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H Toyoda

Publications and source records attributed to H Toyoda.

At least 19 recordsLinked to original sources
PubMed2000

Search for second and third generation leptoquarks including production via technicolor interactions in p&pmacr; collisions at radicals = 1.8 TeV

We report the results of a search for second and third generation leptoquarks using 88 pb(-1) of data recorded by the Collider Detector at Fermilab. Color triplet technipions, which play the role of scalar leptoquarks, are investigated due to their potential production in decays of strongly coupled color octet technirhos. Events with a signature of two heavy flavor jets and missing energy may indicate the decay of a second (third) generation leptoquark to a charm (bottom) quark and a neutrino. As the data are found to be consistent with standard model expectations, mass limits are determined.

Journal Article↗
PubMed2000

Structural analysis of glycosaminoglycans in animals bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila homologues of vertebrate genes encoding glycosaminoglycan biosynthetic enzymes.

Mutations that disrupt developmental patterning in Drosophila have provided considerable information about growth factor signaling mechanisms. Three genes recently demonstrated to affect signaling by members of the Wnt, transforming growth factor-beta, Hedgehog, and fibroblast growth factor families in Drosophila encode proteins with homology to vertebrate enzymes involved in glycosaminoglycan synthesis. We report here the biochemical characterization of glycosaminoglycans in Drosophila bearing mutations in sugarless, sulfateless, and tout-velu. We find that mutations in sugarless, which encodes a protein with homology to UDP-glucose dehydrogenase, compromise the synthesis of both chondroitin and heparan sulfate, as would be predicted from a defect in UDP-glucuronate production. Defects in sulfateless, a gene encoding a protein with similarity to vertebrate N-deacetylase/N-sulfotransferases, do not affect chondroitin sulfate levels or composition but dramatically alter the composition of heparin lyase-released disaccharides. N-, 6-O-, and 2-O-sulfated disaccharides are absent and replaced entirely with an unsulfated disaccharide. A mutation in tout-velu, a gene related to the vertebrate Exostoses 1 heparan sulfate co-polymerase, likewise does not affect chondroitin sulfate synthesis but reduces all forms of heparan sulfate to below the limit of detection. These findings show that sugarless, sulfateless, and tout-velu affect glycosaminoglycan biosynthesis and demonstrate the utility of Drosophila as a model organism for studying the function and biosynthesis of glycosaminoglycans in vivo.

Amidohydrolases↗
PubMed2000

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard.

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.

Carbohydrate Sequence↗
PubMed2000

Destabilization of tumor necrosis factor-alpha mRNA by 5-alpha dihydrotestosterone in Jurkat cells.

The effect of a male steroid hormone, 5DHT, on the expression of TNF-alpha was examined using a human leukemia T cell line, Jurkat. Cells were treated with 5DHT in the presence or absence of PHA, and RNA was isolated followed by a reverse transcriptase - mediated PCR (RT-PCR) to measure the steady state levels of TNF-alpha mRNA. The treatment of cells with 5DHT resulted in a 50% of decrease in the level of TNF-alpha mRNA compared to that in untreated conditions (basal level). A similar level of reduction of the message by 5DHT was also observed in PHA-stimulated cells. The reduction of the steady state levels of TNF-alpha mRNA in Jurkat cells was a result of destabilization of the gene as demonstrated by actinomycin D treatment; a half-life of TNF-alpha message in 5DHT treated cells and non-treated cells was 1 hr and 2.5 hr, respectively, whereas that in 5DHT/PHA and PHA-treated cells was 3hr and 6hr, respectively.

DNA Primers↗
PubMed2000

Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes.

Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.

Amphiregulin↗
PubMed2000

Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo.

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.

Animals↗
PubMed2000

The effect of retreatment with interferon-alpha on the incidence of hepatocellular carcinoma in patients with chronic hepatitis C.

BACKGROUND: Interferon (IFN) has been reported to have beneficial long term effects that reduce the occurrence of hepatocellular carcinoma (HCC), even in patients who do not have complete responses to IFN. The authors evaluated the effect of retreatment with IFN-alpha on the long term prognoses of those with incomplete responses to their initial IFN-alpha treatment. METHODS: Among 271 patients with incomplete responses to initial IFN-alpha treatment who had received sufficient dose and duration (a total dose of more than 350 megaunits administered over a period longer than 12 weeks) between October 1989 and September 1997, 63 patients received retreatment and 208 did not. The authors retrospectively compared the incidence of HCC between patients who received retreatment and those who did not. RESULTS: There were no significant differences in the clinical characteristics between these two groups. The cumulative incidence of HCC was significantly lower among the patients who had retreatment, and retreatment with IFN-alpha was the only factor that correlated with the lower incidence of HCC in multivariate analysis. The results were similar when the 12 patients with complete responses to retreatment were excluded from the analysis. CONCLUSIONS: Retreatment with IFN-alpha appeared to have the additional effect of suppressing the development of HCC in patients who had incomplete responses to the initial treatment, even when the hepatitis C virus was not cleared (i.e., a complete response was not achieved) with retreatment. Further prospective study is required.

Adolescent↗
PubMed2000

Comparison of characteristics between patients with GB virus C/hepatitis G virus (GBV-C/HGV) RNA and those with GBV-C/HGV E2-antibody in patients with hemophilia.

We compared characteristics of patients with GB virus C/hepatitis G virus (GBV-C/HGV) RNA to those of patients with GBV-C/HGV E2-antibody. GBV-C/HGV RNA and GBV-C/HGV antibody were assayed in 83 persons with hemophilia using a reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. GBV-C/HGV RNA was detected in 19 (22.9%) patients and GBV-C/HGV antibody was detected in 17 (20.5%). The background characteristics between the patient groups did not differ with respect to age, severity of hemophilia based on the frequency of use of blood product, and both the initial age at the first use and years since the first use of blood products. There were no differences in coinfection with hepatitis C virus (HCV) and/or human immunodeficiency virus, except that infection with HCV subtype 1a was more prevalent in patients with GBV-C/HGV RNA (P = 0.0229). Human lymphocyte antigen (HLA) typing was conducted in 18 patients with GBV-C/HGV RNA and 15 patients with GBV-C/HGV E2-antibody; 13 of the patients with GBV-C/HGV antibody had either HLA DQ7, DR15, or DR8, whereas only 4 of the patients with GBV-C/HGV RNA did (P < 0. 001). It is concluded that the presumed age at the time of GBV-C/HGV infection, the frequency of exposure to GBV-C/HGV, and the time since the GBV-C/HGV infection were not associated with recovery from infection with GBV-C/HGV. Coinfection with HCV subtype 1a may be related to persistent GBV-C/HGV viremia, whereas HLA DQ7, DR15, or DR8 may be related to the clearance of GBV-C/HGV after infection.

Adolescent↗
PubMed2000

A hairy root culture of melon produces aroma compounds.

Musk melon is the favorite fruit with a high market value in Japan, and the fragrance is one of the major factors determining the fruit quality of melon. In this study, mutant melon hairy roots which had been induced by means of the T-DNA insertion mutagenesis were found to produce volatile compounds with the fruity fragrance of mature melon. The volatile compounds were extracted and identified by GLC-mass spectrometry. Some essential oils such as (Z)-3-hexenol, (E)-2-hexenal, 1-nonanol, and (Z)-6-nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. The productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits.

Cucurbitaceae↗
PubMed2000

Cyclin D1 expression is useful as a prognostic indicator for advanced esophageal carcinomas, but not for superficial tumors.

The purpose of the present study was to define the overexpression of cyclin D1 in superficial and advanced esophageal carcinomas and to investigate whether the expression of this molecule indicates a poor prognosis. This study included 41 patients with superficial esophageal carcinomas (Tis and T1) and 48 patients with advanced esophageal carcinomas (T2, T3, and T4). The expression of cyclin D1 in surgically resected specimens was evaluated immunohistochemically with a monoclonal antibody. Positive immunoreactivity was found in 31 of 89 cases (35%). Overexpression of cyclin D1 did not correlate with TNM classification or histologic type. Of the 48 patients with advanced esophageal carcinomas, 32 patients with cyclin D1-negative tumors survived longer than did 16 patients with cyclin D1-positive tumors (P = 0.0017). In contrast, we observed no survival difference between patients with cyclin D1-positive and -negative superficial esophageal carcinoma. These results suggest that cyclin D1 indicates a poor prognosis in cases of advanced esophageal carcinoma but not in cases of superficial esophageal carcinoma.

Biomarkers, Tumor↗
PubMed2000

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

Animals↗
PubMed1999

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans.

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.

Anticoagulants↗
PubMed1999

Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.

We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.

Enzyme Inhibitors↗
PubMed1999

PF1070A, a novel and potent inducer of the synthesis of metallothionein.

Using mouse Ltk(-) cells (L13-17 cells) that had been transfected with a plasmid in which the lacZ gene had been ligated downstream of 1.4 kbp of the sequence of the promoter of the mouse gene for metallothionein-I (MT-I) as a reporter gene, we examined 268 organic compounds for the ability to activate this promoter. We found that PF1070A, an antibiotic produced by Humicola sp., efficiently activated the MT promoter and caused marked enhancement of beta-galactosidase activity in L13-17 cells. The extent of activation by PF1070A was almost equivalent to that by of zinc ions, the most effective known inducer of the synthesis of MT. PF1070A also caused marked elevation of the levels of the mRNA for MT and of MT itself in L13-17 cells. A similar result was obtained in human HeLa-S3 cells. When PF1070A was added to the culture medium simultaneously with cadmium ion or dexamethasone, the level of expression of the reporter gene was markedly elevated, compared to the level of expression induced by each agent independently. The effect of PF1070A was reduced considerably by deletion of nucleotides at positions -150 and -149 from the site of initiation of transcription in the promoter region of the MT gene and also by deletion of the seven bases located at positions -49 to -43. Since no known cis element was found in these two regions, PF1070A might be a new type of inducer of MT synthesis that promotes expression of the gene for MT via a mechanism completely different from those exploited by other known agents. These results also suggest the presence of a system for control of transcription of the gene for MT that has not previously been recognized. Both cadmium ions and bismuth ions induce the synthesis of MT by acting on the metal response element (MRE). Bismuth ions had no significant effect on the promoter activity that had already reached a maximum level in response to treatment with the optimal concentration of cadmium ion. By contrast, PF1070A further and markedly increased the promoter activity. This result suggests that it is possible to increase the concentration of MT in tissue using PF1070A as an inducer even in cases where the MRE-mediated activation of the MT promoter has already been induced by the accumulation of cadmium, as is the case in a clinical setting. PF1070A may prove to be an excellent inducer of MT synthesis that is effective and clinically applicable. Moreover, use of PF1070A in combination with salts of heavy metals might be useful in controlling expression of a transfected gene that is regulated by the MT promoter since PF1070A can activate the MT promoter to an extent that cannot be achieved with heavy metal ions alone, when PF1070A is used in combination with zinc ions at a concentration of the latter considerably below the toxic level.

Animals↗