Biomedical subjects

Y Kurosawa

Publications and source records attributed to Y Kurosawa.

At least 19 recordsLinked to original sources

PubMed2003

Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein.

Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.

Archaeal Proteins↗

PubMed2003

Serodiagnosis of Japanese encephalitis among Nepalese patients by the particle agglutination assay.

Japanese encephalitis (JE) is a serious health problem in the southwestern region of Nepal. Serological diagnostic kits for routine diagnostic use in this region have not been available. This study was performed to examine if the particle agglutination (PA) assay for Japanese encephalitis virus (JEV) IgM could be applicable to the samples collected in Nepal and also to evaluate the accuracy of clinical diagnosis of JE. One hundred and ninety-three blood samples were collected from the patients clinically diagnosed with JE or other infectious diseases in the JE-endemic, southwestern region of Nepal, in 2000. The PA assay was performed on these 193 serum samples and the results were compared with those by IgM-capture ELISA. Eighty-six samples were IgM-positive by the PA assay, and 71 of 86 were also positive by IgM-capture ELISA (sensitivity, 99%; specificity, 88%; positive predictive value, 0.82; negative predictive value, 0.99). These results suggest that the PA assay is a simple, reliable and useful diagnostic test to support clinical diagnosis in rural hospitals of Asia including Nepal.

Agglutination Tests↗

PubMed2001

Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Amino Acid Sequence↗

PubMed2001

Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease gene.

Huntington's disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human exon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100-week-old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)-positive cells have been found to be increased in the substantia nigra (SN), globus pallidus (GP), and striatum (St) in the brains of 40-week-old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP-positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats.

Age Factors↗

PubMed2001

Activation of autoreactive T cells that help nucleobindin-injected mice produce anti-DNA antibodies.

Nucleobindin (Nuc) is a DNA- and calcium-binding protein that was originally identified as an anti-DNA antibody-enhancing factor in MRL/MpJ-lpr/lpr (MRL/lpr) mice. In MRL/lpr mice, both expression of Nuc mRNA in enlarged lymph nodes and serum concentration of Nuc protein are shown to increase as disease progresses. Administration of recombinant (r) Nuc to young MRL/MpJ-+/+ and normal BALB/c mice leads to augmentation or induction of IgG anti-double-stranded (ds) DNA autoantibodies. In this study, spleen cells prepared from MRL/lpr mice were found to show a proliferative response to rNuc, indicating existence of T cells that are specific to this autoantigen in peripheral lymphoid tissues. Furthermore, CD4+ T cell lines were generated from a BALB/c mouse that had been producing anti-dsDNA antibodies as a result of repeated injections of rNuc. These T cell lines were confirmed to be autoreactive, because they proliferated in culture with syngeneic but not with allogeneic spleen cells without addition of any exogenous antigens. Their proliferation was enhanced by rNuc, and inhibited by an anti-MHC class II monoclonal antibody. Upon in vivo inoculation, these T cells provided help for rNuc-injected BALB/c mice to produce anti-DNA antibodies. These results suggest that Nuc is able to activate autoreactive peripheral T cells through an MHC-class II pathway leading to acceleration or induction of anti-DNA antibody production when it is excessively produced in lupus-prone mice or experimentally administered into normal mice.

Animals↗

PubMed2001

Genetic relationship amongst the major non-coding regions of mitochondrial DNAs in wild boars and several breeds of domesticated pigs.

We completed phylogenetic analysis of the major non-coding region of the mitochondrial DNA (mtDNA) from 159 animals of eight Euro-American and six East Asian domesticated pig breeds and 164 Japanese and five European wild boars. A total of 62 mtDNA haplotypes were detected. Alignment of these regions revealed nucleotide variations (including gaps) at 73 positions, including 58 sites with transition nucleotide substitutions, and two transversion substitutions. Phylogenetic analysis of the sequences could not organize domestic pig breeds into discrete clusters. In addition, many of the haplotypes found in members of diverged clustering groups were found primarily in Euro-American pig breeds, indicating extensive introgression of Asian domestic pigs into European breeds. Furthermore, phylogenetic analysis allocated the DNA sequences of non-coding regions into two different groups, and the deepest branchpoint of this porcine phylogeny corresponded to 86 000-136 000 years before present. This time of divergence would predate the historical period when the pig is thought to have been domesticated from the wild boar.

Animals↗

PubMed2001

Fluvastatin inhibits O2- and ICAM-1 levels in a rat model with aortic remodeling induced by pressure overload.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression is suggested to play an important role in the pathogenesis of vascular remodeling. The aim of the present study was to investigate the effects of the 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitor fluvastatin on superoxide anion (O2-) production and ICAM-1 expression in a rat model with vascular remodeling induced by pressure overload. Two weeks after aortic banding, marked increases in O2- production and ICAM-1 protein levels were observed in the aorta. O2- formation and ICAM-1 immunoreactivity were mainly increased in the endothelium and adventitia of the aorta in banded rats. Oral administration of fluvastatin prevented both these changes and the development of perivascular fibrosis and increased the expression of endothelial nitric oxide synthase. Cholesterol and lipid peroxide levels in serum did not change in the banded rats. Thus the beneficial effects of fluvastatin seen in this study as well as its cholesterol-lowering effect may contribute to attenuate the atherosclerotic process.

Animals↗

PubMed2001

Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise.

The purpose of this study was to examine the validity of the quantitative measurement of muscle oxidative metabolism in exercise by near-infrared continuous-wave spectroscopy (NIRcws). Twelve male subjects performed two bouts of dynamic handgrip exercise, once for the NIRcws measurement and once for the (31)P-magnetic resonance spectroscopy (MRS) measurement as a standard measure. The resting muscle metabolic rate (RMRmus) was independently measured by (31)P-MRS during 15 min of arterial occlusion at rest. During the first exercise bout, the quantitative value of muscle oxidative metabolic rate at 30 s postexercise was evaluated from the ratio of the rate of oxyhemoglobin/myoglobin decline measured by NIRcws during arterial occlusion 30 s after exercise and the rate at rest. Therefore, the absolute values of muscle oxidative metabolic rate at 30 s after exercise [VO(2NIR(30))] was calculated from this ratio multiplied by RMRmus. During the second exercise bout, creatine phosphate (PCr) resynthesis rate was measured by (31)P-MRS at 30 s postexercise [Q((30))] under the same conditions but without arterial occlusion postexercise. To determine the validity of NIRcws, VO(2NIR(30)) was compared with Q((30)). There was a significant correlation between VO(2NIR(30)), which ranged between 0.018 and 0. 187 mM ATP/s, and Q((30)), which ranged between 0.041 and 0.209 mM ATP/s (r = 0.965, P < 0.001). This result supports the application of NIRcws to quantitatively evaluate muscle oxidative metabolic rate in exercise.

Adenosine Triphosphate↗

PubMed2001

Angiotensinogen gene variation and hypertension in a cohort study in Japanese.

Many recent case-control studies have suggested a significant relationship between M235T (the substitution of threonine for methionine at position 235 codon) polymorphism of the angiotensinogen (AGT) gene and hypertension. To investigate whether the M235T polymorphism of AGT gene affects the incidence of hypertension, a retrospective cohort study was performed among Japanese workers. The subjects were Japanese workers at an occupational site in Shimane Prefecture in Japan. The baseline data were set at the received regular health examination in 1992, and a retrospective cohort study was performed for analyzing the incidence of hypertension in 1998. The rates of M235M (MM), M235T (MT) and T235T (TT) genotypes were 4%, 32% and 64%, respectively. The relative risks of MT and TT against MM for the incidence of hypertension by single variance analysis were 1.47 [95% confidence interval (CI) 0.50 - 4.33] and 1.35 (95% CI 0.47 - 3.90), respectively. The relative risks of MT and TT against MM for the incidence of hypertension, adjusted for sex, age, body mass index, fasting glucose and cigarette smoking, drinking and exercise in 1992, were 1.49 (95% CI 0.49 - 4.53) and 1.25 (95% CI 0.42 3.74), respectively. The data from this study suggest that the M235T polymorphism of AGT gene has a weak role in the manifestation of hypertension. Further comprehensive studies are needed to resolve this issue.

Adult↗

PubMed2001

[Study on the effects of walking on functional fitness in aged patients with the hand-arm vibration syndrome].

PURPOSE: This article reports the effectiveness of mild walking exercise for maintaining functional fitness in aged patients with the hand-arm vibration syndrome (HAVS). SUBJECTS AND METHODS: Fifty-two patients suffering from vibration syndrome, for which they received annual compulsory examination from December 1998 to March 1999 at the San-in Rosai Hospital, were examined. They all were male, with a mean age (standard deviation) of 69.1 (7.3) years, and were randomly allocated to an intervention group (N = 26) and a control group (N = 26). The goal of the intervention was to achieve and maintain at least 30 minutes of walking a day. Functional fitness was assessed by a sitting and standing test, a zigzag walking test, a hand working test with a pegboard for dexterity, and a self-care working test proposed by the Physical Fitness Research Institute, Meiji Life Foundation of Health and Welfare. RESULTS: There were no significant differences between the two groups regarding baseline characteristics. The proportions of subjects with regular exercise habits after the intervention were 84.6% (22/26) and 53.8% (14/26) in the intervention and control groups, respectively, the difference being significant. The total scores for functional fitness were improved in the intervention, while decline was noted in the control group. Sitting and standing and self-care working ability were also improved in the intervention group as against the deterioration with age in the control group. CONCLUSIONS: Our findings show to some extent that intervention using mild exercise, walking for 30 min, is effective for aged patients with HAVS to maintain and improve functional fitness.

Aged↗

PubMed2000

Effects of imidapril and captopril on streptozotocin-induced diabetic nephropathy in mice.

We investigated whether the prevention of the development of diabetic nephropathy by angiotensin-converting enzyme inhibitors is associated with decreases in renal angiotensin-converting enzyme activity and/or blood pressure in diabetic mice. C57Bl/6 mice were injected with streptozotocin (200 mg/kg, i.v.) and randomized to receive either imidapril (1 and 5 mg/kg) or captopril (10 and 50 mg/kg) or vehicle by gavage for 28 days. Each assay was performed on 8-10 mice from each treatment. At 28 days after the start of drug treatment, imidapril and captopril significantly reduced blood pressure of the diabetic mice, and this effect of captopril was stronger than that of imidapril. On the other hand, inhibition of renal angiotensin-converting enzyme activity by imidapril was stronger than that by captopril. Imidapril and captopril dose-dependently inhibited urinary albumin excretion to similar extents, but they failed to inhibit the renal hypertrophy and elevation of creatinine clearance. Total renal angiotensin-converting enzyme activity was significantly reduced in diabetic mice, but immunohistochemical localization of angiotensin-converting enzyme was intensive in the vasculature and glomeruli of the diabetic kidney. In conclusion, both effects on blood pressure and angiotensin-converting enzyme activity may be involved in the prevention of development of diabetic nephropathy by imidapril and captopril in streptozotocin-induced diabetic mice. The data suggest that the degrees of contribution of their effects on blood pressure and renal angiotensin-converting enzyme activity to the inhibition of urinary albumin excretion may be different between the two angiotensin-converting enzyme inhibitors.

Albuminuria↗

PubMed2000

Quantification of ischemic muscle deoxygenation by near infrared time-resolved spectroscopy.

The purpose of this study was to quantify muscle deoxygenation in human skeletal muscles using near infrared time-resolved spectroscopy (NIRTRS) and compare NIRTRS indicators and blood saturation. The forearm muscles of five healthy males (aged 27-32 yrs.) were monitored for changes in hemoglobin saturation (SO2) during 12 min of arterial occlusion and recovery. SO2 was determined by measuring the temporal profile of photon diffusion at 780 and 830 nm using NIRTRS, and was defined as SO2-TRS. Venous blood samples were also obtained for measurements of SvO2, and PvO2. Interstitial PO2(PintO2) was monitored by placing an O2 electrode directly into the muscle tissue. Upon the initiation of occlusion, all parameters fell progressively until reaching a plateau in the latter half of occlusion. It was observed at the end of occlusion that SO2-TRS (24.1 +/- 5.6%) agreed with SvO2 (26.2 +/- 6.4) and that PintO2 (14.7 +/- 1.0 Torr) agreed with PvO2 (17.3 +/- 2.2 Torr). The resting O2 store (oxygenated hemoglobin) and O2 consumption rate were 290 microM and 0.82 microM s-1, respectively, values which reasonably agree with the reported results. These results indicate that there was no O2 gradient between vessels and interstisium at the end of occlusion.

Adult↗

PubMed2000

Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

Animals↗

PubMed2000

Geographical variation of mandible size and shape in the Japanese wild pig (Sus scrofa leucomystax).

The mandibles of the Japanese wild pigs (Sus scrofa leucomystax) from various geographical locations, which have been stored in the University of Tokyo and National Science Museum, Tokyo were examined. The mandibles from northern localities were larger in size than those from southern localities. The Oita population was significantly smaller than the Honshu populations. The Mie population was smaller than the Hyogo population in length from the angle of the mandible. In the multivariate analysis, the Kyushu populations could be obviously distinguished from the Honshu populations in the principal component charts. However, each Honshu population did not show the locality-specific distribution of the principal component plots.

Animals↗

PubMed1999

Acute myeloid leukemia with hypergranular cytoplasm accompanied by t(X;11)(q24;q23) and rearrangement of the MLL gene.

This report describes a unique case of acute myeloid leukemia with hypergranular cytoplasm and t(X;11)(q24;q23). The breakpoint on 11q23 was identified within the MLL gene. The hypergranular cytoplasm of leukemic cells and the associated coagulopathy resembled a characteristic of acute promyelocytic leukemia, despite the absence of RARalpha gene rearrangement in this case. The Xq24 site possibly played a role in this atypical blast phenotype.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1999

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.

Amino Acid Sequence↗

PubMed1999

Functional and binding studies of insurmountable antagonism of 606A, a novel AT1-receptor antagonist in rabbit tissues.

Angiotensin AT1-receptor antagonists can be classified into two types, surmountable and insurmountable ones, based on the way they inhibit angiotensin II (AII)-induced vasoconstriction. To elucidate the causes of the difference, we studied how several antagonists associate with and dissociate from AT1-receptor sites by using rabbit adrenal cortical membrane. Four antagonists, 606A, EXP3174, CV11974, and E4177, showed equipotent competitive antagonism when they were added simultaneous with [125I]-AII in binding experiments. However, in AII-induced contraction studies with rabbit aorta, 606A, EXP3174, and CV11974 inhibited the contraction noncompetitively, whereas E4177 inhibited competitively. The longer the pretreatment period with EXP3174 or CV11974, the more effectively the antagonists suppressed AII-induced contraction. However, the suppression of contraction by 606A and E4177 changed little with the length of the pretreatment period. AII-induced contraction of 606A- or E4177-treated aorta recovered easily by washout, but that of CV11974-treated aorta was hard to recover by washout. These results obtained in the aorta were consistent with their characteristics observed in the AII binding study in the rabbit adrenal cortical membrane in cases of EXP3174 and CV11974. The differences between association rate with and dissociation rate from the AT1 receptor of E4177 and 606A were slight, in spite of the clear difference between their action in the contraction study. Because of the variations observed with the four compounds, mechanisms of insurmountable antagonism may not be uniform among AT1-receptor antagonists.

Angiotensin II↗

PubMed1999

Conservation and diversification of MHC class I and its related molecules in vertebrates.

The elucidation of the complete peptide-binding domains of the highly polymorphic shark MHC class I genes offered us an opportunity to examine the characteristics of their predicted protein products in the light of the latest advance in the structural studies of the MHC class I molecules. The results suggest that the fundamental characteristics in the T-cell recognition of the MHC class I molecule/peptide complex are expected to have been established at the early stage of the vertebrate evolution. The elucidation of the typical classical class I molecules from fishes and also of some MHC class I-related molecules may help us-to explore the common denominator of the ancient class I molecules.

Amino Acid Sequence↗