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Biomedical subjects

T Asano

Publications and source records attributed to T Asano.

At least 109 records · Page 6Linked to original sources
PubMed2001

[Diagnosis of Alzheimer's disease using brain SPECT with three-dimensional stereotactic surface projections].

We compared the diagnostic usefulness of three-dimensional stereotactic surface projection (3D-SSP) with that of standard transaxial images in brain SPECT in patients with Alzheimer's disease (AD). The subjects consisted of 69 patients with AD and 60 patients with non-AD, including vascular dementia, Parkinson's disease with dementia, frontotemporal dementia, other dementing diseases and neuropsychiatric diseases. Standard transaxial section and 3D-SSP SPECT images with N-isopropyl-p-[123I] iodoamphetamine were blindly interpreted by three examiners and were classified into the following three patterns: typical AD, atypical AD, and not indicative AD patterns. The 3D-SSP images demonstrated reductions of cerebral blood flow in the parieto-temporal association cortex and posterior cingulate gyrus more clearly and easily than the standard transaxial images. The diagnostic sensitivity and specificity were 93% and 85% with 3D-SSP and 83% and 82% with standard transaxial section respectively. 3D-SSP was especially useful for early or atypical AD which showed no characteristic perfusion abnormalities on standard transaxial images. These results suggest that SPECT with 3D-SSP provides an sensitive as well as accurate tool for the diagnosis of AD.

Aged↗
PubMed2000

Total synthesis of the antiviral marine natural product (-)-hennoxazole A.

[structure:see text] The marine natural product hennoxazole A was synthesized by a convergent approach. The diastereoselective Mukaiyama aldol reaction with beta-alkoxy aldehyde was used to construct the tetrahydropyran segment, and the preparation of the nonconjugated triene moiety was accomplished via S(N)2 displacement of allylic bromide with vinyllithium and Takai's iodoolefination followed by palladium-catalyzed cross coupling with MeMgBr. The final steps involve an amide coupling using DEPC and oxazole synthesis via a oxidation/cyclodehydration process.

Animals↗
PubMed2000

Cuticular pro-phenoloxidase of the silkworm, Bombyx mori. Purification and demonstration of its transport from hemolymph.

Pro-phenoloxidase (proPO) in insects is implicated in the defense against microbes and wounding. The presence of proPO in the cuticle was suggested more than 30 years ago, but it has not been purified. The extract of cuticles of the silkworm, Bombyx mori, was shown to contain two proPO isoforms (F-type and S-type proPOs, which have slightly different mobilities in polyacrylamide gel electrophoresis under nondenaturing conditions). The two isoforms were purified to homogeneity. From hemolymph of the same insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits. The isoforms in the hemolymph and cuticle were activated by a specific activating enzyme. The resulting active phenoloxidases exhibited almost the same substrate specificities and specific activities toward o-diphenols. The substrate specificities and the susceptibilities to inhibitors, including carbon monoxide, indicated that the purified proPO isoforms were not zymogens of laccase-type phenoloxidase. The proPO in hemolymph was shown to be transported to the cuticle. This demonstration was corroborated by the failure to detect proPO transcripts by Northern analysis of total RNA from epidermal cells. In reversed-phase column chromatography, cuticular and hemolymph proPOs gave distinct elution profiles, indicating that some yet to be identified modification occurs in hemolymph proPO and results in the formation of cuticular proPO. There was little transportation of cuticular proPO to the cuticle when it was injected into the hemocoel. The nature of the modification is described in the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11113-11125).

Amino Acid Sequence↗
PubMed2000

Transepithelially transported pro-phenoloxidase in the cuticle of the silkworm, Bombyx mori. Identification of its methionyl residues oxidized to methionine sulfoxides.

Pro-phenoloxidase (proPO) in insects is activated through the action of a protease cascade triggered by minute amounts of microbial cell wall components. It is an important molecule for the defense against invading microorganisms and for the repair of wounds. In the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11100-11112), a proPO isoform, proPO-HS, in the hemolymph of the silkworm, Bombyx mori, is reported to be transported to the cuticle. The transported proPO isoform was recovered from the cuticle and named proPO-CS. The elution profiles of proPO-CS and proPO-HS in reversed-phase high performance liquid chromatography (HPLC) were found to be different, giving a basis to the inference that proPO-CS is a modified form of proPO-HS. In the present study, we investigated the nature of the modifications occurring in proPO-CS, in which proteolytically and chemically cleaved fragments originating from the subunits of proPO-CS and proPO-HS were analyzed by reversed-phase HPLC, amino acid sequencing, and mass spectrometry. A subunit of the heterodimeric proPO-CS was found to contain five or six methionine sulfoxides, and another subunit was found to contain one methionine residue oxidized to the sulfoxide. All of the oxidized methionyl residues were identified. Other than oxidation of the methionyl residues, no additional modification of proPO-CS was found. In the model structure of each subunit of proPO-CS constructed by protein modeling with the known structures of the horseshoe crab, Limulus polyphemus, hemocyanin type II subunit as templates, the methionine residues identified as methionine sulfoxide had high degrees of accessibility to the solvent. The implication of the oxidation at the methionine residues is discussed in relation to the mechanism of transepithelial transport of proPO from the hemolymph to the cuticle.

Amino Acid Sequence↗
PubMed2000

Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 kinase/Akt.

BACKGROUND: Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Reactive oxygen species induce apoptosis in many types of cells including cardiomyocytes. Because insulin has been reported to have protective effects, we examined whether insulin prevents cardiomyocytes from oxidative stress-induced apoptotic death. METHODS AND RESULTS: Cultured cardiomyocytes of neonatal rats were stimulated by hydrogen peroxide (H(2)O(2)). Apoptosis was evaluated by means of the TUNEL method and DNA laddering. Incubation with 100 micromol/L H(2)O(2) for 24 hours increased the number of TUNEL-positive cardiac myocytes (control, approximately 4% versus H(2)O(2), approximately 23%). Pretreatment with 10(-)(6) mol/L insulin significantly decreased the number of H(2)O(2)-induced TUNEL-positive cardiac myocytes (approximately 12%) and DNA fragmentation induced by H(2)O(2). Pretreatment with a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin, and overexpression of dominant negative mutant of PI3K abolished the cytoprotective effect of insulin. Insulin strongly activated both PI3K and the putative downstream effector AKT: Moreover, a proapoptotic protein, BAD:, was significantly phosphorylated and inactivated by insulin through PI3K. CONCLUSIONS: These results suggest that insulin protects cardiomyocytes from oxidative stress-induced apoptosis through the PI3K pathway.

Animals↗
PubMed2000

Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading.

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.

3T3 Cells↗
PubMed2000

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells.

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

Regulator of G protein signaling 8 (RGS8) requires its NH2 terminus for subcellular localization and acute desensitization of G protein-gated K+ channels.

Functional roles of the NH(2)-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied. The deletion of the NH(2)-terminal region of RGS8 (DeltaNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Galpha binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and DeltaNRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active Galpha(o) resulted in translocation of RGS8 protein to the plasma membrane. In contrast, DeltaNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Galpha(o). When coexpressed with G protein-gated inwardly rectifying K(+) channels, DeltaNRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K(+) current observed in the presence of RGS8, however, was not induced by DeltaNRGS8. Thus, we, for the first time, showed that the NH(2) terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.

Active Transport, Cell Nucleus↗
PubMed2000

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits.

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.

ATP-Binding Cassette Transporters↗
PubMed2000

Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity.

The aim of this study was to investigate the mechanisms of the pathogenesis of hyperviscosity following cerebral ischemia. Focal ischemia was produced by embolic occlusion of the right middle cerebral artery (MCA) in rats for 1 hour, followed by recirculation. Twenty-four hours after MCA occlusion, fasudil, a protein kinase inhibitor, was administered intraperitoneally. Blood samples were taken from the abdominal aorta, and viscosity was measured using a cone-plate viscometer. The viscosity of whole blood in the ischemic attack group was significantly increased compared with the sham operated group 24 hours after MCA occlusion. Fasudil dose-dependently and significantly decreased the blood viscosity, and reduced to the normal range after administration of 10 mg/kg of fasudil (sham-operated rats, 5.17+/-0.05 cP; pre dose/ischemic rats, 6.05+/-0.08 cP; post dose/ischemic rats, 5.23+/-0.14 cP; 37.5 sec(-1)). Our findings suggest that cerebral ischemia induces a potent, systemic and long-lasting hyperviscosity, and that the inhibition of protein kinases, especially rho kinase, is efficacious in preventing this hyperviscosity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Absence of association between a common mutation in the methylenetetrahydrofolate reductase gene and preeclampsia in Japanese women.

An association between preeclampsia (PE) and a common missense mutation of the methylenetetrahydrofolate reductase gene (MTHFR), a C to T substitution at nucleotide 677 (C677T), which converts an alanine to a valine residue, has been reported in Italian and Japanese populations. We examined 101 cases of hypertension in pregnancy (HP), including 73 cases of PE, and 215 normal pregnancy controls to confirm the association in Japanese women. No significant differences of the frequency of the T677 allele frequency or percentage of T677 homozygotes were detected among the various types of cases: HP (0.38, 12%, respectively), severe HP (0. 40, 12%), PE (0.38, 11%), severe PE (0.41, 11%), primiparous HP (0. 40, 12%), primiparous PE (0.44, 18%), nonelderly HP (0.39, 13%), nonelderly PE (0.40, 14%), nonobese HP (0.38, 12%), nonobese PE (0. 39, 10%), HP without homozygous T235 of the angiotensinogen gene (TT of AGT) (0.38, 15%), PE without TT of AGT (0.38, 15%), and controls (0.38, 15%). The results indicate that T677 of MTHFR may not be a risk factor for PE in Japanese population.

Adult↗
PubMed2000

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Animals↗
PubMed2000

ESR investigation on the breather mode and the spinon-breather dynamical crossover in Cu benzoate.

A "breather excitation" is observed directly by electron spin resonance in the quantum spin chain Cu benzoate, in which an unexpected field-induced gap has recently been found. The nonlinear field dependence of the resonance field agrees well with the formula based on a quantum sine-Gordon model. The power-law temperature dependence of the linewidth is observed in the gapless spinon regime while the width decreases exponentially for the gapped breather regime. In the intermediate range, a distinct anomaly is found, which is the manifestation of "the spinon-breather dynamical crossover."

Journal Article↗
PubMed2000

p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity.

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.

3T3 Cells↗
PubMed2000

Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation.

Thiazolidinediones, synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), are reported to have direct beneficial effects on diabetic nephropathy without lowering blood glucose levels in human and rat. We hypothesized these effects of thiazolidinediones might be derived from PPARgamma activation of kidney cells, and we examined the expression of PPARgamma and the effect of PPARgamma agonists, troglitazone and 15-deoxy-delta-prostaglandin J2 (15d-PGJ2), on the proliferation and differentiation in rat mesangial cells. A single band of mRNA of PPARgamma with a predicted size was detected in reverse transcription-polymerase chain reaction products (RT-PCR) using established PCR probes of PPARgamma. PPARgamma protein in rat mesangial cells was identified as PPARgamma1 by a Western blot. In a gel mobility shift assay to determine a binding activity of PPARgamma, the nuclear protein from rat mesangial cells bound to a (32)P-labeled oligonucleotide probe, including PPAR response elements. A synthetic and a natural ligand of PPARgamma, troglitazone and 15d-PGJ2, decreased thymidine incorporation in a dose dependent manner. After 7 days incubation with troglitazone and 15d-PGJ2, alpha-smooth muscle actin expression, a marker of mesangial cell de-differentiation, was decreased significantly compared to that of control. These results indicate that PPARgamma1 is expressing in rat mesangial cells, and PPARgamma1 activation with its agonists modulates the proliferation and differentiation of cultured rat mesangial cells.

Animals↗
PubMed2000

Identification and characterization of a novel inositol polyphosphate 5-phosphatase.

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.

Amino Acid Sequence↗
PubMed2000

Magnetic dilution in the geometrically frustrated SrCr(9p)Ga(12-9p)O19 and the role of local dynamics: A muon spin relaxation study

We investigate the spin dynamics of SrCr(9p)Ga(12-9p)O19 for p below and above the percolation threshold p(c) using muon spin relaxation. Our major findings are as follows: (i) At T-->0 the relaxation rate is T independent and approximately p(3), (ii) the slowing down of spin fluctuation is activated with an energy U, which is also a linear function of p(3) and lim U as p-->0 = 8 K; this energy scale could stem only from a single ion anisotropy, and (iii) the p dependence of the dynamical properties is identical below and above p(c), indicating that they are controlled by local excitation.

Journal Article↗