Search PubMed⌕ Search

Biomedical subjects

Y Tani

Publications and source records attributed to Y Tani.

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

Therapeutic effect of novel anti-human Fas antibody HFE7a on graft-versus-host disease model.

In order to evaluate anti-human Fas antibody, we have established a new graft-versus-host disease (GVHD) model wherein splenocytes of human Fas transgenic mice (hFas-TgM) were transferred to immune-deficient SCID mice. In this model, although host SCID cells are not activated by or responsive to graft hFas-TgM cells, graft hFas-TgM cells are activated by and responsive to host SCID cells and thus cause GVHD symptoms. SCID mice that received hFas-TgM splenocytes had increased human Fas-positive lymphocytes in lymph nodes, decreased in body weight, and developed skin diseases, including rash and alopecia. Administration of novel anti-human Fas antibody HFE7A, which did not induce liver toxicity after administration to mice, decreased the level of the human Fas-positive lymphocytes, blocked the decrease of body weight, and suppressed development of skin diseases in this model. These results indicate that induction of apoptosis to activated graft cells with nontoxic anti-Fas antibody could reduce GVHD symptoms.

Acute Disease↗
PubMed2001

Intelligent yeast strains with the ability to self-monitor the concentrations of intra- and extracellular phosphate or ammonium ion by emission of fluorescence from the cell surface.

Saccharomyces cerevisiae strains that respond to environmental changes and transmit the information by emission of fluorescence from the cell surface were constructed. The technique of cell surface engineering enabled the yeast cells to display enhanced cyan blue fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) on the surface under the control of promoters that sense environmental changes. Two model promoters were examined in this study. For monitoring the intra- and extracellular concentrations of phosphate ion, the PHO5 promoter was chosen to display ECFP. The MEP2 promoter was used to display EYFP to sense the concentrations of ammonium ion. Fluorescence was observed by fluorescence microscopy and immunofluorescence microscopy, and the intensity was measured by a flow cytometer. The relationship between ion concentration inside and outside the cells was evaluated by the change in the rate of fluorescence. This S. cerevisiae system enables environmental changes to be transmitted as intra- and extracellular information using a suitable promoter functioning at real time and in a non-invasive manner.

Bacterial Proteins↗
PubMed2001

Chemical force microscopy of self-assembled monolayers on sputtered gold films patterned by phase separation.

Patterned self-assembled monolayers (SAMs) were formed on gold films and observed by friction force microscopy (FFM) and adhesive force mapping with pulsed-force mode atomic force microscopy (PFM-AFM). The substrate gold films were prepared by sputtering gold on flat surfaces of osmium-coated cover glass with surface roughness, Ra, of 0.3 nm. The patterned samples with the CH3 and COOH terminated regions were prepared using the Langmuir-Blodgett (LB) method, partial removal of the LB film by ultrasonication, and SAM formation. The CH3 and COOH terminated regions of the patterned SAMs in air and in water were observed by mapping friction and adhesive forces with FFM and PFM-AFM, respectively, using gold-coated AFM tips chemically modified with a thiol compound terminating in CH3 or COOH. The adhesive forces measured in air increased in the order of CH3/CH3, CH3/COOH (or COOH/CH3) and COOH/COOH, while those in water increased in reverse order. The enormous high adhesive force observed in water for CH3/CH3 was attributed to hydrophobic interaction between the CH3 tip and the CH3 terminated sample surface. With CH3 tip, the lower friction force was observed, however, in water on the CH3 terminated region than on the COOH terminated region. This experimental finding raises a question as to what is the effective normal load in friction measurements in water.

Journal Article↗
PubMed2001

Study of microcontact printed patterns by chemical force microscopy.

Patterned self-assembled monolayers (SAMs) on sputtered gold films prepared by microcontact printing (microCP) were studied by mapping adhesive forces with pulsed-force-mode atomic force microscopy. A stamp for microCP was fabricated by pouring polydimethylsiloxane (PDMS) over a photolithographically prepared master. The patterned SAMs were prepared by two methods. One is called the wet-inking method, in which inking was done by placing a thiol ethanol solution for 30 s on the stamp and then removing the excess ink solution under a stream of nitrogen. The other is called the contact-inking method, in which a pad made of PDMS was dipped overnight in a thiol ethanol solution and then the stamp was placed on the inker pad impregnated with the thiol ethanol solution. The second step for pattern formation was the same for both of the two different microCP methods. Namely, the gold surfaces stamped with alkanethiols were further reacted with a thiol terminating in COOH in ethanol. The resulting patterns with CH3- and COOH-terminated regions were analyzed by imaging the adhesive forces with the chemically modified gold coated AFM tips with a SAM of CH3 or COOH terminal functional groups.

Journal Article↗
PubMed2001

RHC/c genotyping based on polymorphism in the promoter region of the RHCE gene.

Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between RHC/c and RHD were found at positions -468, -304, -58, and -46. On the basis of the nucleotide differences at positions -468 (RHCE vs. RHD) and -292 (RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than either of the methods alone, and therefore, useful and valuable for RHC/c genotyping.

Journal Article↗
PubMed2001

Type 2-biased expression of cytokine genes in lung granulomatous lesions induced by Nippostrongylus brasiliensis infection.

Infections with helminthic parasites occasionally induce pulmonary diseases with possible involvement of immunological mechanisms. In rats infected with the nematode Nippostrongylus brasiliensis, pulmonary granulomatous lesions develop and persist after the larvae have migrated through the lungs. To determine the pathogenesis of this lesion, we examined cytokine gene expression in the lungs using RT-PCR and in situ hybridization. Two weeks after infection, when fully developed lesions appeared, levels of IL-3 and of type2 cytokines IL-4, IL-5, IL-6 and IL-13 gene expression were markedly enhanced in whole lung homogenates. Those of IL-2 and IFN-gamma were also slightly increased 2 weeks postinfection. IL-12 mRNA level did not change after 2 weeks but was slightly increased after 4 weeks. Levels of IL-10 and proinflammatory cytokine TNF gene expression did not show significant changes, although a slight increase was observed in IL-1beta message after 2 weeks. In situ hybridization studies showed that lung granulomatous lesions were composed mainly of lymphoid cells expressing IL-3, IL-4 and IL-13 mRNA, but not IFN-gamma mRNA. IL-5 mRNA-expressing cells were fewer in number than these cells. RMCP II immunohistochemistry revealed that mast cells increased in number in the lung granulomas. From these results, it was concluded that the nematode infection-associated lung granuloma was a type 2 lesion.

Animals↗
PubMed2001

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.

BACKGROUND AND OBJECTIVES: Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti-D antibodies (mAbs) has shown that weak D red cells display type-specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment. MATERIALS AND METHODS: We amplified DNA from four Japanese weak D samples using the polymerase chain reaction (PCR), and directly sequenced the amplified DNA. Using site-directed mutagenesis, we constructed three vectors expressing mutant RHDs-- G212C, V270G (weak D type 1) and G358A (type 2)--in K562 cells. The expression of RhD antigens was examined by flow cytometry using mAbs. RESULTS: A new mutation resulting in a conversion at amino acid residue 212 (Gly to Cys) was detected in a Japanese weak D sample. K562 cells transduced with mutant RhD cDNA reacted weakly in a type-specific manner with mAbs. CONCLUSIONS: The mutations--G212C (new weak D type), V270G (weak D type 1) and G358A (type 2)-- in transmembranous regions had obvious effects on the D epitopes recognized by mAbs. The results of this study provide direct evidence that these mutations can account for weak D phenotypes.

Antibodies, Monoclonal↗
PubMed2001

Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTestTM in breast carcinoma by image analysis.

HercepTestTM (DAKO A/S, Glostrup, Denmark) is an immunohistochemical assay that detects HER2/neu gene products, and evaluates the overexpression status of the HER2/neu protein in determining eligibility for the Trastuzumab (HerceptinR, Genentech, San Francisco, CA, USA) therapy. However, practically, interobserver variability of the HER2/neu interpretation of the immunostained results has caused marked disagreement with regard to the intensity of tumor staining. In this study, we quantitated HER2/neu expression by image analysis, and applied this analyzing system to help to minimize interobserver variability of the interpretation of the HercepTestTM. All the immunostained results were scored semiquantitatively on a range of 0 to 3+ in accordance with the criteria described as per the manufacturer's instructions, and quantitatively evaluated using an image analyzing system with image processing software. Among the 92 cases, 15 were scored as 3+, six were 2+, and 32 were 1+ under intraobservers agreement. When the cases were quantitated, a high correlation was shown between the signal area extracted by image analysis and the corresponding score of staining intensity with the HercepTestTM. By converting the quantitatively extracted data into a scoring system based upon the criteria, the outcome demonstrated a strong concordance with the scoring data obtained from immunostaining. The results indicated that a quantitative scoring system performed by simple image analysis may provide to improve interobserver agreement of the interpretation of the HercepTest TM in clinical practice.

Adult↗
PubMed2001

Concordance in judgments among c-erbB-2 (HER2/neu) overexpression detected by two immunohistochemical tests and gene amplification detected by Southern blot hybridization in breast carcinoma.

We compared the c-erbB-2 protein overexpression status detected by the HercepTestTM (DAKO A/S, Grostrup, Denmark) with another conventional immunohistochemistry system using an anti-c-erbB-2 rabbit polyclonal antibody (Nichirei Co., Tokyo, Japan) and with the c-erbB-2 gene amplification status detected by Southern blot hybridization in 101 surgically resected breast carcinomas. According to the criteria for overexpression, recommended by the manufacturer, c-erbB-2 overexpression by HercepTestTM was detected in 24 cancers (24%), comprising six score-2 tumors and 18 score-3 tumors. The level of agreement in judgment of the HercepTestTM, among three independent observers, was excellent (kappa = 0.845). C-erbB-2 overexpression by Nichirei's antibody and c-erbB-2 gene amplification were detected in 21% and 16% of cases, respectively, and their concordance with HercepTestTM scores of 2-3 was 89% and 90%, respectively. In the score-3 cases by Hercep TestTM only, the concordance rates with overexpression by Nichirei's immunohistochemistry and with gene amplification were slightly higher, 94% and 93%, respectively. Score-2 cases by HercepTestTM were mostly judged as negative overexpression by Nichirei's antibody and as no amplification by Southern blot hybridization. The present results showed that HercepTestTM score-3 detected c-erbB-2 overexpression almost optimally as well as the conventional methods and the score-3 breast carcinomas had clinical and biological implications. Further examination would be necessary to decide the significance of breast cancers of HercepTestTM score 2.

Blotting, Southern↗
PubMed2001

Decreased expression of Bcl-x protein during hepatocarcinogenesis induced exogenously and endogenously in rats.

Dysregulations of apoptosis have been widely recognized as important events in multi-stage carcinogenesis. Bcl-x, a member of the Bcl-2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl-x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short-term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg / kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S-transferase (GST-P)-positive, putative preneoplastic lesions were induced, and Bcl-x protein expression was decreased in 24.7% of such lesions. The incidence of GST-P-positive lesions with decreased Bcl-x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm(2), respectively. In GST-P-positive lesions larger than 0.3 mm(2), both apoptosis induction and cell proliferation activity were enhanced when Bcl-x protein expression was decreased. In the long-term exogenous models, rats were given 10 mg / kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg / kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl-x protein expression. In the endogenous model, rats were fed a choline-deficient, L-amino acid-defined diet for 16 or 80 weeks and sacrificed. Bcl-x protein expression was decreased both in GST-P-positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl-x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.

Alkylating Agents↗
PubMed2001

Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells.

Bufalin, an Na(+)-K(+)-ATPase inhibitor, simultaneously induced cell differentiation and apoptosis in human monocytic leukemia THP-1 cells. In this study, we investigated the regulatory role of protein kinase C (PKC) isozymes in bufalin-induced cell differentiation and apoptosis. A PKC-specific but isozyme-nonselective inhibitor, Ro-31-8220, and a cPKC selective inhibitor, Gö-6976, caused significant attenuation of bufalin-induced interleukin-1beta (IL-1beta) gene expression, a mature monocytic marker, indicating that cPKC participates in the bufalin-induced cell differentiation. On the other hand, cPKCbeta- and nPKCdelta-defective THP-1/TPA cells displayed strong resistance to the bufalin-induced DNA ladder formation. Rottlerin, an nPKCdelta-specific inhibitor, partially attenuated preapoptotic effects of bufalin, such as the limited proteolysis of nPKCdelta and poly(ADP-ribose) polymerase and the cell staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, suggesting that nPKCdelta is involved, at least in part, in bufalin-induced apoptosis. In contrast, Gö-6976 and rottlerin significantly augmented bufalin-induced apoptosis and differentiation, respectively. The findings suggest that bufalin-induced cell differentiation and apoptosis are interlinked and that distinct PKC isozymes are involved in the fate of the cell.

Apoptosis↗
PubMed2001

Glycolaldehyde-forming route in Bacillus subtilis in relation to vitamin B6 biosynthesis.

Glycolaldehyde (GA) was shown to be a precursor of vitamin B6 (B6), and to be formed from glycolate by glycolaldehyde dehydrogenase (GADH) in Escherichia coli. In this study, we show the glycolaldehyde-forming route in B6 biosynthesis in Bacillus subtilis. In the crude extract of B. subtilis, the oxidizing activity of GADH was detected. However, coexisting NADH/NADPH oxidase activity interfered with the determination of the reducing (GA-forming) activity of GADH. NADH/NADPH oxidase was purified and identified as the product of ahpF. In an ahpF disruptant, NADH/NADPH activity was almost eliminated, but the reducing activity of GADH was not detected. We also investigated another possible GA-forming enzyme, glyoxal reductase (GR). GR was partially purified and identified as the product of yvgN. yvgN disruptant did not require B6, and retained the ability to synthesize the same amount of B6 as the wild-type strain. From these results, we concluded that neither GADH nor GR is involved in B6 biosynthesis in B. subtilis.

Journal Article↗
PubMed2001

Stereoselective oxidation of alkylbenzenes by fungi.

Oxygenase is useful when oxygen is to be introduced at a nonactivated carbon-hydrogen bond to give an optically active center. To obtain such an enzyme from microorganisms, we screened soil samples for organisms that assimilated methylethylketone as their sole carbon source. Yeasts and molds that converted ethylbenzene and propylbenzene into their respective oxygenated products during incubation together as resting cells were isolated. One particularly potent strain was identified as Fusarium moniliforme. The fungus oxidized the side chains of ethylbenzene and propylbenzene selectively at the benzylic position. The products were found to be 1-phenylethanol and 1-phenylpropanol, respectively, by GC-MS and HPLC with a chiral column, with 100% enantiomeric excess of the (R-(+)-form.

Journal Article↗
PubMed2001

Degradation of dibenzothiophene by sulfate-reducing bacteria cultured in the presence of only nitrogen gas.

To remove sulfur compounds in petroleum, we isolated sulfate-reducing bacteria that could degrade dibenzothiophene in the presence of only nitrogen gas. Among the 19 strains isolated, some could grow in the presence of 10% (v/v) kerosene and of which two strains were identified as Desulfomicrobium escambium and Desulfovibrio longreachii. Gas chromatography of the ethyl-acetate extract of bacterial cultures, in which 10% or more of the dibenzothiophene initially present was degraded, gave five unknown peaks as the presumable degradation products. Thus, desulfurization of dibenzothiophene could be carried out without oxygen or hydrogen in a pathway different from the anaerobic one already reported, in which biphenyl is detected as the main product.

Journal Article↗
PubMed2001

Optimal conditions for production of (R)-1-phenylpropanol by Fusarium moniliforme strain MS31.

Resting cells of Fusarium moniliforme strain MS31 produced (R)-1-phenylpropanol from propylbenzene. The components of the medium and the reaction conditions were adjusted to increase the specific activity of the hydroxylating enzyme involved. Glucose and sodium nitrate were selected as carbon and nitrogen sources, respectively. The substrate, propylbenzene, inhibited fungal growth and the activity of the enzyme. Acetoin added to the medium increased both growth and activity of the enzyme, and hydroxylation of propylbenzene increased by 1.4-fold. Maximum bioconversion of propylbenzene by resting cells of the fungus was at 25-30 degrees C and pH 7.0 with cells at concentration of 40 mg (dry) per milliliter of reaction mixture. Conversion was accelerated as soon as propylbenzene was added; slowing 2 h later. In the end, F. moniliforme strain MS31 produced (R)-1-phenylpropanol with an enantiomeric excess of 98% at the concentration of 16 mM (2.2 mg.ml(-1)).

Journal Article↗
PubMed2001

Conversion of various aromatic compounds by resting cells of Fusarium moniliforme strain MS31.

Resting cells of Fusarium moniliforme strain MS31 convert propylbenzene to 1-phenylpropanol with high regio- and stereospecificity. To elucidate the scope of substrate acceptability by the fungus, we used various aromatic compounds for the bioconversion. The fungus hydroxylated various alkylbenzenes at the benzylic position to produce optically active alcohols. Butylbenzene was converted to nonbenzylic alcohols. In all cases, the R absolute configuration of products was more abundant. Aromatic compounds with linear side chains and (1-methylethyl)benzene were converted to their corresponding alcohols with an enantiomeric excess of 94% to 100%. Further oxidation of the alcohols was detected, but it was weak.

Journal Article↗
PubMed2001

Enlarged and astaxanthin-accumulating cyst cells of the green alga Haematococcus pluvialis.

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2',7'-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

Journal Article↗