Lewy bodies: purification from diffuse Lewy body disease brains.
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Antibodies↗
Biomedical subjects
Y Ihara
Publications and source records attributed to Y Ihara.
Senescence-accelerated mouse. Neurochemical studies on aging.
Senescence-accelerated mouse (SAMP8) is known as a murine model of accelerated aging and memory dysfunction. The binding activity of [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK-11195) as a neurochemical marker of gliosis markedly increased with aging in the cerebral cortex and hippocampus of SAMP8. Immunoreactivity for glial fibrillary acidic protein (GFAP) was also enhanced. A beta-amyloid precursor protein (APP)-like immunoreactivity and 27-kDa-carboxyl terminal fragments of APP increased in SAMP8 brain. In addition, anti-APP antibody stained reactive astrocytes surrounding spongy degeneration in brain stern of SAMP8. These results suggest that astrocytosis and production of APP-derived fragments occur markedly in SAMP8 brains.
Aging↗
Aberrant glycosylation of E-cadherin enhances cell-cell binding to suppress metastasis.
Introduction of the beta1-4 N-acetylglucosaminyltransferase (GnT-III) gene was reported to suppress metastasis in highly metastatic B16-hm murine melanoma cells (Yoshimura, M., Nishikawa, A. , Ihara, Y., Taniguchi, S., and Taniguchi, N.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8754-8758). In this study, the effect of GnT-III gene transfer on E-cadherin was studied, since E-cadherin acts as a suppressor of metastasis. E-cadherin expression at cell-cell contacts of B16-hm cells expressing high GnT-III activity was greater than controls without affecting transcription. Lectin blotting showed that E-cadherin from GnT-III transfectants was glycosylated by ectopically expressed GnT-III. The glycosylated E-cadherin exhibited the delayed turnover and the decreased release from cell surface, as compared with the native E-cadherin, resulting in the elevated expression at the cell-cell border of GnT-III transfectants. Furthermore, cell-cell aggregation was enhanced in GnT-III transfectants, indicating that the glycosylated E-cadherin is biologically functional. These results suggest that the glycosylated E-cadherin contributes to the suppression of metastasis by the introduction of GnT-III gene into melanoma cells.
Animals↗
Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against tau protein.
A monoclonal antibody (AP422) specific for phosphoserine 422 in microtubule-associated protein tau has been produced. It strongly labels paired helical filament (PHF) tau from Alzheimer's disease brain in a phosphorylation-dependent manner. By contrast, AP422 only labels a small fraction of fetal tau and a very small fraction of tau from adult brain. The amount of tau phosphorylated at Ser-422 in normal brain is minor relative to that phosphorylated at sites recognized by other phosphorylation-dependent anti-tau antibodies of known epitope. It follows that AP422 is the most specific anti-tau antibody available for detecting the neurofibrillary lesions of Alzheimer's disease. We also show that Ser-422 in tau is a good in vitro substrate for mitogen-activated protein kinase, but not for glycogen synthase kinase-3 or neuronal cdc2-like kinase.
Alzheimer Disease↗
Bisecting N-acetylglucosamine on K562 cells suppresses natural killer cytotoxicity and promotes spleen colonization.
beta 1-4 N-acetylglucosaminyltransferase (GnT-III) catalyzes the formation of bisecting N-acetylglucosamine (GlcNAc) in the biosynthesis of N-linked oligosaccharides. To examine the effect of bisecting GlcNAc on the natural killer (NK) cytotoxicity, the GnT-111 gene was introduced into NK-sensitive K562 cells that have no detectable GnT-III activity. We obtained three clones stably expressing high GnT-III (positive transfectants). Introduction of the GnT-III gene resulted in an increase of bisecting GlcNAc and a decrease of external sialic acid as well as tri- and tetraantennary sugars, as judged by flow cytometry. Compared to controls, the NK cytotoxicity was completely blocked against positive transfectants. The binding of effector cells to positive transfectants was also decreased. After s.c. injection into nude mice, positive transfectants produced spleen colonization, although no spleen lesions were formed by control cells. In nude mice depleted of NK cells by anti-asialo GM1 antibody, both positive transfectants and controls produced spleen colonization equally. These results indicate that K562 cells expressing GnT-III are resistant to NK cytotoxicity, resulting in spleen colonization in nude mice.
Acetylglucosamine↗
Telomere shortening in peripheral blood cells was related with aging but not with white blood cell count.
Telomeres in somatic cells are progressively shortened with aging. We investigated the relationship between the telomere length and other factors which may affect the frequency of cell divisions, in peripheral blood cells. Shortening of telomeric repeats was correlated with aging (p < 0.0001), but not with white blood cell count, neutrophil count, and smoking habit. Not only the number of cell divisions, but also some other factors, such as upregulation level of telomerase activity concomitant with the cell division in hematopoietic progenitor cells, might affect the length of telomeric repeats in blood cells.
Adult↗
Tau is widely expressed in rat tissues.
The microtubule-associated protein tau, a major component of paired helical filaments in Alzheimer's disease, had been thought to be a neuron-specific protein. We investigated various rat tissues using both reverse transcriptase-coupled polymerase chain reaction and immunoblotting. tau was found to be widely expressed in many tissues besides the nervous system: at relatively high levels in the heart, skeletal muscle, lung, kidney, and testis and at low levels in the adrenal gland, stomach, and liver. In terms of the tau isoform expression, tissues fall into three classes: those expressing predominantly small tau, those expressing predominantly big tau, and those expressing both at comparable levels. The phosphorylation state of tau varied among the tissues, as shown by differences in the extents of changes in the reactivities with Tau 1 and electrophoretic mobilities after dephosphorylation. It is notable that tau in many nonneural tissues was highly phosphorylated at Ser396 (according to the numbering of the 441-residue human tau isoform). Thus, tau is widely expressed in rat tissues.
Animals↗
Remodeling of cell surface glycoproteins by N-acetylglucosaminyltransferase III gene transfection: modulation of metastatic potentials and down regulation of hepatitis B virus replication.
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Animals↗
A fluorescent assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase activity, involving high performance liquid chromatography.
An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.
Animals↗
Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli.
Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.
Amino Acid Sequence↗
Proliferating cell nuclear antigen (PCNA) expressed in human leptomeninges.
Here we report on the presence of proliferating cell nuclear antigen (PCNA) in human leptomeninges from 35 normal subjects with ages ranging from 57 to 94 years. Strong immunoreactivity with PC10 (a monoclonal antibody to PCNA) was detected in the nuclei of meningothelial cells, smooth muscle cells of leptomeningeal vessels, and ependymal cells. An immunoblot of leptomeningeal homogenate with PC10 showed the presence of a single band at 35 KD, the expected molecular mass of PCNA. Ki-67, another marker for cell proliferation, was undetectable in human leptomeninges. These observations point to isolated PCNA expression in tissue in which cells are not actively proliferating.
Aged↗
Endothelin-1-like immunoreactivity and endothelin receptors in the human placenta from normotensive and hypertensive pregnancies.
The levels of endothelin-1-like immunoreactivity (ET-1-LI) and characteristics of endothelin receptors in the chorionic villous tissue of human placenta were determined. The ET-1-LI level in chorionic villous tissue obtained from normal term placenta was 2,450 +/- 940 pg/g wet weight (mean +/- SD, n = 4). Further analysis using gel permeation chromatography and reverse-phase high performance liquid chromatography showed that the main ET-1-LI constituent of ET-1-LI in this tissue was ET-1. Scatchard analysis of [125I]ET-1 binding to the membrane fraction of chorionic villous tissue obtained from term placenta showed high affinity receptor sites with an apparent dissociation constant (Kd) of 23.6 +/- 11.1 pM and a Bmax value of 388 +/- 238 fmol/mg protein (n = 5). The same binding study with [125I]ET 3 showed a Kd of 13.9 +/- 3.8 pM and a Bmax value of 176 +/- 78 fmol/mg protein (n = 5). These results suggest that both ET-A and ET-B receptors (ET-AR and ET-BR) are expressed in chorionic villous tissue. This finding was further confirmed by Northern blot analysis showing the expression of both ET-AR and ET-BR mRNAs in this tissue. ET-1-LI in the umbilical venous plasma of the newborns from women with pregnancy-induced hypertension (PIH) (38.3 +/- 10.4 pg/mL, n = 5) was significantly (P < 0.05) higher than that in the normal newborns from normotensive pregnant women (26.3 +/- 5.2 pg/mL, n = 12). However, in placental chorionic villous tissue obtained from PIH women, both ET-1-LI level and ET binding profile were not different from those in chorionic villous tissue from normotensive pregnant women. These results suggest that the abundant ET-ET receptor system is present in the placental chorionic villous tissue and that this system is not the major factor of the pathogenesis of placental dysfunction occurring in PIH because these systems are similar in normotensive and hypertensive pregnancies.
Chorionic Villi↗
Identification of two missense mutations in the GIP receptor gene: a functional study and association analysis with NIDDM: no evidence of association with Japanese NIDDM subjects.
Gastric inhibitory polypeptide (GIP) potently stimulates insulin secretion from pancreatic islets in the presence of glucose as an incretin. Because the insulinotropic effect of GIP is reduced in NIDDM, it should be clarified whether defects in the GIP receptor gene contribute to the impaired insulin secretion in NIDDM. Using genomic DNA samples from Japanese NIDDM and non-NIDDM subjects, we have investigated the entire coding region of the GIP receptor gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). We have identified two missense mutations, Gly198-->Cys (Gly198Cys) in exon 7 and Glu354-->Gln (Glu354Gln) in exon 12. Investigation of the function of GIP receptor with either of these mutations reveals a half-maximal stimulation value of GIP-induced cAMP response in Chinese hamster ovary cells expressing the GIP receptor with Gly198Cys of 6.3 +/- 1.2 x 10(-10) mol/l (n = 3), which was considerably higher than that of the normal GIP receptor, 9.4 +/- 3.8 x 10(-12) mol/l GIP (n = 3), whereas that of the GIP receptor with Glu354Gln was not significantly different from that of the normal GIP receptor. To assess the possible role of the GIP receptor gene in genetic susceptibility to NIDDM, we have examined the allelic frequencies of Gly198Cys and Glu354Gln in NIDDM and control subjects. Association studies show no relationship between NIDDM and either of the two mutations.
Alleles↗
Transient ischemia depletes free ubiquitin in the gerbil hippocampal CA1 neurons.
We investigated ubiquitin immunoreactivity in the post-ischemic gerbil hippocampus using a panel of ubiquitin antibodies. Immunostaining for ubiquitin in the hippocampus was strongly dependent on the antibodies used. With rabbit polyclonal antibody U-5379, immunoreactivity disappeared from the hippocampus in the early reperfusion period and reappeared in the dentate granule cells and CA3 pyramidal cells but never in the CA1 pyramidal cells. In contrast, rat monoclonal antibody DF2 and mouse monoclonal antibody MAB1510 showed sustained immunoreactivity in the CA1 during the 48-hour reperfusion period. On the immunoblots of gerbil brain homogenates, three antibodies, U-5379, DF2 and MAB1510, exhibited similar specificities; all three labeled free ubiquitin most strongly. Immunoprecipitation disclosed that, under nondenaturing conditions, U-5379 bound exclusively free ubiquitin, whereas DF2 and MAB1510 had little affinity for free ubiquitin but appeared to have more affinity for conjugated ubiquitin. Immunoabsorption of these antibodies with free ubiquitin confirmed the above result. It is most likely that U-5379 recognized free ubiquitin in the tissue, whereas DF2 and MAB1510 recognized preferentially conjugated ubiquitin. Thus, transient ischemia depletes free ubiquitin but not conjugated ubiquitin in the CA1. This depletion may be caused by impaired conversion from conjugated to free ubiquitin and/or failure of de novo ubiquitin synthesis.
Animals↗
Amyloid beta protein deposition in normal aging has the same characteristics as that in Alzheimer's disease. Predominance of A beta 42(43) and association of A beta 40 with cored plaques.
Two distinct species of amyloid beta protein (A beta) with different C-termini, A beta 42(43) and A beta 40, are deposited in senile plaques (SP) of Alzheimer's disease (AD), with the former being far predominant. To investigate whether A beta 42(43) also predominates over A beta 40 in normal aging, we examined by immunocytochemistry the C-termini of A beta in SP in the brains of non-demented aged individuals and compared the results with those in AD. Virtually all SP were A beta 42(43)-positive; of these 12% in non-demented aged individuals and 25% in AD patients (mean of three areas examined) were also A beta 40-positive. In both the AD and non-demented groups, 2/3 of the A beta 40-positive SP were typical cored SP. These results indicate that A beta 42(43) is the predominant species deposited in SP in normal aging, and there is no qualitative difference in terms of the C-terminus of A beta in the parenchymal amyloid deposition between normal aging and AD.
Aged↗
Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease.
Lewy bodies (LBs) are the pathological hallmarks of degenerating neurons in the brains of patients with Parkinson's disease and diffuse Lewy body disease. We developed a novel purification procedure for LBs using sucrose density separation followed by fluorescence-activated particle sorting, and we raised > 15 monoclonal antibodies to LBs purified from diffuse Lewy body disease brains. The monoclonal antibody that stained the largest number of LBs most intensely did not recognize ubiquitin in free or monoubiquitinated forms nor the ubiquitin conjugating enzymes, but it did react with polyubiquitin chains as well as with high molecular weight polyubiquitinated LB-derived proteins. Thus, these results suggest that LBs contain polyubiquitin chains. Although polyubiquitination of LB proteins may trigger ubiquitin-proteasome proteolytic pathways, the incomplete activation of these pathways could play a mechanistic role in the formation of LBs in neurodegenerative diseases.
Aged↗
Predominant deposition of amyloid-beta 42(43) in plaques in cases of Alzheimer's disease and hereditary cerebral hemorrhage associated with mutations in the amyloid precursor protein gene.
Amyloid (A beta) deposition was investigated in cases of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis, Dutch type, due to mutations in the amyloid precursor protein (APP) gene using the end-specific monoclonal antibodies BA27 and BC05 that recognize A beta 40 or A beta 42(43), respectively. In cases of APP717 mutation the predominant A beta species within plaques terminate at A beta 42(43) with relatively little A beta 40 being present. The total amount of A beta deposited as A beta 42(43) is significantly greater than in sporadic Alzheimer's disease, consistent with the suggestion that this mutation might influence the processing of APP so as to produce more of the highly aggregatable form, A beta 1-42. In cases of APP670/671 mutation the major peptide in plaques is also A beta 42(43), although the proportion of plaques containing A beta 40, and the total A beta load is similar to that in sporadic Alzheimer's disease. As in sporadic Alzheimer's disease, the vascular amyloid in APP670/671 and APP717 and in cases of hereditary cerebral hemorrhage with amyloidosis, Dutch type is predominantly A beta 40 in this latter disorder, however, parenchymal deposits are exclusively A beta 42(43). Although the various APP mutations may influence the type, quantity, and location of A beta deposited, the predominant, and possibly the initial, species deposited in the brain parenchyma is A beta 42(43).
Adult↗
Transfection of N-acetylglucosaminyltransferase III gene suppresses expression of hepatitis B virus in a human hepatoma cell line, HB611.
beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked oligosaccharides and forms a bisecting GlcNAc structure. Although the biological meaning of the bisecting GlcNAc structure remains unclear, it is known that the attachment of a bisecting GlcNAc inhibits further processing of oligosaccharides by other glycosyltransferases. To investigate whether or not structural changes of oligosaccharides affect secretion and gene expression of hepatitis B virus (HBV), we introduced the GnT-III gene into a human hepatoma cell line, HB611, which secreted HBV-related proteins into the medium. Positive transfectants were cloned by hygromycin resistant selection. Three clones have high activities of GnT-III and secreted lower levels of HBV-related proteins into the medium in comparison with other clones. These clones showed marked suppression of HBV-related mRNAs and an increased binding with E-PHA as judged by lectin blot. Expression of beta actin, alpha fetoprotein, albumin, and prealubmin was not correlated with GnT-III activity in all the seven clones. Treatment of these cells with tunicamycin or swainsonine resulted in enhanced expression of HBV-related mRNA. These results indicate that some glycoproteins whose oligosaccharide structures are changed by over-expression of GnT-III suppress HBV gene expression.
Blotting, Northern↗