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H Yoshizaki

Publications and source records attributed to H Yoshizaki.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of RalA GTPase by phosphatidylinositol 3-kinase as visualized by FRET probes.

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

Cell Membrane↗
PubMed2001

Cross-clade neutralizing antibody production against human immunodeficiency virus type 1 clade E and B' strains by recombinant Mycobacterium bovis BCG-based candidate vaccine.

The recombinant Mycobacterium bovis BCG (rBCG) vector-based vaccine secreting the V3 principal neutralizing epitope of human immunodeficiency virus type 1 (HIV-1) Japanese strain was reported to induce both humoral and cellular immune responses effectively [Proc. Natl. Acad. Sci. USA. 92 (1995) 10693]. The antigen-secreting rBCG system was applied to the V3 epitope of clade E HIV-1 in this study. The V3 sequence of 19 amino acids (aa) and 15aa fused with mycobacterial alpha-antigen was not secreted while 12aa and 11aa sequences were successfully secreted from BCG cells. Serum IgG from guinea pig which was immunized with 12aa epitope-secreting recombinant BCG neutralized the WHO reference strain as well as primary field isolates of clade E virus. The serum IgG could also neutralize Thai B (clade B') strains which possessed a conserved GPGQ motif in their V3 sequences. These data suggest that the rBCG construct secreting the 12aa epitope is implicated in the development of a prophylactic vaccine in Thailand in which both clade E and B' viruses are prevalent.

AIDS Vaccines↗
PubMed2001

Collaborative study on rat sperm motion analysis using CellSoft Series 4000 semen analyzer.

A collaborative study was conducted to determine useful and sensitive rat sperm motion parameters in a CellSoft Series 4000 semen analyzer to detect the effects of compounds on sperm motion. The effects on the sperm motion parameters were investigated using alpha-chlorohydrin, boric acid, ethinylestradiol, ethyl methanesulfonate, nitrazepam, nitrobenzene, ornidazole, sulfasalazine or valproic acid which are well known to induce reproductive or testicular toxicities. All compounds used in this study decreased percentage of motile sperm (% motile). Curvilinear velocity (VCL), maximum and mean amplitude of lateral head displacement (ALH max and ALH mean) were decreased by treatment with all compounds except for valproic acid. Treatment with alpha-chlorohydrin, ornidazole or sulfasalazine under mid-dosage regimens decreased only these parameters. Beat cross frequency (BCF) was increased by treatment with sulfasalazine. There were some treatments which caused either decreased or increased changes irrespective of dosage regimen in linearity, average radius, percentage of circular-swimming sperm out of motile sperm (circular/motile) and percentage of circular-swimming sperm out of all sperm (circular/all). Based on these results, we concluded that % motile, VCL, ALH max and ALH mean are considered useful and sensitive parameters for evaluating the effects of compounds on sperm motion. A parameter of BCF can be useful to detect the effects of specific compounds on sperm motion. Linearity, average radius, circular/motile and circular/all are not considered useful or sensitive indicators to detect the effects of compounds on sperm motion.

Animals↗
PubMed2001

Evidence of primary beta-cell destruction by T-cells and beta-cell differentiation from pancreatic ductal cells in diabetes associated with active autoimmune chronic pancreatitis.

OBJECTIVE: Diabetes associated with autoimmune chronic pancreatitis (ACP) is a subtype of diabetes that is responsive to corticosteroid treatment of progressive endocrine and exocrine dysfunction. However, little is known about pathological changes of islet and exocrine pancreas in ACP. RESEARCH DESIGN AND METHODS: We examined pancreatic specimens obtained on biopsy from four diabetic men with ACP (mean [range]: age 62 years [48-78], duration of ACP 3 months [1-5], duration of diabetes 1 month [0-3]) morphologically, immunohistochemically, and morphometrically. RESULTS: The pancreatic specimens in all cases exhibited inflammatory cell infiltration surrounding ductal cells and extensive fibrosis. Some islets were infiltrated with mononuclear cells with disrupted beta-cells. The subsets of T-cells infiltrated to the islets were mainly CD8(+). Islet beta-cell volume was decreased; the mean percentage area of beta-cells in the islets in four cases with ACP were 16% (range 13-20) (P = 0.0015 vs. type 2 diabetic patients, 48% [27-73], n = 8; P = 0.0002 vs. nondiabetic control subjects, 58% [39-77], n = 7). Preserved ductal cells were surrounded predominantly by CD8(+) or CD4(+) T-cells. Some cytokeratin 19-positive ductal cells contained insulin and glucagon, representing upregulated differentiation of islet cells from ductal cells. Insulin promoter factor-1 (IPF-1) was hyperexpressed in insulin-containing ductal cells. CONCLUSIONS: Diabetes associated with ACP is caused by T-cell-mediated mechanisms primarily involving islet beta-cells as well as pancreatic ductal cells. In ACP, ductal islet precursor cells were associated with IPF-1 hyperexpression, suggesting a critical role of IPF-1 on islet cell differentiation and eventual beta-cell restoration.

Acute Disease↗
PubMed2000

Significance of Asn-77 and Trp-78 in the catalytic function of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26.

The primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases. To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction. From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method. Among 31 negative clones selected from 3,000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78. To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis. Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity, but the K(m) values for both allylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type. On the other hand, three Trp-78 mutants, W78I, W78R, and W78D, showed 5-20-fold increased K(m) values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate. However, these mutants showed moderate levels of enzymatic activity and comparable K(m) values for isopentenyl diphosphate to that of the wild-type. These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.

Alkyl and Aryl Transferases↗
PubMed1999

Availability of sperm examination for male reproductive toxicities in rats treated with boric acid.

Sperm morphological examination, computer-assisted sperm analysis (CASA) and histopathologic examination of the testis and epididymis were performed for male rats treated orally with boric acid for 3 weeks at dosage levels of 50, 150 and 500 mg/kg/day, and treated males were mated with untreated females. None of the males treated with 500 mg/kg/day could impregnate untreated females. The fertility index showed a tendency to decrease in males treated with 150 mg/kg/day. At necropsy, the pre-implantation loss rate in females mated with males treated with 150 mg/kg/day was higher than the control values. Upon epididymal sperm analysis using the CASA system, all parameters including the number of sperm and sperm motions were found to be affected in males treated with 500 mg/kg/day, and the number of sperm, percent motile, velocities and amplitude of lateral head displacement (ALH) were affected in males treated with 150 mg/kg/day. Upon sperm morphological examination, head and tail abnormalities were observed in males treated with 150 and 500 mg/kg/day. In the histopathological examination, atrophy of the seminiferous tubules and multinucleated giant cells in the testes were observed in males treated with 500 mg/kg/day.

Animals↗
PubMed1998

Baicalin, an inhibitor of HIV-1 production in vitro.

The flavonoid baicalin markedly inhibits replication of human immunodeficiency virus type 1 (HIV-1) in a concentration-dependent manner in normal peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA) in vitro. The effect was more pronounced when the cells were pretreated with baicalin. Furthermore, baicalin inhibits HIV-1 replication in PHA-stimulated PBMC from asymptomatic HIV-1-seropositive carriers. The 50% inhibitory concentration for HIV-1 replication was approximately 0.5 microg/ml. At the concentration of 2 microg/ml of baicalin, copy numbers of HIV-1 proviral DNA were approximately 50 times less than in untreated controls. In a cell-free infection system, baicalin inhibited the activity of HIV-1 reverse transcriptase (RT), but not the activity of human DNA polymerases alpha and gamma (DNA polymerase beta was slightly inhibited), suggesting that the anti-HIV-1 effect of baicalin may at least partly be due to inhibition of HIV-1 RT.

Anti-HIV Agents↗
PubMed1998

Protein C activation by recombinant thrombomodulin in plasma.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM), which partially improved the amino acid sequence of human urinary thrombomodulin (UTM), was expressed in C127 cells. GAG-UTM accelerates protein C activation by thrombin and also thrombin inhibition by antithrombin III (ATIII) in the buffer system. Both accelerating activities of GAG-UTM are more potent than those of unmodified recombinant UTM (r-UTM) without a GAG chain. As ATIII in plasma also inhibits protein C activation by a thrombin-thrombomodulin complex, we studied whether GAG-UTM accelerates protein C activation in plasma. GAG-UTM suppressed the generation of thrombin in activating plasma protein C stronger than r-UTM. By Western blot analysis using anti-protein C antibody, activated protein C was generated by GAG-UTM more than by r-UTM. From these results, the acceleration of activated protein C formation by GAG-UTM was confirmed in plasma too.

Blotting, Western↗
PubMed1998

Increased anticoagulant activity of recombinant thrombomodulin modified with glycosaminoglycan.

Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activated protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.

Angiotensin III↗
PubMed1998

Importance of sialic acid in recombinant thrombomodulin in terms of pharmacokinetics and separation of desialyzed glycoprotein.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.

Animals↗
PubMed1998

Effect of posture change on control of ventilation.

To clarify the control mechanism of ventilation during posture change, ventilatory parameters, PETCO2, and ventilatory response to CO2 were examined in 11 healthy male subjects at supine (0 degrees) and 75 degrees head-up tilt positions. Minute expiratory ventilation (V.E), tidal volume (VT), respiratory frequency (f), end-tidal and transcutaneous PCO2 and CO2 output (V.CO2), and ventilatory response to CO2 were measured during a steady state condition. V.E (V.A) and VT increased significantly at 75 degrees tilt with significant decrease in PETCO2 from 40.1 mmHg (0 degrees) to about 36.1 mmHg (75 degrees). Transcutaneous PCO2 also decreased during tilt, by 3.3 mmHg. Physiological dead space (VD/VT) and V.CO2, however, remained unchanged, and ventilatory equivalent (V.E/V.CO2, V.A/V.CO2) increased significantly. The CO2-ventilatory response curve shifted upward (or leftward) without significant change in the response slope. At 75 degrees tilt, EMG activity of gastro-cnemius muscle increased. These findings suggested that PETCO2 decreased because of increased V.E (V.A) with a leftward shift of CO2-ventilatory response curve. Various signals such as afferents from lower extremities might have net stimulatory effects on a CO2-ventilation control system to reset the controlled level of PETCO2 to a lower range, but without significant change in CO2-ventilatory response during upright position.

Adult↗
PubMed1997

Correlation of titer of antibody to principal neutralizing domain of HIVMN strain with disease progression in Japanese hemophiliacs seropositive for HIV type 1.

With the use of the principal neutralizing determinant (PND) peptide-based ELISA to measure anti-PND antibodies that specifically bound synthetic peptides derived from HIVIIIB, HIVMN, HIVRF, HIVSC, HIVWJM-2, HIVAf1l.con, or HIVAf2.con, type-specific antibodies to the HIVMN peptide were studied in 350 serum specimens from Japanese with hemophilia A who had been injected with known unheated factor VIII concentrates until 1985 and had been infected with HIV-1 subtype B. These antibodies were not found in any of the seronegative sera of hemophiliacs, patients with autoimmune diseases, or normal healthy controls. Further, all hemophiliacs rapidly progressing to AIDS and death among the 95 hemophiliacs in a restricted Nara area had antibody titers of less than 20 and their low levels preceded the rapid progression to the disease state. In contrast, slowly progressing hemophiliacs maintained an antibody titer of more than 100 from the initial stages of viral infection and remained asymptomatic. Sequence analysis of the V3 regions of HIV-1 indicated that the hemophiliacs who maintained a high anti-PNDMN antibody level showed a conserved MN sequence. In contrast, the HIV-infected hemophiliacs with nonreactivity in the ELISA showed sequence changes in the neutralizing epitopes of HIVMN. The dynamic of the serum anti-PNDMN antibody titer appear to be a characteristic indicator of the progression of the HIV-infected status in Japanese hemophiliacs seropositive for HIV-1.

Adult↗
PubMed1997

Nucleotide sequence variation of human T-lymphotropic virus type II in Vietnam.

A high rate of human T-lymphotropic virus type II (HTLV-II) infection has been documented in intravenous drug abusers (IVDAs) in South Vietnam. We have investigated the molecular characteristics of the virus and have shown that one HTLV-II subtype is predominant in Ho Chi Minh City. This molecular subtype, HTLV-IIb, was identified in a number of South Vietnamese by nucleotide sequence analysis of the long terminal repeat (LTR) region. HTLV-IIa was not found. These findings suggest that HTLV-IIb is endemic in IVDAs in South Vietnam, although IVDAs in urban areas in North America are predominantly infected with HTLV-IIa.

Adult↗
PubMed1996

Human T-lymphotropic virus type II in Japan.

Serum specimens were assayed for human T-lymphotropic virus type II(HTLV-II) infection in 1,500 individuals known to be seropositive for HTLV-I and 30,000 blood donors in Japan. All HTLV-I-positive individuals were negative for HTLV-II. However, one of the blood donors was clearly seropositive for HTLV-II. Further, the donor was shown to be positive for HTLV-IIb. Here we report at least one case with HTLV-II in Japan and discuss the origin of the infection.

Adult↗