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H Ihara

Publications and source records attributed to H Ihara.

At least 19 recordsLinked to original sources
PubMed2001

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

Acetylglucosamine↗
PubMed2001

The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

Bacillus subtilis↗
PubMed2001

Bezafibrate attenuates the overexpression of plasminogen activator inhibitor-1 messenger RNA by a combination of mono-unsaturated fatty acid and insulin in hepG2 cells.

The effects of bezafibrate (PPAR alpha activator) and troglitazone (PPAR gamma activator) on the expression of plasminogen activator inhibitor type-1 (PAI-1) in HepG2 cells were investigated. Exposure of the cells for 24 hours to either oleic acid or insulin showed no obvious effects on PAI-1 synthesis, whereas the combination of the two agents induced a 2.3-fold increase in PAI-1 synthesis, which was accompanied by a 3-fold increase in both the 2.2 kb and 3.2 kb forms of PAI-1 mRNA. This up-regulation of PAI-1 synthesis was attenuated by bezafibrate in a dose-dependent manner (1-100 microM) with 30% reversal at 100 microM. In contrast, troglitazone further stimulated PAI-1 synthesis to 140% of the level obtained in the presence of both oleic acid and insulin. This attenuation by bezafibrate and enhancement by troglitazone required the presence of both oleic acid and insulin. It is interesting that PAI-1 expression was affected so differently by these two PPAR activators.

Bezafibrate↗
PubMed2001

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin.

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.

Animals↗
PubMed2001

The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction.

We demonstrated that a region in the stem of N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the alpha-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.

Animals↗
PubMed2001

The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin.

The enzyme GnT-III (beta 1,4-N-acetylglucosaminyltransferase III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) residue on glycoproteins. Our previous study described that the transfection of GnT-lll into mouse melanoma cells results in the enhanced expression of E-cadherin, which in turn leads to the suppression of lung metastasis. It has recently been proposed that the phosphorylation of a tyrosine residue of beta-catenin is associated with cell migration. The present study reports on the importance of bisecting GlcNAc residues by GnT-lll on tyrosine phosphorylation of beta-catenin using three types of cancer cell lines. An addition of bisecting GlcNAc residues to E-cadherin leads to an alteration in cell morphology and the localization of beta-catenin after epidermal growth factor stimulation. These changes are the result of a down-regulation in the tyrosine phosphorylation of beta-catenin. In addition, tyrosine phosphorylation of beta-catenin by transfection of constitutively active c-src was suppressed in GnT-III transfectants as well as in the case of epidermal growth factor stimulation. Treatment with tunicamycin abolished any differences in beta-catenin phosphorylation for the mock vis à vis the GnT-lll transfectants. Thus, the addition of a specific N-glycan structure, the bisecting GlcNAc to E-cadherin-beta-catenin complex, down-regulates the intracellular signaling pathway, suggesting its implication in cell motility and the suppression of cancer metastasis.

Acetylglucosamine↗
PubMed2001

The differential effect of angiotensin II and angiotensin 1-7 on norepinephrine, epinephrine, and dopamine concentrations in rat hypothalamus: the involvement of angiotensin receptors.

Angiotensin 1-7 has been recently claimed the active member of the angiotensins' family. In the present study we compared the effect of angiotensin II and angiotensin 1-7 on the concentration of dopamine, serotonin, epinephrine, and norepinephrine and some of their metabolites in the rat hypothalamus, where the levels of angiotensins are particularly high. Intracerebroventricular injection of angiotensin II, but not angiotensin 1-7, time-dependently elevated the levels of both epinephrine (p < 0.05) and norepinephrine (p < 0.05) in the hypothalamus and both effects could be prevented by intracerebroventricular injection of either AT(1) (candesartan), AT(2) (PD123319) or AT(1-7) (A-779) receptor antagonist. Neither angiotensin II nor angiotensin 1-7 produced any changes in the level of dopamine, dihydroxyphenylacetic acid, homovanilic acid, serotonin, 5-hydroxyindoleacetic acid, or tryptophan at any time point in comparison with the control groups. However, AT(1) but not AT(2) receptor blockade, unmasked the stimulatory effect of angiotensin 1-7 on dopamine concentration in the hypothalamus. Thus, angiotensin II and its active metabolite angiotensin 1-7 regulate selectively, albeit differentially, adrenergic, noradrenergic and dopaminergic systems in the hypothalamus, the effects that involve AT(1), AT(2) and AT(1-7) angiotensin receptors.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2001

[A case of pheochromocytoma with von Recklinghausen's and review of 67 Japanese cases].

A 19-year-old woman with von Recklinghausen's disease was referred to our hospital because of right adrenal pheochromocytoma. The tumor was detected incidentally with the abdominal ultrasonography when she complained epigastralgia to the home doctor who treated her hypertension. Plasma and urinary catecholamines level were elevated. The tumor was removed by laparoscopy assisted adrenalectomy without pneumoperitoneum. The resected specimen was 35 x 60 x 75 mm in size and weighed 70 g. Pathological diagnosis was adrenomedullary pheochromocytoma. Postoperative course was uneventful. She has been well with no signs of recurrence after 7.5 years. We reviewed 67 Japanese patients previously reported as von Recklinghusen's disease with pheochromocytoma. Of the 60 patients whose details were described, 16.7% had metastases and pathological malignancy from pheochromocytoma.

Adrenal Gland Neoplasms↗
PubMed2000

Retention versatility of silica-supported comb-shaped crystalline and non-crystalline phases in high-performance liquid chromatography.

Silica-supported comb-shaped polymer (Sil-ODA18) provided a specific selectivity towards aromatic and non-aromatic solutes. Selectivity of Sil-ODA18 and ODS (polymeric and monomeric) columns towards polycyclic aromatic hydrocarbons and geometric isomers have been discussed in respect of molecular shapes and sizes. The retention versatility of this phase arises from the orientation change on transition from the ordered crystalline to the less ordered non-crystalline state. Selectivity in the crystalline and non-crystalline regions of Sil-ODA18 is similar to that of polymeric and monomeric ODS phases, respectively. Sil-ODA18 column also exhibited the characteristic temperature dependency of liquid-crystalline phases, i.e., an increase in retention with increasing temperature on transition from crystalline to isotropic state.

Chromatography, High Pressure Liquid↗
PubMed2000

Differential effects of nicotine against stress-induced changes in dopaminergic system in rat striatum and hippocampus.

A number of studies have shown an increase in nicotine self-administration among smokers when exposed to stress. Since it is well known that nicotine or stress alter the dopaminergic system, we examined the effect of chronic nicotine administration on the dopamine level and its metabolism in the striatum and the hippocampus during stressful conditions in rats. Nicotine (0.4 mg/kg, i.p. for 14 days) increased the dopamine level in the striatum (P<0. 05) and decreased it in the hippocampus (P<0.05) in comparison with the effect of saline. Three hours of water-immersion restraint stress sharply elevated the dopamine level (P<0.05) and reduced the 3-methoxytyramine level (P ranged from 0.05 to 0.001 depending on the area and time point) in both brain regions studied, while dihydroxyphenylacetic acid and homovanilic acid levels were not altered. Nicotine pretreatment attenuated some of these changes in a region- and time-dependent manner. However, stress induced a decrease in dopamine turnover in the hippocampus (P<0.05) but not in the striatum, and nicotine failed to prevent this effect. Stress-induced alterations gradually returned toward normal during the 48-h observation period, and in some cases this was facilitated by nicotine. Thus, we demonstrated differential, region- and time-dependent protective effects of chronic nicotine administration against stress-induced changes in dopamine levels and release in brain regions critically affected by stress.

Animals↗
PubMed2000

An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine.

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.

Artifacts↗
PubMed2000

Analysis of the human sleep electroencephalogram by the correlation dimension.

Sleep electroencephalograms (EEG) of healthy male subjects were analyzed by the correlation dimension. The mean correlation dimensions significantly decreased from stage 'awake' to stages 1, 2 and 3 and increased during rapid eye movement sleep. The mean correlation dimensions of the sleep EEG in the second half of the night were significantly higher than those in the first half of the night. These significant changes were also seen during sleep stage 2.

Adult↗
PubMed2000

Coagulation-associated enhancement of fibrinolytic activity via a neutralization of PAI-1 activity.

Total fibrinolytic activity in the vasculature is finely tuned by the balance between tissue plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). Although PAI-1 targets plasminogen activators, it also reacts with other serine proteases such as thrombin and factor Xa. The latter was shown to interact with PAI-1 only when a physiological concentration of calcium ions (Ca++) is present. Through such interaction, thrombin and Ca++-bound factor Xa shortened fibrin clot lysis times in a purified system by neutralizing PAI-1 activity. Both unfractionated heparin and vitronectin were shown to enhance the clot lysis further. Together with the cleavage and inactivation of PAI-1 by human neutrophil elastase, which was reported previously from our laboratory, such neutralization of PAI-1 activity by these serine proteases was shown to be strongly involved in the coagulation-associated enhancement of fibrinolytic activity.

Anticoagulants↗
PubMed2000

Genomic structure and promoter analysis of the human alpha1, 6-fucosyltransferase gene (FUT8).

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1,6-fucosyltransferase (alpha1,6FucT) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the asparagine-linked GlcNAc residue of complex N-glycans via alpha1,6-linkage. We have cloned the genomic DNA which encodes the human alpha1,6FucT gene ( FUT8 ) and analyzed its structure. It was found that the gene consists of at least nine exons spanning more than a 50 kbp genomic region, and the coding sequence is divided into eight exons. The translation initiation codon was located at exon 2, and thus exon 1 encodes only 5'-untranslated sequences. Transcription initiation site of FUT8 was determined by 5'-rapid amplification of the cDNA end and a primer-extension analysis using the total RNA isolated from SK-OV-3 cells, which have a high level of alpha1,6FucT activity. We then characterized the FUT8 promoter region by a reporter gene assay. The luciferase reporter assay indicated that the 5'-flanking region of exon 1, which covered about 1 kbp, conferred the promoter activity in SK-OV-3 cells. This region contains potential binding sites for some transcription factors, such as bHLH, cMyb, GATA-1, as well as a TATA-box, but not a CCAAT motif. 5'-Untranslated sequences found in ESTs and the cDNA for the FUT8 suggest the presence of an additional exon(s) at the upstream of the first exon identified in this study, and therefore, the transcription of the gene would be regulated by multiple promoters.

Base Sequence↗
PubMed2000

Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III.

The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.

Acetylgalactosamine↗
PubMed2000

Group and case study of the dysexecutive syndrome in alcoholism without amnesia.

OBJECTIVES: To test the dysexecutive syndrome (DES) hypothesis of chronic alcoholism by the neuropsychological group and case study approaches. METHODS: A comprehensive neuropsychological assessment, including the "behavioural assessment of dysexecutive syndrome", a battery of tests recently designed to be "ecologically valid", was administered to 17 patients with chronic alcoholism without amnesia to examine executive functions, intelligence, and memory. In terms of each neuropsychological measure, reciprocal analyses of group means and individual case profiles were conducted: for the first contrasting the alcoholic patients with 17 age matched healthy subjects; and for the second making intersubject and intrasubject comparison of the patients, according to percentile basis impairment indices obtained from the control subjects. RESULTS: Despite relatively unimpaired memory and intelligence, the patients as a whole had the impairment of a wide range of executive domains, extending to "everyday" problem solving as well as more elementary aspects of executive functions, such as visuospatial performance, mental set shifting, and the inhibition of habitual behaviour. The profile analysis divided individual patients into four groups: the representative DES characterised by a clear dissociation between impaired executive functions and preserved intelligence and memory; the group of a modified dysexecutive pattern in which memory as well as executive functions were impaired with intelligence preserved; the group of general cognitive deterioration; and the group of unimpaired cognitive functioning. About two thirds of the patients were categorised into either the first or the second type of DES. CONCLUSION: DES characterised by the even more pronounced impairment of executive functions than of intelligence and memory afflicts a considerable proportion of patients with chronic alcoholism. Due to its subtlety, this would be potentially left out, unless appropriate behavioural measures were administered. This condition may prevent patients with alcoholism from achieving full recovery and benefiting from rehabilitation.

Activities of Daily Living↗
PubMed2000

Removal of parvovirus B19 from hemoglobin solution by nanofiltration.

Parvovirus B19 (B19), which may contaminate red cell components for blood transfusion, is known to be resistant to several viral inactivation methods. To increase the safety of hemoglobin solutions as a source of red cell substitutes, we investigated the removal of parvovirus B19 from hemoglobin solution using nanofiltration. The hemoglobin solution spiked with parvovirus B19 was tangentially filtered using the BMM-35 filter (mean pore size of 35 nm) followed by BMM-15. The parvovirus B19 of 10(8.5) PT50 (median PCR titer)/10 microL was not changed after the BMM-35 filtration. However, the BMM-15 filtration decreased the parvovirus B19 from 10(8.3-8.7) PT50 to 10(1.3-2.2) PT50, indicating more than 6 log10 reduction. When the initial parvovirus B19 of 10(6.0) PT50 was subjected to the BMM-15 filtration, the residual virus was 10(-0.3-0.5) PT50 orundetected in some fraction of the filtrate. Hemoglobin recovery was 70.4 +/- 3.4%. The ratio of methemoglobin was not changed during the filtration. These findings indicate that the BMM-15 filtration is a promising approach to prepare a safer hemoglobin solution for red cell substitutes.

Blood Substitutes↗