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Biomedical subjects

T Toyoda

Publications and source records attributed to T Toyoda.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Tuberculosis of the elderly (above the age of 75) in national hospitals].

In Japan tuberculosis is becoming rapidly the disease of the elderly. We studied the background, the type and level of hospital cares needed, and the outcome of patients with pulmonary tuberculosis (sputum smear and/or culture positive) above the age of 75 who were admitted to 8 national hospitals during the period from January 1 to December 31, 1997. The study included 150 patients (male: 109, female: 41, mean age: 81.6), of whom 25% needed care in a single-bed room, 84.3% had underlying diseases (cardiovascular diseases: 43.3%, malignant diseases: 20.9%, neuro-psychiatric diseases: 13.4%), and 47.6% needed cares mainly in feeding and excretions. 92 patients (62.6%) improved and 45 patients (30.6%) died, of whom the cause of death was directly related to tuberculosis in 42. The mean hospital stay was 4.7 months. However, in 42 patients whose cause of death was related to tuberculosis, 66% died within 3 months, while in 102 patients who were discharged 71% stayed more than 3 months. The same comparison was done in 508 patients with bacteriologically proven tuberculosis above the age of 75 admitted to National Tokyo Hospital during the period from 1990 to 1999. The result was almost the same, among 133 patients died in the hospital 60% died within 3 months, while in 375 patients who were discharged hospital stay was more than 3 months in 70%. In the near future, the elderly will occupy more than 25% of the beds of the tuberculosis ward in Japan and most of them have underlying diseases other than tuberculosis. Because tuberculosis, once the disease of the young, is becoming rapidly the disease of the elderly, it is imperative for us to make necessary adjustments to meet this inevitable trend.

Aged↗
PubMed2000

Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme.

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

Animals↗
PubMed2000

Defective terminal differentiation and hypoplasia of the epidermis in mice lacking the Fgf10 gene.

Here, we characterized the skin and hair phenotype of mice lacking the fibroblast growth factor 10 gene (Fgf10), a newly identified member of the fibroblast growth factor family. Histological examination of Fgf10(-/-) newborn mouse skin revealed abnormalities in epidermal morphogenesis. The number of proliferating cells in the basal layer was decreased, the granular layer was hypoplastic and lacked distinctive keratohyaline granules and tonofibrils. The expression of loricrin, a marker of epidermal differentiation, was dramatically reduced. Despite the presence of Fgf10 transcripts in normal hair follicles, abnormalities of hair development were not observed in Fgf10(-/-) skin. These data suggest that Fgf10 is required for embryonic epidermal morphogenesis but is not essential for hair follicle development.

Animals↗
PubMed2000

Epitope mapping of the influenza A virus RNA polymerase PA using monoclonal antibodies.

To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101-400 and three bound to that of amino acids 518-600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein.

Animals↗
PubMed2000

Mapping of functional domains on the influenza A virus RNA polymerase PB2 molecule using monoclonal antibodies.

Monoclonal antibodies against the PB2 of A/Puerto Rico/8/34 (A/PR/ 8/34) (H1N1) were prepared in order to define the functional domains of the RNA polymerase of influenza virus. The fifteen monoclonal antibodies that were generated were divided into 4 groups on the basis of ELISA binding to PB2 or its peptide fragments. Six Group I antibodies that bound to the PB2 N-terminal region (amino acids 1-104) did not inhibit transcription by the viral ribonucleoprotein complex. A single Group II antibody recognizing the region of amino acids 206-259 inhibited ApG-primed transcription. Groups III and IV antibodies bound to the C-terminal region of amino acids 660-759. Of these, Group III antibodies inhibited transcription. The present results identify multiple monoclonal antibody binding domains in PB2, two of which, when bound by antibodies, negatively affect viral RNA transcription.

Amino Acid Sequence↗
PubMed2000

Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments.

Polypeptide release factor one from Thermus thermophilus, ttRF1, was purified and subjected to crystallization. Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction. Stable fragments of ttRF1 suitable for crystallization were screened by limited proteolysis. Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry. They were N-terminal fragments generated by proteolysis at amino acid positions 211, 231 and 292. The corresponding recombinant polypeptides, ttRF1(211), ttRF1(231) and ttRF1(292), were overproduced and subjected to crystallization. Of these polypeptides, ttRF1(292) gave rise to crystals that belong to P3(1) (or P3(2)) space group with unit cell parameters a = b = 64. 5 A, c = 86.6 A and diffract up to 7 A resolution.

Amino Acid Sequence↗
PubMed2000

Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch.

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

Alleles↗
PubMed2000

Influenza virus-induced encephalopathy: clinicopathologic study of an autopsied case.

BACKGROUND: Rapid progressive encephalopathy with a high fever, consciousness loss and recurrent convulsions has been occasionally reported in children during influenza pandemics in Japan since 1995. We examined a 2-year old girl with hemorrhagic shock and encephalopathy syndrome associated with acute influenza A virus infection (A/Nagasaki/76/98; H3N2), to answer several questions for which no histologic or virologic data exist. METHODS: A clinicopathologic study using immunohistochemical staining and viral genome detection by reverse transcriptase polymerase chain reaction (RT-PCR) was performed with this autopsied case. RESULTS: The virus antigen was positive in CD8+ T lymphocytes from the lung and spleen. The virus infected a very limited part of the brain, especially Purkinje cells in the cerebellum and many neurons in the pons, without inducing an overt immunologic reaction from the host. The RT-PCR used for detecting the hemagglutinin gene demonstrated positive bands in all frozen tissues and cerebrospinal fluid taken at autopsy and not in samples obtained on admission. CONCLUSIONS: The pathologic change induced by the direct viral invasion cannot be responsible for all of the symptoms, especially for the rapid and severe clinical course of the disease within 24-48 h after the initial respiratory symptoms. Together with the rapid production of several inflammatory cytokines, the breakdown of the blood-brain barrier may induce severe brain edema and can be a major pathologic change for the disease. Any therapeutic strategy to control this multistep progression of the disease could be effective.

Antigens, Viral↗
PubMed2000

Stabilization of human recombinant erythropoietin through interactions with the highly branched N-glycans.

Human erythropoietin (EPO) produced in Chinese hamster ovary cells (CHO-EPO) is a hydrophobic protein stabilized by the highly branched complex-type N-glycans. To characterize the stabilizing effect of the N-glycans, the properties of enzymatically N-glycan-modified CHO-EPO species were compared spectrophotometrically. CD and fluorescence spectra following the protein unfolding induced by guanidine hydrochloride or pH revealed that the inner regions including the galactose residues of the N-glycans stabilize the protein conformation. The decrease in the conformational stability caused by enzymatic trimming of the N-glycans was associated with the exposure of the hydrophobic protein surface areas accessible to 1-anilino-8-naphthalenesulfonic acid (ANS) binding. Further, the ANS binding and heat denaturation of Escherichia coli-expressed EPO (nonglycosylated EPO) were depressed in dilute solutions (1 mM or so) of free N-glycans of the complex type. These results, together with the finding that the N-glycans of CHO-EPO make little contact with the aromatic amino acid residues exposed on the protein surface, indicate that the inner regions including the galactose residues of the intramolecular N-glycans stabilize the protein conformation by clinging to the hydrophobic protein surface areas mainly made up of nonaromatic hydrocarbon groups.

Animals↗
PubMed2000

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies.

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.

Amino Acid Sequence↗
PubMed2000

Induction of tolerance against ischemia/reperfusion injury in the rat brain by preconditioning with the endotoxin analog diphosphoryl lipid A.

OBJECT: Inflammatory responses and oxygen free radicals have increasingly been implicated in the development of ischemic brain injury. In some cases, an attenuation of inflammation or free-radical injury can provide tissue protection. Diphosphoryl lipid A (DPL) is a detoxified derivative of a lipopolysaccharide (endotoxin) of Salmonella minnesota strain R595, which is capable of stimulating the immune system without eliciting direct toxic effects. In this study the authors examined the influence of preconditioning with DPL on ischemia/reperfusion injury in rats. METHODS: Sprague-Dawley rats were injected intravenously with either DPL or vehicle. Twenty-four hours later, some animals were tested for superoxide dismutase (SOD) activity. Others were subjected to a 3-hour period of focal cerebral ischemia and, after a reperfusion period of 24 hours, were killed. Infarction volume, SOD activity, and myeloperoxidase (MPO) activity were assayed in the postischemic animals. Pretreatment with DPL produced significant reductions in cerebral infarction and MPO activity in the ischemic penumbra. A significant enhancement of basal SOD activity was observed 24 hours after DPL treatment (that is, before ischemia), and a further enhancement of SOD activity was seen in the ischemic penumbra 24 hours after reperfusion. CONCLUSIONS: These data provide the first evidence of a neuroprotective effect of preconditioning with DPL in an in vivo model of cerebral ischemia. Although the precise mechanisms through which DPL exerts its neuroprotective influence remain to be established, an inhibition of the complex inflammatory response to ischemia and an enhancement of endogenous antioxidant activity are leading candidates.

Animals↗
PubMed2000

No association of borna disease virus with psychiatric disorders among patients in northern Kyushu, Japan.

There is controversy over the prevalence of Borna disease virus (BDV) antibodies and its RNA in the peripheral blood mononuclear cells (PBMCs) of psychiatric patients, and the contribution of BDV to human psychiatric disorders. We examined 299 plasma and 229 PBMC samples. No plasma samples were positive for BDV-p40, p24 or gp18 antibodies by western blot analysis. The prevalence of BDV RNA in the psychiatric (schizophrenic) patients (1.8%) was not significantly different from that in the healthy volunteers (0.6%). The nucleotide sequences of BDV p40 and p24 were highly conserved with those of BDV He/80. Our results suggested that there is a lack of association between BDV infection and psychiatric disorders among the patients in Northern Kyushu, Japan.

Adolescent↗
PubMed1999

Substitution of amino acid residue in influenza A virus hemagglutinin affects recognition of sialyl-oligosaccharides containing N-glycolylneuraminic acid.

Sialic acids are essential components of cell surface receptors used by influenza viruses. To determine the molecular mechanisms of viral recognition of two major species of sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we tested the binding reactivity of nine human H3 influenza A viruses to sialylglycolipids containing type II sugar chain and different molecular species of terminal sialic acids. All human H3 viruses tested except A/Memphis/1/71 bound both Neu5Ac and Neu5Gc. Nucleotide sequence analysis suggests that amino acids at 143, 155, and 158 are linked to the viral recognition of Neu5Gc.

Amino Acid Sequence↗
PubMed1999

Molecular mapping of influenza virus RNA polymerase by site-specific antibodies.

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1-159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501-617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1-159); the central region (amino acids 305-559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1-222) of PB1; and amino acids 301-517 and 601-716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402-559, where one of the cap-binding domains mapped previously overlapped.

Animals↗
PubMed1999

Sequence, expression in Escherichia coli, and characterization of lysophospholipase II.

Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form lysophospholipase named lysophospholipase II from mouse embryo. The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of lysophospholipase I previously cloned from rat liver (H. Sugimoto et al., J. Biol. Chem. 271 (1996) 7705-7711). The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24794, and showed 64% identity to that of lysophospholipase I with the Gly-X-Ser-X-Gly esterase/lipase consensus. The lacZ fusion protein expressed in E. coli cells exhibited lysophospholipase activity and reacted with antibody raised against previously purified pig gastric lysophospholipase II (H. Sunaga et al., Biochem. J. 308 (1995) 551-557), but not with antibody against rat liver lysophospholipase I. The expressed enzyme was purified to a specific activity of 0.15 micromol/min per mg by DEAE-Sepharose A-500 chromatography. The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid. Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that lysophospholipase II transcript as well as lysophospholipase I transcript was widely distributed in mouse tissues.

Amino Acid Sequence↗