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Biomedical subjects

K Yoshioka

Publications and source records attributed to K Yoshioka.

At least 181 records · Page 10Linked to original sources
PubMed1997

Adiabatic compressibility of flagellin and flagellar filament of Salmonella typhimurium.

The partial specific volume and adiabatic compressibility of flagellin, its F40 fragment deprived of the disordered terminal regions, from Ala-1 to Arg-65 and from Ser-451 to Arg-494, and the flagellar filament of Salmonella typhimurium were determined from the density and the sound velocity measurements at 15 degrees C. The partial specific volumes were 0.728 cm3/g, 0.745 cm3/g, and 0.734 cm3/g, and the partial specific adiabatic compressibilities were 4.0 x 10(-12) cm2/dyn, 6.7 x 10(-12) cm2/dyn, and 4.7 x 10(-12) cm2/dyn, for flagellin, F40, and the filament, respectively. The smaller values of flagellin than those of F40 are reasonably explained by the presence of disordered terminal regions, which are supposed to be highly hydrated by water molecules. The volume increase upon polymerization of flagellin into the filament is also confirmed by depolymerization under a high pressure. The smaller volume and compressibility of the filament compared with those of F40 suggest an extensive hydration of the filament on its complex surface structure, which surpasses the effect on the volume and compressibility by a possible increase in the cavity volume at intersubunit interfaces upon polymerization.

Circular Dichroism↗
PubMed1997

Inducible nitric oxide synthase expression and erythropoietin production in human hepatocellular carcinoma cells.

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.

Base Sequence↗
PubMed1997

Long-term potentiation and depression in layer III and V pyramidal neurons of the cat sensorimotor cortex in vitro.

Synaptic plasticity of the cat sensorimotor cortex was examined intracellularly in vitro. After tetanic stimulation of the white matter, layer III and V pyramidal neurons showed long-term potentiation (LTP) of EPSPs in high incidence without GABA(A) antagonist. The incidence and magnitude of LTP were very conspicuous in layer V cells. After an NMDA receptor antagonist application, the synaptic potentiation was blocked completely in layer III but not in layer V cells. Long-term depression (LTD) of the evoked EPSPs was also induced by the same stimulation in some layer III cells, where a transient hyperpolarization of the membrane potential was observed during tetanus.

2-Amino-5-phosphonovalerate↗
PubMed1997

2.1 kb 5'-flanking region of the brain type dystrophin gene directs the expression of lacZ in the cerebral cortex, but not in the hippocampus.

Duchenne muscular dystrophy is a muscle-wasting disease accompanied by a variable, but often significant degree of mental retardation, possibly due to the absence of dystrophin. However, the function of brain type dystrophin remains insufficiently clear. With this background, in order to study the cell-specific regulation of brain type dystrophin expression in mice, we generated transgenic mice carrying the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene, fused to the coding region of the bacterial lacZ gene. Three transgenic mice lines showed lacZ expression in the cerebral cortex. However, lacZ expression was not detected in the CA region of the hippocampus. These results suggest that the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene contains the regulatory element required for its expression in the cerebral cortex, but not in the hippocampus.

Animals↗
PubMed1997

Peroxynitrite induces the conversion of xanthine dehydrogenase to oxidase in rabbit liver.

Effect of peroxynitrite (ONOO-) on the conversion of xanthine dehydrogenase to oxidase in rabbit liver was examined. ONOO- (25-200 microM) induced the conversion of xanthine dehydrogenase to oxidase in a dose-dependent manner. The addition of hydroxyl radical scavengers (mannitol and dimethyl sulfoxide) gave no alteration in the ONOO(-)-induced conversion of xanthine dehydrogenase to oxidase, implying that the action of ONOO- is not due to hydroxyl radicals which may be formed from ONOO-. The experiment utilizing dithiothreitol also revealed that the action of ONOO- might be due to oxidation of sulfhydryl group of xanthine dehydrogenase. These results suggest that ONOO- has the potential to convert xanthine dehydrogenase to oxidase, and that this effect may be correlated with cytotoxic actions of ONOO-.

Animals↗
PubMed1997

Imaging Plasmodium falciparum-infected ghost and parasite by atomic force microscopy.

Atomic force microscopy was used to image the membrane cytoskeleton network of normal and P. falciparum-infected ghosts. The membrane cytoskeleton network was examined in air-dried ghost preparations from normal and infected cells. We found that the spectrin network was changed in infected ghosts. The thickness of the normal red cell membrane was about 15.05 +/- 2.27 nm, while the thickness of the P. falciparum-infected membrane was found to be 22.97 +/- 3.84 nm. The ghost containing ring stage parasites exhibited areas of particle-like protrusions ranging in size from 0.2 to 0.7 micron. The surface of the P. falciparum parasite was also imaged in air-dried samples, showing the existence of a large protrusion extending from the parasite surface.

Animals↗
PubMed1997

Urinary transforming growth factor-beta in patients with glomerular diseases.

We measured the urinary levels of active transforming growth factor-beta (TGF-beta) by enzyme-linked immunosorbent assay in 12 healthy controls and 42 patients with various glomerular diseases, including mesangial proliferative (IgA nephritis, Henoch-Schönlein purpura nephritis, and IgA-negative mesangial proliferative glomerulonephritis) and non-proliferative (minimal change nephrotic syndrome and focal glomerulosclerosis) types. Urinary TGF-beta, expressed as a ratio to urinary creatinine (ng/mg creatinine), was elevated in patients with IgA nephritis and focal glomerulosclerosis, and was significantly higher than in patients with other types of glomerular diseases and healthy controls. There was a significant correlation between urinary TGF-beta levels and the grade of interstitial fibrosis. Among patients with proliferative-type disease, urinary TGF-beta was significantly correlated with the grade of mesangial matrix increase and the magnitude of proteinuria. The relationship between urinary TGF-beta levels and the immunostaining intensity of TGF-beta in the glomeruli was not significant. These results indicated that urinary TGF-beta reflects the grade of interstitial fibrosis in glomerular diseases and also the mesangial matrix increase in proliferative-type glomerulonephritis. Measuring TGF-beta levels in the urine might be helpful in monitoring patients with some types of glomerular disease.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

Tachykinin receptors on motoneurons in the spinal cords of neonatal rats, gerbils and hamsters.

As a step to clarify the profiles of tachykinin receptors in the mammalian central nervous system, we examined the effects of various tachykinin receptor agonists and antagonists on motoneurons in isolated spinal cord preparations from rats, gerbils and hamsters. After treatment with tetrodotoxin, potential changes were recorded extracellularly from lumbar ventral roots at 27 degrees C. Bath-application of tachykinin NK1, NK3 receptor agonists produced depolarizing responses of ventral roots. In contrast, selective NK2 agonists exerted no or only marginal depolarizing action. Neurokinin A (NKA), however, exerted a distinct depolarizing action on motoneurons. Tachykinin NK1 receptor antagonists antagonized the actions of SPOMe and NKA in a competitive manner. The present results suggest that tachykinin NK1 and NK3 receptors are present on spinal motoneurons of newborn rats, gerbils and hamsters, and that NKA acts on the NK1 receptors.

Animals↗
PubMed1997

Enzymatic inactivation of enkephalin neurotransmitters in the spinal cord of the neonatal rat.

The possible involvement of enzymatic degradation in the inactivation of enkephalins in the spinal cord of neonatal rats was investigated electrophysiologically and biochemically. In an isolated spinal cord-saphenous nerve preparation, electrical stimulation of the saphenous nerve evoked a slow depolarization lasting 20-30 s of the ipsilateral L3 ventral root. This slow depolarization was depressed by a mixture of peptidase inhibitors, consisting of actinonin (10 microM), thiorphan (0.6 microM), bestatin (10 microM), arphamenine B (10 microM) and captopril (10 microM). Naloxone (0.5 microM) not only reversed this effect of the mixture of peptidase inhibitors but also potentiated the slow depolarization beyond the pre-control level. In an isolated spinal cord preparation, electrical stimulation of a lumbar dorsal root evoked a slow depolarization of the contralateral ventral root of the same segment. This slow depolarization was depressed by application of [Met5]enkephalin in a dose dependent manner. This effect of [Met5]enkephalin was markedly potentiated by addition of the mixture of peptidase inhibitors. Among the five peptidase inhibitors, actinonin, thiorphan or bestatin alone potentiated the depressant effect of [Met5]enkephalin, whereas arphamenine B and captopril did not. Membrane fractions prepared from neonatal rat spinal cords showed degrading activities for [Met5]- and [Leu5]enkephalins and these activities were inhibited by the mixture of peptidase inhibitors. Among the five peptidase inhibitors, actinonin and thiorphan markedly inhibited the [Met5]enkephalin-degrading activity while bestatin was less effective. Arphamenine B and captopril were ineffective. The present results suggest that enzymatic degradation by peptidases plays a role in the termination of the transmitter action of enkephalins in the neonatal rat spinal cord. The present results, together with our previous results on the enzymatic degradation of tachykinins in a study in which we used the same preparations, suggest that similar but distinct combinations of peptidases are involved in the inactivation of enkephalin and tachykinin neurotransmitters.

Animals↗
PubMed1997

Role of reactive oxygen species in cis-dichlorodiammineplatinum-induced cytotoxicity on bladder cancer cells.

This study was undertaken to investigate the intracellular induction of reactive oxygen species (ROS) by cis-dichlorodiammineplatinum (CDDP) and the augmentation of their cytotoxicity in bladder cancer cells (KU7) by enhancement of ROS generation by the glutathione (GSH) depletors buthionine sulphoximine (BSO) and diethylmaleate (DEM). CDDP-induced cytotoxicity in KU7 cells and its modulation by GSH depletors were determined using spectrophotometric measurement with crystal violet staining. The effects of GSH depletors on intracellular GSH levels were confirmed using the GSH reductase-DTNB recycling method. Intracellular ROS generation induced by CDDP with or without GSH depletors was estimated from the amount of intracellular dichlorofluorescein (DCF), an oxidized product of dichlorofluorescein (DCFH), which was measured with an anchored cell analysis and sorting system. The cytotoxic effects of CDDP (IC50 15.0 +/- 2.5 microM) were significantly enhanced by BSO (IC50 9.3 +/- 2.6 microM, P < 0.01) and DEM (IC50 10.3 +/- 0.3 microM, P <0.01). BSO and DEM produced a significant depletion in intracellular GSH levels (9.6 +/- 0.4 nmol 10(-6) cells, 17.9 +/- 1.0 nmol 10(-6) cells) compared with the controls (30.5 +/- 0.6 nmol 10(-6) cells). Intracellular DCF production in KU7 cells treated with CDDP (1.35 +/- 0.33 microM) was significantly enhanced by the addition of BSO (4.43 +/- 0.33 microM) or DEM (3.12 +/- 0.22 microM) at 150 min. These results suggest that ROS may play a substantial role in CDDP-induced cytotoxicity and that GSH depletors augment its cytotoxicity through an enhancement of ROS generation in bladder cancer cells.

Antimetabolites↗
PubMed1997

Serum levels of IL-10, IL-15 and soluble tumour necrosis factor-alpha (TNF-alpha) receptors in type C chronic liver disease.

We previously reported that the number of TNF-alpha-producing cells was increased in the liver of patients with type C chronic liver disease. To understand further the pathophysiology of this change, we examined serum levels of two soluble TNF receptors, TNF-alphaRI (p55) and -alphaRII (p75), and IL-10, all of which act as TNF-alpha buffer, and IL-15, a novel cytokine sharing many immunological activities with IL-2, using ELISA methods. We studied control individuals and patients with type C chronic liver disease, including asymptomatic hepatitis C virus (HCV) carriers with persistently normal serum ALT values, and those with chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Both types of sTNF-alphaR closely correlated with disease progression. Patients with LC and HCC had significantly elevated levels for sTNF-alphaRII compared with the other patient groups and controls. Serum IL-10 levels were significantly greater in all chronic liver disease groups than in controls. With respect to IL-15, the values were high in CH, LC and HCC compared with those of controls. Notably, HCC patients showed highest values for both IL-10 and IL-15, with significant differences from the other patient groups. Serial determinations revealed that interferon (IFN) treatment for CH patients resulted in the suppression of circulating IL-10 and IL-15 levels along with decrease in serum aminotransferase values. Both cytokines remained at decreased levels after cessation of therapy in patients who went into clinical and virological remission. On the other hand, treatment did not affect serum levels of sTNF-alphaRs. These findings indicate that serum levels of these molecules correlated with disease progress in chronic HCV infection, and that IL-10 and IL-15 may reflect the degree of inflammation in the liver. It is also suggested that both cytokines may be related to the development of HCC.

Adult↗
PubMed1997

Production of interleukins 10 and 12 by peripheral blood mononuclear cells (PBMC) in chronic hepatitis C virus (HCV) infection.

We previously reported that interferon-gamma (IFN-gamma) production by PBMC in response to HCV core protein was increased in patients with type C chronic liver disease. To understand better the pathophysiology of this disease, we evaluated production of IL-10 and IL-12 by PBMC from 41 patients with chronic HCV infection, including asymptomatic HCV carriers with persistently normal serum ALT values. IL-10 is known to inhibit many effector functions of the immune system, suppressing Th1-type cell development, while IL-12 stimulates differentiation of Th1-type cells, facilitating cell-mediated immunity. IL-10 production was determined by culturing lymphocytes with concanavalin A (Con A), while IL-12 was produced by monocytes in the presence of Staphylococcus aureus Cowan 1 (SAC) with or without recombinant HCV core protein, respectively. The cytokine levels in culture supernatants were measured by ELISA. Spontaneous IL-10 production was greater in patients with chronic hepatitis (CH) (229 +/- 119 pg/ml, P < 0.01) and liver cirrhosis (LC) (185 +/- 88 pg/ml, P < 0.05) than in controls (119 +/- 27 pg/ml), while it was decreased during IFN treatment (70 +/- 25 pg/ml). Both HCV core protein and Con A enhanced IL-10 production by cells from HCV-infected patients. IL-12 was not detectable in medium alone cultures, and SAC-induced IL-12 production did not differ between various patient groups and controls. Simultaneous addition of HCV protein resulted in an increase of IL-12 production in chronic liver disease compared with SAC-alone cultures. Addition of IL-10 to the cultures equally suppressed IFN-gamma production for both controls and patient groups, but the enhancing effect of IL-12 on IFN-gamma production was significantly less in LC than in controls and other patient groups. The findings suggest that secretion of IL-10/IL-12 by cells from control individuals and various patient groups may be different, and that the cytokines might show different effects on IFN-gamma production by some cells.

Adult↗
PubMed1997

A 900 bp genomic region from the mouse dystrophin promoter directs lacZ reporter expression only to the right heart of transgenic mice.

In order to study the regulatory mechanism of developmental and tissue-specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZ gene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.

Animals↗
PubMed1997

Humoral immune response to the hypervariable region of hepatitis C virus differs between genotypes 1b and 2a.

Antibody to the hypervariable region (HVR) of HCV is thought to have neutralizing activity. The HCV genotype is known to affect the clinical course of infection. The antibody response to HVR and its relationship to the virologic and clinical characteristics were investigated in 21 patients with chronic hepatitis C. HVRs amplified by polymerase chain reaction from serum HCV were expressed as glutathione S-transferase fusion proteins. From 8 to 34 clones per serum sample were obtained (375 clones total), and the anti-HVR antibody in serum was assessed by Western blot. Both the incidence of fusion proteins positive for anti-HVR antibody and the activity of antibody were significantly higher in 6 patients with genotype 2a than in 15 patients with genotype 1b. This result suggests that the quantity of humoral response to HVR affects the clinical outcome of infection with these two HCV genotypes.

Adult↗
PubMed1997

Difference in affinity for DNA between HMG proteins 1 and 2 determined by surface plasmon resonance measurements.

High mobility group (HMG) proteins 1 and 2 contain two similar but non-identical repeats of DNA-binding domains and an acidic C-terminal. The proposed functions of HMG proteins 1 and 2 imply a probable difference in their DNA-binding abilities. The primary studies by gel retardation assay showed that HMG2 has higher affinity than HMG1 for supercoiled and linear DNA. The DNA-binding of HMG2 appeared strong enough to allow exchange with HMG1 molecule already bound to DNA, while the DNA-binding region of HMG1 showed higher affinity than that of HMG2. In order to compare more quantitatively the affinities, surface plasmon resonance (SPR) measurements using a BIAcore instrument were conducted. The kinetic data indicated that the Kd for the complex of HMG2 with DNA is smaller than that of HMG1, in contrast to the situation for the DNA-binding region of these proteins. The sequence between the second DNA-binding domain and the acidic C-terminal of HMG proteins is required for tight DNA-binding. Also, the acidic C-terminal strongly modulates the DNA-binding ability of each protein. The usefulness of SPR measurement for quantitative analysis of affinity and regions involved in DNA-binding under conditions nearly identical to those in solution is discussed.

Animals↗