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Biomedical subjects

Y Ihara

Publications and source records attributed to Y Ihara.

At least 37 records · Page 2Linked to original sources
PubMed2001

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid.

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.

Antithrombin III↗
PubMed2001

Doxorubicin-induced DNA intercalation and scavenging by nuclear glutathione S-transferase pi.

Glutathione S-transferase (GST) functions in xenobiotic biotransformation and drug metabolism. Increased expression of GSTpi, an isozyme of GST, has been found in cancer cells resistant to doxorubicin hydrochloride (DOX) or cis-diamminedichloroplatinum (II) (CDDP), and this increase was believed to be correlated with drug resistance of cancer cells. GST is mainly expressed in the cytoplasm; GSTpi in the nucleus has been reported in cancer cells, but the meaning of this result is not known. Here, we studied changes in the amount of nuclear GSTpi after exposure of cancer cells to anticancer drugs, and role of the nuclear GSTpi in drug resistance. We found nuclear GSTpi in cancer cells resistant to DOX, and the amount of nuclear GSTpi was enhanced by treatment of the cancer cells with DOX or CDDP. We also found that a mushroom lectin, an inhibitor of nuclear transport, inhibited the nuclear transfer of GSTpi, suggesting the existence of a specific transport system for the nuclear transfer of GSTpi. Nuclear GSTpi protected DNA against damage by anticancer drugs. These results suggest a possible role of GSTpi in the acquisition of resistance to anticancer drugs by cancer cells.

Antineoplastic Agents↗
PubMed2001

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase.

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

Journal Article↗
PubMed2001

Human TSC-22 gene: no association with type 2 diabetes.

OBJECTIVE: In order to elucidate whether or not genetic variations of TSC-22 (TGF [transforming growth factor]-beta-stimulated clone 22), which was originally identified as a TGF-beta-responsive leucine zipper protein in murine osteoblastic cells, are associated with type 2 diabetes, the genomic organization of the human TSC-22 gene was determined and the association between its polymorphisms and type 2 diabetes was examined. RESULTS: The human TSC-22 gene spans approximately 5 kilobase pairs and is encoded in three exons. Two single nucleotide polymorphisms (SNPs) were identified in the coding region of the first exon, two other SNPs in the first intron, and one SNP in the putative promoter region. There were, however, no significant differences in the frequency of these polymorphisms between patients with type 2 diabetes and non-diabetic control subjects. CONCLUSION: It is unlikely that the TSC-22 gene is a locus responsible for type 2 diabetes.

Aged↗
PubMed2001

Roles of ATP-sensitive K+ channels in cell survival and differentiation in the endocrine pancreas.

To determine the roles of the ATP-sensitive K+ (K(ATP)) channels in endocrine pancreas more directly, two types of genetically engineered Kir6.2 mice were developed: mice expressing a dominant-negative form of Kir6.2 specifically in beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2-/- or Kir6.2 null mice). The Kir6.2G132S Tg mice show severe impairment of K(ATP) channel function only in the beta-cells, whereas Kir6.2 null mice are completely defective in K(ATP) channel function in all of the cells in which Kir6.2 is a constituent of the K(ATP) channels, because of the disruption of Kir6.2. Both types of mice show abnormal architecture of the pancreatic islets. The number of beta-cells in Kir6.2G132S Tg mice decreases markedly with age, whereas that in Kir6.2-/- mice decreases slightly. alpha-Cells, which are normally present only in the periphery of pancreatic islets, also appear in the center of the islets in both Kir6.2G132S Tg and Kir6.2-/- mice. Interestingly, the number of peptide YY (PYY) and glucagon-positive cells is markedly increased in Kir6.2 null mice, whereas the number of PP cells and delta-cells is not altered. Apoptotic cells are detected by the TdT-mediated dUTP nick-end labeling (TUNEL) method at a high frequency in both Kir6.2G372S Tg and Kir6.2-/- mice compared with the respective controls. Thus, studies of Kir6.2G372S Tg and Kir6.2-/- mice indicate that K(ATP) channels play an important role in cell survival and differentiation in the endocrine pancreas.

Adenosine Triphosphate↗
PubMed2000

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells.

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.

Amyloid beta-Peptides↗
PubMed2000

Mutant presenilin 2 transgenic mice. A large increase in the levels of Abeta 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin.

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid beta protein (Abeta) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Walter, J., Grünberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313-322). Here, using a new extraction protocol, we quantitated the Abeta40 and Abeta42 levels in the Tris-saline-insoluble fraction. The insoluble Abeta levels were found to be higher than the soluble Abeta levels, and the insoluble Abeta42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble Abeta42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutant PS2 transgenic mice contained remarkably increased levels of Abeta42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of Abeta42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.

Amino Acid Substitution↗
PubMed2000

Widespread expression of alpha-synuclein and tau immunoreactivity in Hallervorden-Spatz syndrome with protracted clinical course.

Hallervorden-Spatz syndrome (HSS) is a rare autosomal recessive disorder clinically characterized by extrapyramidal signs and progressive dementia. In a typical case, the clinical symptoms become apparent during late childhood, and usually the course is protracted over a decade or more. We recently had an opportunity to study the brains of two cases of HSS with a clinical course of over 30 years. Case 1 was a 44-year-old female and case 2 was a 37-year-old male. Grossly, the brains showed severe fronto-temporal lobar atrophy with abundant spheroids and mild iron deposits in the globus pallidus, associated with features of motor neuron disease. In addition, there was diffuse sponginess in the atrophic cortex as well as widespread Alzheimer's neurofibrillary tangles (NFTs) and Lewy bodies (LBs) in the cortical and subcortical regions, including the spinal cord. Ultrastructurally, NFTs were composed of paired helical filaments, and LBs of central dense cores with radiating fibrils. Discrete immunostaining was demonstrated in NFTs and neuropil threads with various antibodies against phosphorylated tau, and in LBs with antibody against alpha-synuclein. In addition, diffuse, overlapping immunoreactivity of alpha-synuclein and phosphorylated tau was seen within the cytoplasm of many neurons. However, when LBs and NFTs coexisted within the same neurons, they were clearly segregated. The findings of our present cases as well as those reported in the literature may indicate that simultaneous and extensive occurrence of abnormal phosphorylation of tau and accumulation of alpha-synuclein may constitute cardinal pathological features of HSS with protracted clinical course.

Adult↗
PubMed2000

Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules.

Collapsin response mediator protein-2 (CRMP-2) is a member of the CRMP/TOAD/Ulip/DRP family of cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP-2 mediates the intracellular response to collapsin 1/semaphorin 3A, a repulsive extracellular guidance cue for axonal outgrowth. The mutation of UNC-33, a Caenorhabditis elegans homolog of CRMP-2, results in abnormality of microtubules in neurites, but the mechanism of CRMP-2 action remains to be clarified. Here, we report that overexpression of human CRMP-2 in Neuro2a cells, a mouse neuroblastoma cell line, results in blebbing of the cytoplasm. Furthermore, some cells exhibited intranuclear inclusions, which were labeled with antibodies to CRMP-2 and tubulin. CRMP-2 was found to be associated with microtubule bundles in the spindles at the metaphase and in the midbodies at the late telophase in mitotic cells. Thus, it is most likely that failure of complete disassembly of the spindle microtubules during mitosis is responsible for the formation of these intranuclear inclusions. We suggest that CRMP-2 functions by regulating the dynamics of microtubules.

Acetylation↗
PubMed2000

Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells.

Several lines of evidence suggest that enhanced oxidative stress is involved in the pathogenesis and/or progression of Alzheimer's disease (AD). Amyloid beta-protein (Abeta) that composes senile plaques, a major neuropathological hallmark of AD, is considered to have a causal role in AD. Thus, we have studied the effect of oxidative stress on Abeta metabolism within the cell. Here, we report that oxidative stress induced by H(2)O(2) (100-250 microM) caused an increase in the levels of intracellular Abeta in human neuroblastoma SH-SY5Y cells. Treatment with 200 microM H(2)O(2) caused significant decreases in the protein levels of full-length beta-amyloid precursor protein (APP) and its COOH-terminal fragment that is generated by beta-cleavage, while the gene expression of APP was not altered under these conditions. A pulse-chase experiment further showed a decrease in the half-life of this amyloidogenic COOH-terminal fragment but not in that of nonamyloidogenic counterpart in the H(2)O(2)-treated cells. These results suggest that oxidative stress promotes intracellular accumulation of Abeta through enhancing the amyloidogenic pathway.

Alzheimer Disease↗
PubMed2000

Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells.

Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.

Animals↗
PubMed2000

Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes.

We have shown recently that oxidative stress by chronic hyperglycemia damages the pancreatic beta-cells of GK rats, a model of non-obese type 2 diabetes, which may worsen diabetic condition and suggested the administration of antioxidants as a supportive therapy. To determine if natural antioxidant alpha-tocopherol (vitamin E) has beneficial effects on the glycemic control of type 2 diabetes, GK rats were fed a diet containing 0, 20 or 500 mg/kg diet alpha-tocopherol. Intraperitoneal glucose tolerance test revealed a significant increment of insulin secretion at 30 min and a significant decrement of blood glucose levels at 30 and 120 min after glucose loading in the GK rats fed with high alpha-tocopherol diet. The levels of glycated hemoglobin A1c, an indicator of glycemic control, were also reduced. Vitamin E supplementation clearly ameliorated diabetic control of GK rats, suggesting the importance of not only dietary supplementation of natural antioxidants but also other antioxidative intervention as a supportive therapy of type 2 diabetic patients.

Animals↗
PubMed2000

Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522.

3F4, a monoclonal antibody raised against partially purified paired helical filaments (PHFs), strongly labeled neurofibrillary tangles and some plaque neurites but barely labeled neuropil threads. The levels of the 65-kDa antigen were significantly increased in the soluble fraction of the brains affected by Alzheimer's disease (AD), as compared with that in the case of control brains. The antigen was previously identified as human collapsin response mediator protein-2 (hCRMP-2) by sequencing the immunoaffinity-purified 65-kDa antigen [Yoshida, H., Watanabe, A., and Ihara, Y. (1998) J. Biol. Chem. 273, 9761-9768]. Here, we show that the 3F4 antigen represents a highly phosphorylated form of CRMP-2. The 3F4-reactive phosphoepitope was localized to the carboxyl-terminal portion of hCRMP-2, and was created by a novel 45-50-kDa protein kinase in rat brain extract. Site-directed mutagenesis of this portion showed that multiple sites of CRMP-2 are differentially phosphorylated within residues 507-522, and that phosphorylation of three sites, Thr-509, Ser-518, and Ser-522, is required for full 3F4 binding. The phosphorylation of this particular portion carboxyl-terminal to the basic region of CRMP-2 may play an important role in regulating its activity, and may be involved in the formation of degenerating neurites in AD brain.

Alzheimer Disease↗
PubMed2000

Cell cycle-dependent regulation of N-acetylglucosaminyltransferase-III in a human colon cancer cell line, Colo201.

The mechanism for cell-cycle-dependent regulation of N-acetylglucosaminyltransferase III (GnT-III) activity was investigated using synchronized culture of Colo201, a human colon cancer cell line. In the synchronized culture, it was found that GnT-III activity rapidly increased in the M phase and the maximal activity was five times higher than the basal level found in the G1 phase. Northern blot and Western blot analyses revealed that the increase in the activity is due not to an increase in expression level of its mRNA but, rather, to the level of protein. Furthermore, it was shown by a pulse-chase experiment that the increased protein level of GnT-III is the result of its prolonged turnover rate. Lectin blotting with erythroagglutinating phytohemagglutinin showed that the content of bisecting N-acetylglucosamine structure in glycoproteins was transiently increased during the M phase in conjunction with the increased activity of GnT-III. These results suggest that GnT-III activity undergoes a cell-cycle-dependent regulation and thereby oligosaccharide structures of N-glycans vary specifically during the M phase of the cell cycle. Thus, it is possible that the cell-cycle-dependent alteration of N-glycans by GnT-III might play a role in biological events, such as the progression of cell cycle and cell division.

Aphidicolin↗
PubMed2000

Beta1,6-N-acetylglucosamine-bearing N-glycans in human gliomas: implications for a role in regulating invasivity.

The metastatic potential of tumor cells has been shown to be correlated with the expression of tri- and tetra-antennary beta1,6-N-acetylglucosamine (beta1,6-GlcNAc)-bearing N-glycans, which are recognized by Phaseolus vulgaris leukoagglutinating lectin (L-PHA). The expression of beta1,6-GlcNAc-bearing N-glycans also has been used as a marker of tumor progression in human breast and colon cancers. In this report, the role of N-glycan branching in regulating glioma migration and invasion was examined. The expression of beta1,6-GlcNAc-bearing N-glycans was found in human glioma specimens, whereas astrocytes from normal adult brain were negative. The expression of N-acetylglucosaminyltransferase V (GnT-V) mRNA, which is responsible for the biosynthesis of beta1,6-GlcNAc-bearing N-glycans, was high in glioma cell lines with robust ets-1 expression. To study the molecular mechanism of GnT-V expression in human glioma cells, an inducible ets-1 gene was stably transfected into SNB-19 cells using a tetracycline repressor system. GnT-V mRNA expression was increased by the induction of c-ets-1, suggesting that the Ets-1 transcription factor directly regulates the transcription of GnT-V. Stable transfection of GnT-V into human glioma U-373 MG cells resulted in changes in cell morphology and focal adhesions and a marked increase in glioma invasivity in vitro. L-PHA has little effect on cell migration. On the contrary, Phaseolus vulgaris erythroagglutinating lectin (E-PHA), which recognizes bisecting beta1,4-GlcNAc-bearing N-glycans, strongly inhibits cell migration (haptotaxis) on a fibronectin substrate in U-373 MG transfectants and other glioma cell lines tested. These results suggest that the increased beta1,6-GlcNAc-bearing N-glycan expression found in malignant gliomas is modulated by GnT-V through the Ets-1 transcription factor, and that the branching of complex type N-glycans plays a major role in glioma invasivity.

Adult↗
PubMed2000

Effect of apolipoprotein E allele epsilon4 on the initial phase of amyloid beta-protein accumulation in the human brain.

Deposition of amyloid ss-protein (Ass), a hallmark of Alzheimer's disease, occurs to some extent in the brains of most elderly individuals. We sought to learn when Ass deposition begins and how deposition is affected by apolipoprotein E allele epsilon4, a strong risk factor for late-onset Alzheimer's disease. Using an improved extraction protocol and specific enzyme-linked immunosorbent assay, we quantified the levels of Ass40 and Ass42 in the insoluble fractions of brains from 105 autopsy cases, aged 22 to 81 years at death, who showed no signs of dementia. Ass40 and Ass42 were detected in the insoluble fractions from all of the brains examined; low levels were even found in the brains of patients as young as 20 to 30 years of age. The incidence of significant Ass accumulation increased age-dependently, with Ass42 levels beginning to rise steeply in some patients in their late 40's, accompanied by much smaller increases in Ass40 levels. The presence of the apolipoprotein E epsilon4 allele was found to significantly enhance the accumulation of Ass42 and, to a lesser extent, that of Ass40. These findings strongly suggest that the presence of epsilon4 allele results in an earlier onset of Ass42 accumulation in the brain.

Adult↗