these techniques eliminate HIV-1 RNA and proviral DNA from semen. METHODS: Spermatozoa were separated from semen samples from HIV-1-positive patients with haemophilia by discontinuous Percoll gradient centrifugation and the 'swim-up' method. The HIV-1 RNA and proviral DNA were measured by a highly sensitive PCR. In another test 5 x 10(6) copies of HIV-1 RNA (LAI strain) were added to semen from healthy donors and then assessed after single and combined procedures. RESULTS: Swim-up processing after Percoll gradient centrifugation reduced HIV-1 RNA and HIV-1 proviral DNA in semen to undetectable levels in the original specimen. Although discontinuous and continuous Percoll gradient centrifugation respectively reduced HIV-1 RNA added to seminal plasma specimens from healthy donors to less than < 1 copy from 10(5) and about 1 copy per 10(3) pre-separation copies, the discontinuous method left detectable HIV-1 RNA and proviral DNA in one out of 12 samples from patients with HIV-1 infection (8%). HIV-1 RNA and proviral DNA were decreased to undetectable levels after adding the swim-up procedure. CONCLUSIONS: Swim-up separation following Percoll gradient centrifugation should offer adequate protection for HIV-1-discordant couples.

Adult↗
PubMed2000

Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast.

A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.

Amino Acid Sequence↗
PubMed2000

Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay.

The tumor suppressor gene PTEN is frequently mutated in diverse human cancers and in autosomal dominant cancer predisposition disorders. Recent studies have shown that the lipid phosphatase activity of PTEN is critical for its tumor suppressor function and that PTEN negatively regulates the phosphatidylinositol 3'-kinase-protein kinase B pathway. Although more than half of PTEN mutations result in protein truncation, a significant fraction of PTEN mutations are missense mutations. To examine whether tumor-derived and germ-line-derived missense mutations inactivate PTEN lipid phosphatase function, we constructed 42 distinct types of PTEN missense mutations and expressed them in Escherichia coli. The purified (His)6-tagged PTEN proteins were tested for their ability to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-triphosphate. In addition, we examined the effect of mutant PTENs on the ability of PTEN to bind to the phospholipid membrane. The results revealed that the majority of PTEN missense mutations [38 of 42 (90%)] eliminated or reduced phosphatase activity and that all of the mutations examined had no effect on the membrane binding activity of PTEN. Our study indicated that phosphoinositide phosphatase activity is important for the tumor suppressor function of PTEN and that there may be other mechanisms of PTEN inactivation that are not monitored by in vitro phosphatase assay and in vitro membrane binding assay.

Escherichia coli↗
PubMed2000

p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains.

Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta. Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta.

Animals↗
PubMed2000

Intrinsic interrelation of lymphatic endothelia with nerve elements in the monkey urinary bladder.

Histochemical staining techniques for 5'-nucleotidase (5'-Nase) and acetylcholinesterase (AChE) were undertaken to localize the lymphatic network and nerve plexus in the monkey urinary bladder. Abundant 5'-Nase-positive lymphatic networks were characterized by increased number of valve-like structures and decreased calibre of blind-ends from the subepithelium to the subserosa. AChE-positive nerve fibers were visible throughout the vesical walls as fine plexuses, the densest being the neuromuscular plexus among the detrusor muscle cells or in each muscle bundle. AChE-positive nerve fibers or terminals were more frequently discernible around blood vessels than around lymphatics, and showed more intimate association with the lymphatics in the muscularis than those in the subepithelium. The nerve terminals in the subepithelium were frequently separated from attenuated lymphatic endothelium by the long processes of fibroblasts or some connective tissue cells. An ultrastructural observation revealed that unmyelinated nerve fibers with numerous neurofilaments and neurotubules run in close apposition to the lymphatic endothelium. Noteworthily, fewer terminal varicosities containing numerous small agranular vesicles (30-50 nm) and mitochondria, partially or completely bare of their Schwann cell covering in the vicinity of the lymphatic endothelium, were found in the subendothelium of initial lymphatics than in collecting ones. These terminals were occasionally identified at a distance of 120-350 nm from the subendothelial aspect of valve-originating roots, although no direct innervation of the vascular muscle cells could be found. A loose fibro-elastic connective tissue was usually interlaced between glial cell covering and lymphatic endothelium. The intrinsic interrelation of the lymphatic wall with the nerve plexus implies that the twisted subendothelial nerve terminals might be involved in intramural lymph drainage of the bladder.

Acetylcholinesterase↗
PubMed2000

Autonomic failure in ALS with a novel SOD1 gene mutation.

The authors report a patient with ALS and a novel SOD1 gene mutation who was in the totally locked-in state and developed autonomic failure followed by sudden cardiac arrest. A neuropathologic study showed widespread multisystem degeneration, including involvement of the autonomic nuclei in the medulla and spinal cord. SOD1 gene analysis detected a missense mutation of V118L in exon 4. These findings show notable phenotypic heterogeneity for SOD1-associated ALS.

Adult↗
PubMed2000

Role of gut cryptopatches in early extrathymic maturation of intestinal intraepithelial T cells.

Lympho-hemopoietic progenitors residing in murine gut cryptopatches (CP) have been shown to generate intestinal intraepithelial T cells (IEL). To investigate the role of CP in progenitor maturation, we analyzed IEL in male mice with a truncated mutation of common cytokine receptor gamma-chain (CRgamma-/Y) in which CP were undetectable. IEL-expressing TCR-gammadelta (gammadelta-IEL) were absent, and a drastically reduced number of Thy-1highCD4+ and Thy-1highCD8alphabeta+ alphabeta-IEL were present in CRgamma-/Y mice, whereas these alphabeta-IEL disappeared from athymic CRgamma-/Y littermate mice. Athymic CRgamma-/Y mice possessed a small TCR- and alphaEbeta7 integrin-negative IEL population, characterized by the disappearance of the extrathymic CD8alphaalpha+ subset, that expressed pre-Talpha, RAG-2, and TCR-Cbeta but not CD3epsilon transcripts. These TCR- IEL from athymic CRgamma-/Y mice did not undergo Dbeta-Jbeta and Vdelta-Jdelta joinings, despite normal rearrangements at the TCR-beta and -delta loci in thymocytes from euthymic CRgamma-/Y mice. In contrast, athymic severe combined immunodeficient mice in which CP developed normally possessed two major TCR-alphaEbeta7+ CD8alphaalpha+ and CD8- IEL populations that expressed pre-Talpha, RAG-2, TCR-Cbeta, and CD3epsilon transcripts. These findings underscore the role of gut CP in the early extrathymic maturation of CD8alphaalpha+ IEL, including cell-surface expression of alphaEbeta7 integrin, CD3epsilon gene transcription, and TCR gene rearrangements.

Animals↗
PubMed2000

p300/CBP acts as a coactivator of the cone-rod homeobox transcription factor.

CRX (cone-rod homeobox) is shown to play an important role on the photoreceptor cell maturation and maintenance. However, little is known about the molecular mechanism how CRX potentiates transcription. Here, we investigated the effect of several coactivators (p300/CBP, SRC-1, TIF2, and AIB1) on the transactivation function of CRX. The transient expression assay with a luciferase reporter gene showed that only p300/CBP potentiates the transactivation function of CRX. Furthermore, by interaction studies in vivo (a mammalian two-hybrid assay) and in vitro (a pulldown assay), p300/CBP was found to directly bind CRX through their C-terminal domains. The C-terminal domain of CRX was mapped as a transactivation domain to associate with p300/CBP. Taken together, our results demonstrate that p300/CBP acts as one of the coactivators of CRX.

Animals↗
PubMed2000

Successful implantation of in vitro-matured, electro-activated oocytes in the pig.

In the present study, we derived parthenogenetic porcine fetuses from in vitro-matured oocytes following a simple activation process in order to evaluate their developmental limitations in-vivo. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h. They were subjected to a single square pulse of direct current for 100 microsec at 1,500 V/cm and then treated with 5 microg/mL cytochalasin B for 4 h to obtain activated diploid oocytes. The diploids were cultured in modified Whitten's medium until transfer. Diploids which had cleaved to the 2- and 3- to 4-cell stages were transferred to oviducts of recipients. Live and/or dead parthenogenetic fetuses were recovered in 6 of 8 trials at 17, 18, 19, 24 and 29 d post activation. The total proportion of fetuses to transferred diploids was 31.3% (62/198). When fetuses were recovered at 19 d post activation, the proportion of development into fetuses was 71% (15/21). Our results, however, suggest that periods of gestation longer than 19 d resulted in a decrease of these proportions to 45% (18/40) at 24 d and to 18% (7/40) at 29 d. The hearts were beating in nearly all of the fetuses recovered at 19, 24 and 29 d post activation. Thus, parthenogenetic porcine diploids developed to at least the stage of limb-bud formation beyond the early heart-beating stage. Abnormalities were also externally visible on some fetuses. Formation of cyst-like structures in the heart and liver, and insufficient development of the head region and acephali were observed in some cases.

Animals↗
PubMed2000

Effect of the photocatalytic activity of TiO(2) on plasmid DNA.

We investigated the photodynamic DNA strand-breaking activity of TiO(2). A solution of super-coiled pBR 322 DNA was irradiated with 5 J/cm(2) of UVA in the presence of TiO(2) and the products were analyzed by using gel electrophoresis. The ratio of open-circular DNA to super-coiled circular DNA was calculated from the resulting peak areas as a DNA strand-breaking index (SBI). The SBI of anatase-structure TiO(2) (band gap=3.23 eV) was greater than that of rutile structure (band gap=3.06 eV), and the level of SBI correlated with the photocatalytic activity for degradation of 2-propanol. The inhibitory effects of active oxygen scavengers, including DMSO, glutathione and histidine, on the DNA strand-breaking activity were examined. All of the scavengers except ascorbic acid showed inhibitory effects, as did several polyhydric alcohols including mannitol, a well-known hydroxyl radical scavenger. These results suggest that the photodynamic DNA strand-breaking activity of TiO(2) is due to active oxygen species, especially hydroxyl radicals. Polyhydric alcohols showed an inverse correlation between the inhibitory effect on DNA strand-breaking activity and the octanol/water partition coefficient (logP).

Ascorbic Acid↗
PubMed2000

Small-Angle X-Ray Scattering Studies on Nucleation Formation of Dextran Precipitation in the Presence of Boron.

In the present study, the nucleation formation process of dextran precipitation in the presence of boron was investigated by small-angle X-ray scattering (SAXS) techniques. The formation mechanism of aggregates or nuclei is very important in the initial step of crystallization. SAXS measurement is an excellent technique for observing these processes. The results of SAXS measurement suggest that boron, as a trace impurity, induces the aggregating of dextran molecules intermolecularly as a process in the nucleation formation of dextran precipitate nuclei and causes precipitation of dextran through an elongation process. These reactions occur readily at low boron concentrations. In fact, boron, which was detected at low levels in dextran solutions, was concentrated in the precipitates. Therefore, an aqueous solution of boron-free dextran should be resistant to precipitation. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo.

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.

Animal Nutritional Physiological Phenomena↗
PubMed2000

The IVS4 + 4 A to T mutation of the fanconi anemia gene FANCC is not associated with a severe phenotype in Japanese patients.

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and a susceptibility to leukemia. There are at least 8 complementation groups (A through H). Extensive analyses of the FA group C gene FANCC in Western countries revealed that 10% to 15% of FA patients have mutations of this gene. The most common mutation is IVS4 + 4 A to T (IVS4), a splice mutation in intron 4, which has been found only in patients of Ashkenazi Jewish ancestry. When we screened 29 Japanese patients (20 unrelated patients and 4 families) using polymerase chain reaction-single strand conformation polymorphism, we found 8 unrelated patients homozygous for IVS4. This is apparently the first non-Ashkenazi-Jewish population for whom this mutation has been detected. The Ashkenazi Jewish patients homozygous for IVS4 have a severe phenotype, in comparison with other FA patients. Our analyses of Japanese patients indicate no significant difference between IVS4 homozygotes and other patients with regard to severity of a clinical phenotype. Thus, ethnic background may have a significant effect on a clinical phenotype in FA patients carrying the same mutation. (Blood. 2000;95:1493-1498)

Adolescent↗
PubMed2000

Michael addition of selenoamides to alpha,beta-unsaturated carbonyl compounds: stereocontrolled synthesis of delta-oxo selenoamides and their reactivity

[reaction: see text] Lithium eneselenolates generated from selenoamides underwent Michael addition to alpha,beta-unsaturated esters and ketones with high diastereoselectivity to give delta-oxo selenoamides in moderate to high yields within a few seconds. Further selective transformations of the delta-oxo selenoamides were also achieved.

Journal Article↗
PubMed2000

ELISA for a complexed antigen with a monoclonal antibody blocking reaction with the free antigen-assay-specific for complexed prostate-specific antigen.

We have developed an enzyme-linked immunoassay (ELISA) for serum complexed-antigen (prostate-specific antigen; PSA)(c-PSA) with simultaneous blocking of free-antigen (PSA)(f-PSA). The assay utilizes three different monoclonal antibodies (MAbs) recognising three distinct PSA epitopes. The detection limit was established as 0.19 microg/l (n=20, mean of zero standard+2S.D.) and the average recovery of f-PSA was 98-100%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2% and 2.8% to 6.3%, respectively. There was a good correlation between serum c-PSA measured by the present ELISA and PSA-alpha(1)-antichymotrypsin complex (PSA-ACT) concentrations (r=0.991). This method should provide a better tool for discriminating between benign and malignant prostatic disease.

Antibodies, Blocking↗
PubMed2000

Involvement of nuclear receptor coactivator SRC-1 in estrogen-dependent cell growth of MCF-7 cells.

Steroid hormones regulate cell growth and function through the transcriptional control of target genes by their cognate nuclear receptors. These receptors bind to ligands and associate with transcriptional cofactors to stimulate transcription. SRC-1, one of the nuclear receptor coactivators, is known to interact with nuclear receptors and enhance transactivation function in a ligand-dependent manner. In this study, to assess the function of SRC-1 in cell growth regulated by nuclear receptor ligands, we established a stable transformant cell line overexpressing human SRC-1 and studied the action of 17beta-estradiol (E(2)) on cell growth as well as the expression of E(2)-responsive genes in MCF-7 cells. We found that SRC-1 overexpression potentiates cell growth stimulated by E(2) in accordance with enhancement of transcriptional activation of exogenous and endogenous E(2)-responsive genes. These findings clearly indicate the importance of nuclear receptor coactivators for the activities of steroid/lipophilic vitamins in cell growth and gene expression.

Adenocarcinoma↗