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Biomedical subjects

Y Ihara

Publications and source records attributed to Y Ihara.

At least 91 records · Page 5Linked to original sources
PubMed1998

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

Aged↗
PubMed1998

Amyloid beta-protein (A beta) accumulation in the putamen and mammillary body during aging and in Alzheimer disease.

Immunocytochemical studies clearly showed that amyloid beta-protein (A beta) deposits are widely distributed in the subcortical regions as well as the cortices of normal aged and Alzheimer disease (AD) brains. To investigate the temporal profile of A beta accumulation in the subcortical region, we quantitated A beta40 and A beta42 levels, using sensitive enzyme immunoassays, in the putamen and mammillary bodies of normal individuals aged 24 to 87 years and of AD patients. In these two regions, A beta42 was the predominant species; in particular, A beta42 was the only A beta species detected in the putamen. In several cases the mammillary body contained only A beta40, but not A beta42. Although the extent of A beta accumulation in the 2 subcortical regions was much less than that in the cortex of the same subject, A beta42 appears to accumulate in both subcortical regions at the same time as in the cortex and leptomeninges. In addition, the A beta42 levels in the putamen or in the mammillary body correlated with those in the occipitotemporal cortex. This strongly suggests that the extent of A beta42 accumulation in the brain is determined not only by the duration of A beta accumulation but also by other unknown regional factors. Western blotting showed that the initial A beta species to accumulate in the putamen or mammillary body varied among individuals. In some cases, an A beta42 stable dimer was the most predominant species, while in other cases a 3 or 4 kD A beta42 monomer was more abundant, suggesting that the clearance rates of the A beta42 stable dimer and monomer are different in vivo.

Adult↗
PubMed1998

Accumulation of tau in autophagic vacuoles in chloroquine myopathy.

Long-term administration of chloroquine to rats induces a vacuolar myopathy, which is specifically termed chloroquine myopathy (CM). In CM, tau mRNA levels were transiently upregulated in the early phase, while tau itself slowly accumulated in the late phase. The temporal profiles of tau mRNA levels and its accumulation were very similar to those of beta-amyloid protein precursor (APP) and its carboxyl-terminal fragments, both of which have been known to be degraded in lysosomes. Immunocytochemistry showed that tau progressively accumulated in the rimmed vacuoles exhibiting increased acid phosphatase activities, and immunoelectron microscopy demonstrated that tau was located within autophagic vacuoles. These results suggest that the accumulation of tau in CM is due to defective tau degradation in the lysosomal compartment in the muscle.

Acid Phosphatase↗
PubMed1998

Simultaneous quantification of APP and tau mRNA by RT-PCR.

We describe here a method for simultaneous relative quantitation of APP and tau expressions in rat muscle using polymerase chain reaction combined with reverse transcription. The target mRNAs were coamplified with an internal control, glyceraldehyde-3-phosphate dehydrogenase (G3PD). The amplified products were measured with a laser image analyzer, and the values were plotted against the cycle number. In the exponential phase of amplification, the linearity and reproducibility of RT-PCR demonstrate identical efficiencies of cDNA synthesis and PCR amplification for APP, tau and G3PD. These results suggest that in the exponential phase of amplification, a relative quantitation of target mRNAs could be accomplished by normalization with internal control. Preliminary experiments indicate that the expressions of both APP and tau in rat muscle were first increased and then decreased by chronic intoxication with chloroquine.

Alzheimer Disease↗
PubMed1998

Astrocytes containing amyloid beta-protein (Abeta)-positive granules are associated with Abeta40-positive diffuse plaques in the aged human brain.

Amyloid beta-protein (Abeta) is the major component of senile plaques that emerge in the cortex during aging and appear most abundantly in Alzheimer's disease. In the course of our immunocytochemical study on a large number of autopsy cases, we noticed, in many aged nondemented cases, the presence of unique diffuse plaques in the cortex distinct from ordinary diffuse plaques by immunocytochemistry. The former were amorphous, very faintly Abeta-immunoreactive plaques resembling diffuse plaques, but they stained for Abeta40 and were associated with small cells containing Abeta-positive granules. A panel of amino- and carboxyl-terminal-specific Abeta antibodies showed that such Abeta40-positive diffuse plaques and cell-associated granules were composed exclusively of amino-terminally deleted Abeta terminating at Abeta40, -42, and -43. Double immunostaining also showed that those Abeta-immunoreactive granules are located in astrocytes and not in microglia or neurons. Immunoelectron microscopy revealed that nonfibrillar Abeta immunoreactivity was located within lipofuscin-like granules in somewhat swollen astrocytes. These findings raise the possibility that astrocytes take up Abeta and attempt to degrade it in lysosomes in the aged brain.

Adult↗
PubMed1998

Quantitation of amyloid beta-protein (A beta) in the cortex during aging and in Alzheimer's disease.

In this study we sought to learn about when and how amyloid beta-protein (A beta) accumulates in the cortex of normal individuals and about the difference in the A beta accumulation between normal aged and Alzheimer's disease (AD) brains. From consecutive autopsy cases and AD cases, hippocampus CA1 and occipitotemporal cortex T4 were sampled for A beta quantitation by the well characterized two-site enzyme immunoassays (EIAs). There was a strong tendency toward A beta 42 accumulation between the ages of 50 and 70 years in T4 and a little later in CA1. The A beta 42 levels were consistently higher in T4 than those in CA1 in any given case. The levels of A beta 42 in AD brains were significantly higher than those in control brains, and the extent of A beta 42 amino-terminal modification was also much greater in AD brains than that in control brains. Even in cases in which no senile plaques were immunocytochemically detected, EIAs clearly showed that significant amounts of A beta 42 already had accumulated. In contrast to A beta 42, A beta 40 showed no apparent age-dependent accumulation, and its high levels were found to be associated with AD.

Adult↗
PubMed1997

Amyloid beta-protein (A beta) associated with lipid molecules: immunoreactivity distinct from that of soluble A beta.

We previously identified a novel amyloid beta-protein (A beta), that binds to GM1 ganglioside, in brains exhibiting the early pathological changes of AD. In this study, we raised monoclonal antibodies, using membrane fractions containing abundant GM1 ganglioside-bound A beta as antigens. Monoclonal antibody 4396, produced in this study, immunoprecipitates A beta42 in the membrane fractions of brains with diffuse plaques, but does not react with soluble A beta42 or GM1 ganglioside. Furthermore, this antibody recognizes the A beta bound to lipid vesicles containing GM1 ganglioside, and unexpectedly, phosphatidylinositol. In contrast, a control anti-A beta monoclonal antibody does not recognize the A beta bound to these lipid vesicles. These results indicate that A beta associated with lipids has an immunoreactivity distinct from that of soluble A.

Alzheimer Disease↗
PubMed1997

Free radicals and superoxide dismutase in blood of patients with Alzheimer's disease and vascular dementia.

We measured hydroxyl radical (.OH) levels in blood, superoxide dismutase (SOD) activity in red blood cells (RBC) relative to both total protein (RBC-SOD/P) and Cu,Zn-SOD protein (RBC-SOD/SOD), SOD activity in plasma (plasma-SOD), and Cu,Zn-SOD protein relative to total RBC protein (Cu,Zn-SOD/P) in 22 patients with probable dementia of the Alzheimer type (DAT group, mean age 74.8+/-9.4 years), 16 with probable vascular dementia (VAD group, mean age 76.9+/-6.7 years) and 19 non-demented controls (control group, mean age 73.5+/-6.2 years). Levels of .OH in the DAT and VAD groups were significantly (P<0.01 and P<0.001, respectively) higher, whereas the values of RBC-SOD/P and RBC-SOD/SOD in these two groups (both P<0.001) and Cu,Zn-SOD/P in the DAT group (P<0.001) were significantly lower than the corresponding control values. Members of the VAD group with risk factors for stroke (RF+ group) showed significantly higher .OH levels than members of the VAD group without risk factors (RF- group; P<0.01) and the control group (P<0.001). RBC-SOD/P and RBC-SOD/SOD values in the RF+ group were significantly (both P<0.01) lower than the corresponding control values. There were no significant differences among the VAD, RF+ and control groups with respect to Cu,Zn-SOD/P values, or between the RF- and control groups for any measured parameter. We conclude that oxidative stress plays a role in the brain damage seen in both DAT and VAD, and that the causes of decreased SOD activity in RBC differ between DAT and VAD patients.

Aged↗
PubMed1997

Inorganic polyphosphate and the induction of rpoS expression.

Inorganic polyphosphate [poly(P)] levels in Escherichia coli were reduced to barely detectable concentrations by expression of the plasmid-borne gene for a potent yeast exopolyphosphatase [poly(P)ase]. As a consequence, resistance to H2O2 was greatly diminished, particularly in katG (catalase HPI) mutants, implying a major role for the other catalase, the stationary-phase KatE (HPII), which is rpoS dependent. Resistance was restored to wild-type levels by complementation with plasmids expressing ppk, the gene for PPK [the polyphosphate kinase that generates poly(P)]. Induction of expression of both katE and rpoS (the stationary-phase sigma factor) was prevented in cells in which the poly(P)ase was overproduced. Inasmuch as this inhibition by poly(P)ase did not affect the levels of the stringent-response guanosine nucleotides (pppGpp and ppGpp) and in view of the capacity of additional rpoS expression to suppress the poly(P)ase inhibition of katE expression, a role is proposed for poly(P) in inducing the expression of rpoS.

Acid Anhydride Hydrolases↗
PubMed1997

Purification and characterization of UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine.

A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.

Animals↗
PubMed1997

Somatodendritic localization of phosphorylated tau in neonatal and adult rat cerebral cortex.

We studied the distribution of phosphorylated tau (p-tau) in neonatal and adult rat cerebrum using monoclonal antibodies that recognize distinct tau epitopes in a phosphorylation-dependent manner. In neonatal cortex, p-tau was detected in the apical dendrites and somata of pyramidal neurons as well as in axons. In adults, p-tau was present predominantly in neuronal somata and dendrites and, to a lesser extent, in axons, whereas tau-1, that recognizes a dephosphorylated tau epitope, immunolabeled neuropil and axons, and not the neuronal somata and dendrites. These results suggest that tau phosphorylated at multiple sites is present in somatodendritic compartments of rat cortical neurons throughout life, and that phosphorylation may be one of the determinants for the compartmentalization of tau in the brain.

Animals↗
PubMed1997

Specific increase in amyloid beta-protein 42 secretion ratio by calpain inhibition.

Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively. Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species. Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD). Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD. However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity. Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells. A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect. These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

Acetylcysteine↗
PubMed1997

Intracellular generation and accumulation of amyloid beta-peptide terminating at amino acid 42.

Amyloid beta-peptide (Abeta) is known to accumulate in senile plaques of Alzheimer's disease (AD) patients and is now widely believed to play a major role in the disease. Two populations of peptides occur terminating either at amino acid 40 or at amino acid 42 (Abeta1-40 and Abeta1-42). Alternative N-terminal cleavages produce additional heterogeneity (Abetax-40 and Abetax-42). Peptides terminating at amino acid 42 are believed to be the major player in sporadic AD as well as familial AD (FAD). Whereas the cellular mechanism for the generation of Abeta terminating at amino acid 40 is well understood, very little is known about the cleavage of Abeta after amino acid 42. By using two independent methods we demonstrate intracellular Abeta1-42 as well as Abetax-42 but less Abetax-40 and Abeta1-40 in kidney 293 cells stably transfected with wild type beta-amyloid precursor protein (betaAPP) or the FAD-associated Val/Gly mutation. Moreover, retention of betaAPP within the endoplasmic reticulum (ER) by treatment with brefeldin A does not block the cleavage at amino acid 42 but results in an increased production of all species of Abeta terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur within the ER. Treatment of cells with monensin, which blocks transport of (betaAPP) within the Golgi causes a marked accumulation of intracellular Abetax-42 and Abetax-40. Therefore these experiments indicate that the gamma-secretase cleavage of Abeta after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletions of betaAPP as well as inhibition of intracellular acidification by NH4Cl does not block intracellular Abeta1-42 or Abetax-42 production.

Amyloid beta-Peptides↗
PubMed1997

Significant downregulation of the major swine xenoantigen by N-acetylglucosaminyltransferase III gene transfection.

Introduction of the beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene into swine endothelial cells (SEC) reduced their susceptibility to normal human serum (NHS) in complement-mediated cell lysis and also suppressed the antigenicity to human natural antibodies as evidenced by flow cytometric analysis, as well as Griffonia simplicifolia 1 isolectin (1B4 lectin) binding to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (the alpha-galactosyl epitope). Western blot analysis indicated that proteins smaller than 66 kDa had diminished reactivity to NHS and 1B4 lectin. GnT-III, a key enzyme involved in branch formation of N-linked sugars, was found to downregulate the expression of xenoantigen, suggesting that this approach may be of value in clinical xenotransplantation in the future.

Animals↗
PubMed1997

Gastric inhibitory polypeptide activates MAP kinase through the wortmannin-sensitive and -insensitive pathways.

The signal transduction pathways of a cloned human gastric inhibitory polypeptide (GIP) receptor have been investigated in CHO cells stably expressing this receptor. Exposure of GIP receptor expressing cells to GIP significantly increased MAP kinase activity. Time course analysis showed that a rapid and marked increase in MAP kinase activation was detected and that this activation reached maximal levels 10 min after the addition of GIP. Dose-response analysis showed that GIP activated MAP kinase activity in a dose-dependent manner with an ED50 value of 5.9 x 10(-10) M of GIP. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), partially inhibited GIP-induced MAP kinase activation, suggesting that GIP activates MAP kinase through two different, wortmannin-sensitive and -insensitive pathways. It has been demonstrated that in CHO cells cAMP attenuates MAP kinase activity by inhibiting Raf-1. Since GIP elevates intracellular cAMP, we examined the effects of cAMP on MAP kinase activation. Interestingly, forskolin, which increased intracellular cAMP levels, significantly inhibited MAP kinase activation by GIP, but did not affect MAP kinase activation by GIP in the presence of wortmannin, suggesting that the wortmannin-sensitive pathway activates an MAP kinase cascade at or above the level of Raf-1 and that the wortmannin-insensitive pathway activates an MAP kinase cascade below the level of Raf-1. These findings demonstrate that the GIP receptor is linked to the MAP kinase cascade via at least two different pathways.

Androstadienes↗
PubMed1997

Overexpression of N-acetylglucosaminyltransferase III disrupts the tyrosine phosphorylation of Trk with resultant signaling dysfunction in PC12 cells treated with nerve growth factor.

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) is a pivotal glycosyltransferase which participates in branch formation by catalysis of the synthesis of a bisecting GlcNAc structure in N-glycans. These structures are thought to be one of the unique features of the N-glycans of neural tissues. To examine the intracellullar role of GnT-III expression and its product in neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells which normally express a low level of GnT-III. In the GnT-III gene-transfected cells, lectin blot analysis showed that some glycoproteins showed increased levels of bisecting GlcNAc structures. Following treatment with nerve growth factor (NGF) the control cells showed neurite outgrowth for differentiation whereas the transfectants showed no morphological response or change in the rate of cell growth. Transient tyrosine phosphorylation of the Trk/NGF receptor was detected at 5-15 min after NGF treatment in control cells, but not detected in the GnT-III gene-transfected cells despite the intact binding of NGF to the cells. Moreover the dimerization of Trk with NGF treatment was not induced in the GnT-III transfectant as compared with the dimerization seen in control cells. These results indicate that overexpression of GnT-III gene in PC12 cells affects some functions of glycoprotein receptors such as Trk by alteration of N-glycan structures, and results in changes in the intracellular signaling pathway of tyrosine phosphorylation modified by NGF.

Animals↗
PubMed1997

Bisecting GlcNAc structures act as negative sorting signals for cell surface glycoproteins in forskolin-treated rat hepatoma cells.

The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:beta-D-mannoside-beta-1, 4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans. We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA). In whole cell lysates, the E-PHA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels. In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface. Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and beta-glucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.

Acetylglucosamine↗