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Biomedical subjects

Y Katakura

Publications and source records attributed to Y Katakura.

At least 19 recordsLinked to original sources

Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray.

To construct yeast strains showing tolerance to high salt concentration stress, we analyzed the transcriptional response to high NaCl concentration stress in the yeast Saccharomyces cerevisiae using DNA microarray and compared between two yeast strains, a laboratory strain and a brewing one, which is known as a stress-tolerant strain. Gene expression dynamically changed following the addition of NaCl in both yeast strains, but the degree of change in the gene expression level in the laboratory strain was larger than that in the brewing strain. The response of gene expression to the low NaCl concentration stress was faster than that to the high NaCl concentration stress in both strains. Expressions of the genes encoding enzymes involved in carbohydrate metabolism and energy production in both strains or amino acid metabolism in the brewing strain were increased under high NaCl concentration conditions. Moreover, the genes encoding sodium ion efflux pump and copper metallothionein proteins were more highly expressed in the brewing strain than in the laboratory strain. According to the results of transcriptome analysis, candidate genes for the creation of stress-tolerant strain were selected, and the effect of overexpression of candidate genes on the tolerance to high NaCl concentration stress was evaluated. Overexpression of the GPD1 gene encoding glycerol-3-phosphate dehydrogenase, ENA1 encoding sodium ion efflux protein, and CUP1 encoding copper metallothionein conferred high salt stress tolerance to yeast cells, and our selection of candidate genes for the creation of stress-tolerant yeast strains based on the transcriptome data was validated.

Gene Expression Regulation, Fungal↗

Symptoms in relation to chemicals and dampness in newly built dwellings.

OBJECTIVES: As the airtightness of dwellings has recently increased, problems associated with indoor air pollution and dampness have become important environmental health issues. The aim of this study was to clarify whether symptoms in residents living in newly built dwellings were related to chemicals and dampness. METHODS: Symptoms of 317 residents were surveyed by standardized questionnaires, and the concentrations of formaldehyde, acetaldehyde, and 17 volatile organic compounds (VOCs) in their homes were measured. Dampness (condensation on window panes and/or walls, and mold growth) was identified by questionnaires given to the householders or their partners. RESULTS: Some VOCs (toluene, butyl acetate, ethylbenzene, alpha-pinene, p-dichlorobenzene, nonanal, and xylene) were significantly related to the symptoms, and the sum of all VOCs (all identified VOCs) was significantly related to throat and respiratory symptoms [odds ratio (OR) for eye symptoms =2.4; 95% confidence interval (CI) 1.0-5.5], although the concentrations of VOCs were relatively low. As for the dampness index, condensation on window panes and/or walls was related to all symptoms, and mold growth was related to all symptoms except skin, throat and respiratory and general symptoms. As the number of dampness signs increased, the ORs increased for the symptoms except general symptoms (OR for nose symptoms = 4.4, 95% CI 1.6-11.9). CONCLUSION: Both VOCs and dampness were significantly related to symptoms. We should take measures to reduce the concentrations of VOCs, dampness and microbial growth in dwellings.

Acetaldehyde↗

Relation between colour vision loss and occupational styrene exposure level.

AIMS: To investigate the relation between colour vision loss and the exposure level of styrene. Exposure level included the current exposure concentration, past cumulative exposure, and the maximum exposure level in the past. METHODS: Colour vision was examined by the Lanthony desaturated panel D-15 test for 76 subjects exposed to styrene in a fibreglass reinforced plastics boat plant (as an exposed group) and 102 non-exposed subjects (as a control group). The current exposure level was expressed by the concentration of atmospheric styrene and end shift urinary mandelic acid (MA) and phenylglyoxylic acid (PGA) levels. The individual cumulative exposure index (CEI) was calculated, based on the exposure frequency and urinary MA concentrations measured for the past eight years. RESULTS: The Colour Confusion Index (CCI) of the exposed group showed a significant difference from the age matched controls. However, only a slight significant relation was found between CCI and the concentration of urinary MA plus PGA. In this study, the exposed group was further divided into two subgroups (as sub-MA+PGA groups) by the median of urinary MA plus PGA of each subject. The dividing line between the subgroups was 0.24 g/g creatinine, which was equivalent to an atmospheric concentration of styrene of about 10 ppm. The CCI values of both the sub-MA+PGA groups were significantly higher than that of the control group. The relation between CCI value and the maximum exposure concentration in the past eight years was examined. It was found that the CCI values of the group with the maximum exposure concentration of styrene over 50 ppm were significantly higher than that of the other groups. CONCLUSIONS: Exposure to styrene would impair colour vision even if the exposure concentration was lower than 10 ppm. Furthermore, if the maximum concentration of styrene exposure transiently exceeded 50 ppm in the past, the styrene related damage might remain. Thus, the safe limit of exposure to styrene and the relation between exposure to styrene and the degree of damage to ocular structure, retina, optic nerve, and brain need to be re-examined.

Adult↗

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.

Animals↗

Effects of prenatal styrene exposure on postnatal development and brain serotonin and catecholamine levels in rats.

Maternal reproductive effects in Wistar rats exposed to 0, 50, or 300 ppm styrene for 6 h/day during gestational days 6 to 20 were evaluated. Their offspring were observed postnatally for neurochemical changes, growth, and physical landmarks of development. Mothers exposed to styrene were compared with pair-fed and ad-lib-fed controls in order to adjust nutrient conditions. Prolongation of the gestational period, food intake, and the number of neonatal deaths or stillbirths in 300-ppm-exposed dams showed evidence of styrene-related effects. Other reproductive parameters, such as litter size, birth weight, and sex ratio, were found to exhibit no effects within the variation range studied. A neurochemical effect was observed in that the 5-HT and HVA concentrations in cerebrum were significantly decreased. Incisor eruption (mandible), eye opening, and the air-righting reflex were delayed in rat pups born to dams receiving 300 ppm styrene exposure compared with the pair-fed and ad lib control groups. Pups born to dams exposed to 50 ppm styrene also had a significantly delayed air-righting reflex compared with ad lib controls. These results suggest that the offspring were susceptible to the effects of styrene on a few developmental landmarks even when nutritional effects were controlled.

Animals↗

Effects of low-level occupational exposure to styrene on color vision: dose relation with a urinary metabolite.

To investigate the threshold effects of chronic low-level occupational exposure to styrene on color vision, we examined color discrimination in 105 male workers exposed to styrene (mean age 37.7 years; mean length of exposure 6.2 years; mean urinary concentration of mandelic acid 0.21 g/L) and in 117 referents (mean age 37.7 years). We also assessed the effects of styrene by examination of the nature of the relation between disorders of nervous function and age, alcohol consumption, and other variables. A standardized questionnaire was adopted to collect information about work history, occupational or nonoccupational solvent exposure, alcohol consumption, and drug use. Color vision was evaluated by the Lanthony desaturated panel D-15 test. The results of the test were expressed as the color confusion index (CCI). There was a dose-dependent relationship between the urinary concentration of mandelic acid and color vision loss. The CCIs of the subgroups whose urinary mandelic acid levels were 0.1-0.2 and >0.2 g/L were significantly higher than those of each referent group (P<0.05 and P<0.01, respectively), but not in the subgroup whose urinary mandelic acid level was lower than 0.1 g/L. Our study suggests that a low level of styrene, presumably 0.1-0.2 g/L, involves the risk of inducing adverse effects on color vision. After confounding factors were adjusted for, the urinary mandelic acid level had a significant positive relationship with color vision.

Adult↗

Meningeal carcinomatosis in patients with breast cancer: report of 8 patients.

BACKGROUND: Meningeal carcinomatosis (MC) is an uncommon but aggressive complication of advanced breast cancer with a recently increasing incidence. Although the prognosis is extremely poor for MC patients, early diagnosis and appropriate treatment are important. SUBJECTS AND METHODS: We reviewed 8 cases of MC from breast cancer at Kyoto University Hospital from 1990 to 1999. The median age was 51.5 years. All patients had widespread systemic metastases when diagnosed with MC. clinical symptoms were categorized into 3 groups: cranial nerve symptoms, spinal nerve symptoms, and other symptoms. Imaging studies were positive for MC in only 4 patients. Initial CSF cytology studies were positive in 4 patients, and repeated CSF cytology yielded positive results in the remaining 4 patients. Thus the median interval between the onset of any clinical symptom of MC and the initiation of treatment was 22.5 days (range 7 to 120 days ). All patients received whole brain radiotherapy (WBRT). Four patients were given intrathecal chemotherapy and/or intrathecal immunotherapy in addition to WBRT. RESULTS: Improvement of cranial nerve symptoms, spinal nerve symptoms, and other symptoms were observed in 3/5, 1/3, and 5/7 patients, respectively. Patients with cranial nerve symptoms who started WBRT within 29 days of the onset of the symptoms showed at least partial recovery whereas patients who started WBRT later showed no recovery. The median survival was 123 days (53 to 310 days). MC was the direct cause of death in 1 of 8 patients. CONCLUSION: When MC is clinically suspected, neither a negative imaging study nor a single negative CSF cytology can rule out MC. Prompt initiation of WBRT with or without intrathecal chemotherapy may be important for recovery from cranial nerve symptoms.

Adult↗

Risk factors for human alveolar echinococcosis: a case-control study in Hokkaido, Japan.

A case-control study to determine the risk factors for human alveolar echinococcosis (AE) was conducted in Hokkaido, Japan. The 134 cases were either patients clinically diagnosed as having AE (N =61) or other individuals found to be seropositive for Echinococcus multilocularis although showing no abnormalities in ultrasound scans (N = 73). Two groups of case-matched controls were employed: one of 670 individuals selected from a list of residents of Hokkaido (group A) and the other of 650 subjects selected from a list of individuals who had been found seronegative in earlier investigations (group B). Because of the long latency of AE, potential risk factors were studied over two different periods: for the 0-10 and 11-20 years before the year of diagnosis/recorded seropositivity. The results of multivariate analyses, in which the cases were compared with each control group, indicated that the rearing of cattle and pigs and the use of well water were significant risk factors and that the use of tap water significantly decreased the risk of an individual having AE. Extension of the piped water supply and health education on improving the disposal of household rubbish (to keep foxes away from houses) should help reduce the risk of AE infection in the study area.

Animal Husbandry↗

The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc.

Reduction in klotho gene expression causes accelerated senescence in klotho mutant mice. We have now found two key substances, phosphorus and zinc, which affect the appearance of klotho phenotypes. Klotho mutant homozygotes fed nonpurified diet with a phosphorus concentration of 1.03 g/100 g showed typical klotho phenotypes. However, most of the klotho phenotypes no longer appeared in male homozygotes fed a 0.4 g/100 g phosphorus diet. These homozygotes were capable of spermatogenesis. In the kidneys of the rescued male homozygotes, klotho protein expression was clearly detected. On the other hand, female klotho mice required supplementation of 0.25 g/100 g zinc orotate to the 0.4 g/100 g phosphorus diet to be rescued. Unlike in the rescued male mice, klotho protein levels in the kidneys of the rescued females were quite low. Wild-type (C3H/He) mice fed 1.5 or 1.0 g/100 g phosphorus diets had lower klotho protein expression in the kidneys than those fed a 0.4 g/100 g phosphorus diet (Kruskal-Wallis test, P < 0.05). These results indicate that dietary phosphorus and zinc modulate the phenotypes of klotho mice, and that klotho expression in the kidneys is regulated not only in klotho mutant mice, but also in wild-type mice.

Aging↗

Constrained optimization of L-lysine production based on metabolic flux using a mathematical programming method.

Constrained optimization for microbial fermentation was studied. For optimization, we used not the maximum principle but a nonlinear programming method because of the need to consider many metabolic reactions. In the case of L-lysine fermentation, the optimization problem in L-lysine production was formulated as a nonlinear programming problem. In general, the state equations based on material balances are represented as differential equations, but such equations which are dependent on time can not be applied to a nonlinear programming problem. Therefore, the state equations were made discrete in a time base, and a new single vector which is not dependent on time was substituted. From these formulae, the objective function and the constraints using nonlinear programming problem were defined as the amount of L-lysine produced, and as a metabolic reaction model and empirical equations, respectively. Computer program was developed to solve this constrained nonlinear programming problem. The applied algorithm of the computer programming was a sequential quadratic programming method (SQP method). When the constrained nonlinear programming problem is solved using the SQP method, the maximum amount of L-lysine produced and the optimal feeding rate of L-threonine could be calculated. From the calculated results, it was clear that introduction of the equality and inequality constraints was easy. L-Lysine at a concentration up to 75.3 g/l could be produced when the fermentation was carried out under optimal conditions.

Journal Article↗

A kinetic model for a biopanning process considering antigen desorption and effective antigen concentration on a solid phase.

A phage display library is a powerful tool for screening ligands such as antibodies and peptides that specifically recognize a target. In this study, we established a kinetic model describing the affinity selection process of phage display libraries and verified the model experimentally. Desorption of target molecules from a solid phase and orientation of the epitopes of adsorbed target molecules are taken into account in this model. The ratio of the effective antigen density to the total antigen density was estimated to be 0.0127(+/-)0.0018 when bovine pancreatic ribonuclease A (RNase A) adsorbed on polystyrene beads was recognized by an anti-RNase A single-chain Fv phage antibody. The model can faithfully describe the recovery of the phage antibody in a round of biopanning based on the effective concentration of RNase A on the beads, the desorption rate constant of RNase A from the beads, the dissociation constant and dissociation rate constant of the phage antibody from RNase A, and the time for blocking, equilibrium and washing in the biopanning process. A recommended biopanning protocol based on the model is also described.

Journal Article↗

Measurement of association rate constant of antibody-antigen interaction in solution based on enzyme-linked immunosorbent assay.

A simple and rapid method based on enzyme-linked immunosorbent assay (ELISA) was developed for measuring the association rate constant of antibody-antigen interactions. An antibody and its antigen are mixed in a solution to initiate the equilibrium reaction. At different time intervals, the amount of the free antibody in the reaction mixture is estimated by an indirect ELISA. The association rate constant was estimated by nonlinear regression against an equation introduced from the derivation of the mass balance of antigen-antibody interaction. This method can determine the association rate constant of antibodies with a dissociation rate constant up to 5 x 10(-3) s(-1). The association rate constant of a single-chain Fv (scFv) to its antigen, bovine pancreatic ribonuclease A (RNase A), determined by the present method agreed well with those determined by the fluorescence polarization method and surface plasmon resonance method. No significant difference in the association rate constant was found between the soluble anti-RNase A scFv and the same scFv displayed on a phage (5.65 +/- 0.54 x 10(4) M(-1) s(-1) and 5.96 +/- 0.56 x 10(4) M(-1) s(-1), respectively).

Journal Article↗

In vitro selection of hematoporphyrin binding DNA aptamers.

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX.

Base Sequence↗

Efficient production of desulfurizing cells with the aid of expert system.

An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 5l jar fermentor with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20mmolkg-drycells(-1)h(-1) and of 45g-drycellsl(-1), respectively. At 100h of cultivation, 1g/l of dibenzothiophene (DBT) was converted to 2HBP within 20h, i.e., 10mmolkg-drycells(-1)h(-1) of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment.

Journal Article↗

Increase in desulfurization activity of Rhodococcus erythropolis KA2-5-1 using ethanol feeding.

Rhodococcus erythropolis KA2-5-1 is one of the best strains for the desulfurization of dibenzothiophene (DBT) via a sulfur-specific pathway in which DBT is converted to the end product, 2-hydroxybiphenyl, by releasing sulfite via DBT-sulfone and 2-(2'-hydroxyphenyl) benzene sulphinate. The objective of this research is to develop a culture method in order to attain a high cell density with a high level of specific desulfurization activity. Compared with glucose or glycerol, ethanol was found to be a preferable carbon source for obtaining a high specific activity (SA) of desulfurization. When the amount of DBT fed was restricted by feeding 2.9 mg-DBT/g-ethanol solution, the maximum SA and final cell concentration were 135.5 (mmol-2HBP/kg-dry cell weight-h) and 37 (g-dry cell weight/l), respectively. On the other hand, when glucose or glycerol was used as a carbon source, the SA was lower than 50 (mM-2HBP/kg-dry cell weight-h) and the final cell concentration was also lower than 27 (g-dry cell weight/l). The activities of the desulfurization enzymes in R. erythropolis KA2-5-1 grown on ethanol were remarkably higher than when the strain was grown on glucose or glycerol. It was also suggested that NADH, which is produced by the biochemical reaction of NAD with ethanol catalyzed by alcohol dehydrogenase, might contribute to the conversion of FMN to FMNH2, which is a coenzyme for the activities of desulfurization enzymes.

Journal Article↗

Control of antibody-antigen interaction using anion-induced conformational change in antigen peptide.

The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions. We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)n and (LLKK)n. In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel alpha-helical structure and the epitope was subsequently 'bent'. In the presence of 41 microM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5. Oligo-DNAs, as anions, dissociated the antibody-peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody-peptide complex at pH 7.5 were 4x10(-8), 1x10(-7) and 6x10(-6) M for decamer, octamer and hexamer DNA, respectively.

Amino Acid Sequence↗

Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris.

A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the alpha-factor signal peptide known for high secretion efficiency. P. pastoris expressed the human lysozyme at about 300 mg/l broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lysozyme). To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed. The calorimetric studies showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (DeltaH). On the basis of structural information on the EAEA-lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues.

Calorimetry, Differential Scanning↗

L-Lysine production by exponential feeding of L-threonine.

The effect of L-threonine feeding in the production phase on L-lysine production by Brevibacterium flavum, which requires L-homoserine or L-threonine for cell growth, was investigated considering the concerted inhibition by L-threonine plus L-lysine, and the metabolism related to lysine production. Exponential feeding of L-threonine increased L-lysine production to 70 g/l about three times that without feeding. From the analysis of the metabolic flux, carbon flux of L-lysine synthesis pathway in the production phase after L-threonine feeding was higher than that in the growth phase. The results show that feeding of an inhibitory substance may increase the production, especially when the substance is necessary for the continuation of cell growth and/or production.

Journal Article↗