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Biomedical subjects

R Watanabe

Publications and source records attributed to R Watanabe.

At least 91 records · Page 5Linked to original sources
PubMed1999

Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis.

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

Amidohydrolases↗
PubMed1999

Inhibitory effect of trehalose dimycolate (TDM) and its stereoisometric derivatives, trehalose dicorynomycolates (TDCMs), with low toxicity on lung metastasis of tumour cells in mice.

We examined the effect of semisynthetic trehalose-6,6 '-dimycolate (TDM) and its synthetic stereoisomeric derivatives (trehalose 6,6'-dicorynomycolates; TDCMs) prepared in oil-in-water (o/w) emulsion on inhibition of lung metastasis produced by highly metastatic murine tumour cells, colon 26-M3.1 carcinoma and B16-BL6 melanoma cells, using experimental and spontaneous metastasis models. Intravenous (i.v.) administration of TDM (100 microg/mouse) 1, 3 or 8 days before tumour inoculation significantly inhibited lung metastasis of colon 26-M3.1 cells, in a dose-dependent manner. Single administration of TDM 1 day after tumour inoculation also showed the therapeutic effect on experimental lung metastasis of colon 26-M3.1 cells. Similarly, multiple administrations of TDM after tumour inoculation resulted in a significant inhibition of spontaneous lung metastasis of B16-BL6 cells (on day 35), although it showed no effect on suppression of tumour growth (on day 21). In comparison of toxicity in vivo among TDM and four TDCMs such as TDCM(2R,3R), TDCM(2S,3R), TDCM(2R,3S) and TDCM(2S,3S), all of the TDCMs appeared to be less toxic than TDM itself. Furthermore, all of the TDCMs were prophylactically as well as therapeutically active for inhibition of lung metastasis of both colon 26-M3.1 and B16-BL6 tumour cells, showing higher inhibitory activity than that of TDM. In particular, TDCMs induced a marked suppression of the growth of B16-BL6 tumour cells in vivo. These results suggest that systemic administration of TDM as well as TDCMs led to inhibition of tumour metastasis and TDCMs are more potential to suppress tumour growth and inhibit tumour metastasis than TDM.

Adjuvants, Immunologic↗
PubMed1999

The MH1 domains of smad2 and smad3 are involved in the regulation of the ALK7 signals.

The biological responses of the transforming growth factor beta (TGF-beta) superfamily are induced by activation of a receptor complex and Smad proteins. We surveyed the TGF-beta superfamily receptors using the degenerate PCR strategy, and found activin receptor-like kinase 7 (ALK7) to be abundantly expressed in fetal rat pancreatic islets. ALK7 is also expressed in adult rat islets and pancreatic beta-cell-derived MIN6 cells. The constitutively active form of ALK7, ALK7(T194D), activated Smad3 and a chimeric Smad protein, Smad3-2, containing the MH1 domain of Smad3 and the MH2 domain of Smad2, and translocated them to nuclei and then induced activation of the human PAI-1 promoter. However, neither Smad2 nor Smad2-3 protein, containing the MH1 domain of Smad2 and the MH2 domain of Smad3 were activated. These results indicate that the ALK7 signal regulates nuclear localization and activation of Smad2 and Smad3, and the MH1 domain of Smad2 has inhibitory effects on the nuclear localization.

Activin Receptors↗
PubMed1999

Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase.

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an approximately 38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an approximately 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of approximately 10,000-fold purified Golgi GCS with the same reagents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.

Amino Acid Sequence↗
PubMed1999

Use of N-[5-(5,7-dimethyl boron dipyrromethene difluoride-sphingomyelin to study membrane traffic along the endocytic pathway.

We have used N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM or 'BODIPY-SM'), a fluorescent analog of sphingomyelin, to study lipid transport along the endocytic pathway of human skin fibroblasts. The unique spectral properties of the BODIPY fluorophore allow the investigator to distinguish various populations of labeled endosomes and lysosomes within the living cell by fluorescence microscopy, and in conjunction with quantitative fluorescence microscopy, to estimate the concentration of these lipids in different intracellular compartments. This methodology is also applicable for visualizing the accumulation of lipids in the endosomes and lysosomes of storage disease fibroblasts.

Biological Transport↗
PubMed1999

Prospective evaluation for upper gastrointestinal tract acute graft-versus-host disease after hematopoietic stem cell transplantation.

The incidence and clinical significance of upper gastrointestinal tract acute graft-versus-host disease (upper GI GVHD) were prospectively evaluated in 44 Japanese patients who underwent allogeneic (n = 26) or autologous (n = 18) stem cell transplantation. Endoscopic examination was routinely performed between days 20 and 50 post-transplant and when symptoms of upper GI and/or acute GVHD of other organs were present. The results were compared with the historical records of 49 allograft and 20 autograft recipients. The diagnosis of upper GI GVHD was confirmed by histologic findings of GVHD and persistent upper GI tract symptoms. The incidence of upper GI GVHD was 46% in the prospective allograft group, higher than in the retrospective group. Upper GI GVHD was not diagnosed in any autograft patients. Twelve of 19 patients with upper GI GVHD had skin GVHD, and two of the 12 had concurrent lower GI GVHD. Upper GI GVHD was successfully treated with steroids and did not progress to symptomatic lower GI GVHD. In addition, upper GI GVHD completely resolved without specific alteration in immunosuppressant therapy in six patients. No risk factors for upper GI GVHD could be identified. The presence of upper GI GVHD did not significantly affect early death rate, incidence of chronic GVHD, and overall survival. In conclusion, by the prospective evaluation of the upper GI tract by endoscopy we could accurately diagnose upper GI GVHD in half our allogeneic recipients. However, upper GI GVHD was successfully controlled with or without additional steroids in all cases and had little impact on transplant outcome.

Adolescent↗
PubMed1999

A family with hereditary factor X deficiency with a point mutation Gla32 to Gln in the Gla domain (factor X Tokyo).

We report a new family with hereditary factor X deficiency. The propositus had a markedly prolonged prothrombin time, a mild prolongation of activated partial thromboplastin time and a clotting time activated by Russell's viper venom. Factor X activity in plasma was 3 u/dl (normal range 56-138 u/dl). Factor X antigen level was 61 u/dl. Molecular analysis revealed a homozygous mutation, Glu (GAG) to Gln (CAG) at residue 32 which normally undergoes gamma-carboxylation within the gamma-carboxyglutamic acid rich domain. The genotypes of family members completely correlated with their factor X activities. It is suggested that the Glu32 to Gln mutation is the molecular basis for the abnormal factor X in this family.

1-Carboxyglutamic Acid↗
PubMed1999

Internalization and sorting of plasma membrane sphingolipid analogues in differentiating oligodendrocytes.

We studied the formation of early endosomes in differentiating oligodendrocytes and type-2 astrocytes, which are derived from common precursor cells in rat neonates, using fluorescent analogues of lactosylceramide (LacCer) and sulfatide labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-pentanoic acid (BODIPY FL C5). These sphingolipid analogues exhibit a concentration-dependent shift in their fluorescence emission maximum from green to red wavelengths that can be used to estimate the relative concentration of an analogue in the intracellular membranes of living cells by quantitative fluorescence microscopy. When oligodendrocytes at various stages of differentiation were incubated with 1 microM BODIPY-sphingolipid at 10 degrees C and washed, yellow/green plasma membrane fluorescence was observed. Quantitative studies confirmed that the amount of BODIPY-LacCer or -sulfatide incorporated into the plasma membrane of a given cell type was identical. When these cells were subsequently warmed to 37 degrees C for 2-10 min to allow internalization to occur, the BODIPY-sphingolipid analogues were distributed in a punctate pattern throughout the cytoplasm. Within individual cells labeled with BODIPY-sulfatide, some endosomes exhibited green fluorescence, whereas others emitted red/orange fluorescence. In contrast, when BODIPY-LacCer was used, only green endosomes were observed. Although this phenomenon could be observed at earlier stages of differentiation, it was most obvious in mature oligodendrocytes, where quantitative measurements of the red/green ratio of individual endosomes suggested about a threefold difference between the concentration of the LacCer and sulfatide analogues in endosomes. These results suggest that "lipid sorting" takes place during endocytosis in mature oligodendrocytes, resulting in selective exclusion of certain lipid species during the internalization process. This sorting event may result in the net addition of lipids to the differentiated oligodendrocyte plasma membrane.

Animals↗
PubMed1999

Contribution of virus-receptor interaction to distinct viral proliferation of neuropathogenic and nonneuropathogenic murine leukemia viruses in rat glial cells.

The efficiency of receptor-mediated entry of pseudotyped virus carrying the surface protein (SU) of clone A8, a neuropathogenic variant of Friend murine leukemia virus (FrMLV), to rat glial cell line F10 was 1 order of magnitude greater than that of pseudotyped virus carrying SU of nonneuropathogenic FrMLV clone 57. Introduction of the gene coding for ecotropic MLV receptor on F10 cells (F10-ecoR) into SIRC cells, which are naturally resistant to FrMLV infection, also revealed the difference in receptor recognition between the A8 and the 57 viruses. Our results show that the difference in receptor utilization between A8-SU and 57-SU only partially explains the 3-order-of-magnitude difference in proliferation between A8 and 57 viruses in F10 cells.

3T3 Cells↗
PubMed1999

Dry eye after haematopoietic stem cell transplantation.

AIMS: To determine the incidence, natural course, and severity of dry eye occurring or worsening after haematopoietic stem cell transplantation (SCT). METHODS: At a tertiary care hospital, 53 patients undergoing allogeneic or autologous SCT followed by at least 180 days of follow up were studied prospectively. Examination included grading of symptoms of dry eye, evaluation of ocular surface, tear break up time, and Schirmer tests with and without nasal stimulation. Meibomian gland secretion was also examined using a slit lamp while applying steady digital pressure. RESULTS: Of the 53 patients, 44 received allografts. Half of these patients (22) developed dry eye or their pre-existing dry eye worsened after SCT, while none of nine autograft recipients did. Onset of dry eye was 171 (SD 59) days after SCT. Two types of dry eye occurred. One (n=10) was severe with ocular surface findings resembling Sjögren's syndrome and reduction of reflex tearing soon after onset. A mild type (n=12) had unimpaired reflex tearing. Meibomian gland dysfunction (MGD) was more frequent and severe in patients with dry eye and chronic graft versus host disease (GVHD), and overall severity of dry eye was greater in patients with MGD and chronic GVHD. CONCLUSIONS: Dry eye after SCT occurred only in allograft recipients, and was not evident in autograft recipients. The severe form of dry eye had a tendency to develop rapidly. Further study on the prediction and treatment of severe dry eye after SCT is necessary.

Adult↗
PubMed1999

Bladder neck suspension using percutaneous bladder neck stabilization to the pubic bone with a bone-anchor suture fixation system: A new extraperitoneal laparoscopic approach.

A 39-year-old woman with type 1 genuine stress urinary incontinence was treated using a novel extraperitoneal laparoscopic bladder neck suspension procedure with a bone-anchor suture fixation system. Operative time and blood loss were 2.5 and 50 g, respectively. The patient showed a remarkable improvement on chain cystography and in subjective and objective well-being with no complications, even 12 months after surgery. Extraperitoneal laparoscopic bladder neck suspension using the bone-anchor suture fixation system is an easy and feasible procedure.

Adult↗
PubMed1999

Single- and double-strand breaks in solid pBR322 DNA induced by ultrasoft X-rays at photon energies of 388, 435 and 573 eV.

We measured strand breaks of pBR322 plasmid DNA irradiated with ultrasoft X-rays using monochromatic synchrotron radiation as a light source. Three photon energies, 388, 435 and 573 eV, a value below and above the nitrogen K-edge and above the oxygen K-edge, respectively, were chosen for the irradiation experiments as they have an equivalent photon transmittance of the sample. Irradiated DNA was analyzed by agarose gel electrophoresis and the numbers of single- and double-strand breaks (ssb and dsb) were determined by measuring the band intensity on the gel after ethidium bromide staining. The action cross-sections for the ssb and dsb slightly increased with the photon energy. The ratio between 388 and 573 eV was about 1.5 for both forms of strand breaks. The absorbed energy required for a strand break was about 60 eV for ssb and 1 keV for dsb, less than one fifth of the values obtained previously in the 2 keV region. On the other hand, the absorbed energies per strand break, as well as the ratio of the action cross-section for the ssb to that for the dsb, were constant regardless of the photon energy used. The K-shell photoabsorption on carbon, nitrogen and oxygen atoms in the DNA molecule, followed by an Auger cascade, induced DNA strand breaks with a constant efficiency in terms of the absorbed energy. These results indicate that the strand breaks of the DNA molecule in the solid state are mainly caused by the photo- and Auger-electrons and the efficiency of the strand breaks little depends on the atoms ejecting these secondary electrons.

DNA↗
PubMed1999

Immunohistochemical study of a patient with diffuse pulmonary corpora amylacea detected by open lung biopsy.

The chest radiographs of an asymptomatic 58-year-old Japanese man with pulmonary corpora amylacea (PCA) revealed bilateral patchy and nodular infiltrates. Lung specimens obtained by open lung biopsy were histochemically and immunohistochemically analyzed. In all sections of dissected lung tissue, hematoxylin and eosin staining revealed homogeneous eosinophilic acellular round bodies (50-100 microm in diameter) containing granular black pigments in the alveolar spaces. Some round bodies were surrounded and phagocytized by alveolar macrophages. The laminated round bodies stained positively with PAS and Congo red. In addition, many of the rounded bodies contained particles which stained positively with Berlin blue. Immunohistochemical staining for epithelial membrane antigen (EMA) as well as PE-10 was distinctively positive. This is a very rare case of diffuse PCA found by open lung biopsy. Immunohistochemical examination suggested that PCA consisted of pulmonary surfactant protein and epithelial membrane antigen.

Biopsy↗
PubMed1999

High incidence of meningeal infiltration by leukemic cells after infection of chimeric virus between neuropathogenic and non-neuropathogenic retroviruses.

Friend murine leukemia virus (F-MuLV) clone A8, previously shown to cause spongiform degeneration in rat brain, induced leukemia within 10 weeks after infection when inoculated into newborn rats. The chimeric virus Rec2, which contains the pol and env genes of 57 virus on the background of A8 and does not cause spongiform degeneration in the central nervous system (CNS), induced leukemic cell infiltration of the CNS, mainly of the meningeal region, in 58.3% of infected rats. In contrast, A8 induced little or no leukemic cell infiltration of the CNS. Other chimeric viruses containing the LTR and the 5' half of the 5' leader sequence of A8 induced aggressive leukemia, and after infection of these viruses, leukemic cell infiltration of the CNS was only observed in less than 20.0% of the rats. These results indicate that the fragment containing the LTR and the 5' half of the 5' leader sequence of A8 is essential for induction of aggressive leukemia in rats but is not sufficient to cause CNS infiltration. We found that leukemic cell infiltration of the CNS is dependent on the sequence of the virus.

3T3 Cells↗
PubMed1999

[The relationship between abilities in interpersonal relations and interpersonal motivations].

Three hundred and six (306) undergraduates participated in a study that examined abilities in interpersonal relations and interpersonal motivations. Results indicated that: (1) Three interpersonal motivations underlay interpersonal attitudes, namely, other-praise acquisition, other-rejection avoidance, and relationship avoidance. (2) Abilities in interpersonal relations, such as social skills and perceived interpersonal competence, influenced relative strengths among the individual's three interpersonal motivations. High levels of abilities in interpersonal relations lead to stronger motivation to acquire praise by others. In contrast, those with low levels of abilities were motivated to avoid interpersonal relations altogether. Those in-between were motivated to avoid rejection by others.

Female↗