Biomedical subjects

Akira Hara

Publications and source records attributed to Akira Hara.

At least 91 records · Page 5Linked to original sources

PubMed2003

Identification of amino acid residues involved in substrate recognition of L-xylulose reductase by site-directed mutagenesis.

L-Xylulose reductase (XR) catalyzes the oxidoreduction between xylitol and L-xylulose in the uronate cycle. The enzyme has been shown to be identical to diacetyl reductase, an enzyme that reduces alpha-dicarbonyl compounds. XR belongs to the short-chain dehydrogenase/reductase family, and shows high sequence identity with mouse lung carbonyl reductase (MLCR), an enzyme that reduces 3-ketosteroids but not sugars. In this study, we have confirmed the roles of Ser136, Tyr149 and Lys153 of XR as the catalytic triad by drastic loss of activity resulting from the mutagenesis of S136A, Y149F and K153M in rat XR. We have also constructed several mutant XRs, in which putative substrate binding residues from rat XR were substituted with those found in the corresponding positions of MLCR, in order to identify amino acids responsible for the different substrate recognition of the enzymes. While single mutants at positions 137, 143, 146, 190 and 191 caused little or moderate change in substrate specificity, a double mutant (N190V and W191S) and triple mutant (Q137M, L143F and H146L) resulted in almost loss of activity for only the sugars. In addition, the triple mutant exhibited 3-ketosteroid reductase activity, which was further enhanced by quintuple mutagenesis of the above five residues. These results suggest the importance of the size and hydrophobicity of the five residues for substrate recognition by XR and MLCR. Furthermore, the mutant enzymes containing a Q137M mutation were stable against cooling, which provides a structural mechanism of the cold inactivation that is a characteristic of the rodent XR.

Aldehyde Reductase↗

PubMed2003

Involvement of phospholipase D in insulin-like growth factor-I-induced activation of extracellular signal-regulated kinase, but not phosphoinositide 3-kinase or Akt, in Chinese hamster ovary cells.

Available evidence suggests the involvement of phospholipase D (PLD) in cell proliferation and survival. Phosphoinositide 3-kinase (PI 3-kinase)/Akt and extracellular signal-regulated kinases (ERKs) are signalling molecules that have essential roles in cell proliferation and survival. We previously demonstrated that sphingosine 1-phosphate (S1P)-induced PLD activation via the G-protein-coupled receptor endothelial differentiation gene (EDG) 3/S1P(3) was involved in S1P-induced stimulation of PI 3-kinase and Akt. In the present study, we examined the involvement of two PLD isozymes, PLD1 and PLD2, in insulin-like growth factor (IGF)-I receptor tyrosine kinase-mediated stimulation of PI 3-kinase/Akt and ERKs. IGF-I and to a lesser degree S1P stimulated PI 3-kinase activity in Chinese hamster ovary cells overexpressing EDG3/S1P(3). IGF-I-induced ERK phosphorylation was suppressed by butan-1-ol, but not butan-2-ol, whereas no effect of butanol was observed in IGF-I-induced Akt activation in S1P(3)-overexpressing Chinese hamster ovary cells. Overexpression of wild-type PLD1 and PLD2 substantially potentiated S1P-, but not IGF-I-, induced activation of PI 3-kinase and Akt, whereas overexpression of the catalytically inactive mutant of PLD1 or PLD2 did not affect the responses to either agonist. On the other hand, overexpression of wild-type PLD1 and PLD2 potentiated IGF-I- and, to much smaller extents, S1P-induced ERK stimulation. ERK activation by IGF-I as well as S1P was dependent on Ras, but Akt activation by IGF-I was not dependent on Ras. These results suggest that PLDs are involved in growth factor regulation of at least two signalling pathways, PI 3-kinase/Akt and ERKs, depending on the class of cell-surface receptors.

Animals↗

PubMed2003

Glucocorticoids and dehydroepiandrosterone sulfate ameliorate ischemia-induced injury of the cochlea.

This study aimed to evaluate the effects of steroidal drugs on the functional recovery of the cochlea after transient ischemia. Albino guinea pigs were subjected to transient cochlear ischemia of 30 min duration, and the threshold shifts of the compound action potential (CAP) from the pre-ischemic values were evaluated 4 h after ischemia. Pre-ischemic administration of a glucocorticoid, prednisolone or methylprednisolone, significantly ameliorated the post-ischemic CAP threshold shifts as compared with control animals at a relatively wide range of doses. Post-ischemic administration of these glucocorticoids also exhibited protective effects. Pre-ischemic administration of dehydroepiandrosterone sulfate significantly decreased the post-ischemic CAP threshold shifts 4 h after ischemia. The present results indicate that glucocorticoids and dehydroepiandrosterone sulfate possess therapeutic effects against ischemic injury of the cochlea, such as idiopathic sudden sensorineural hearing loss.

Action Potentials↗

PubMed2003

Nevoid basal cell carcinoma syndrome: report of an aggressive case with ethmoid sinus invasion.

Nevoid basal cell carcinoma syndrome, also referred to as Gorlin-Goltz syndrome, is a rare autosomal dominant disorder characterized by multiple basal cell carcinomas, jaw cysts, palmar or plantar pits, ectopic calcification of the falx cerebri, and various skeletal developmental abnormalities. A minority of basal cell carcinomas demonstrate aggressive behavior and involve the craniofacial bones in nevoid basal cell carcinoma syndrome. A non-familial case of nevoid basal cell carcinoma syndrome with a basal cell carcinoma of the eyelid invading to the ethmoid sinus is reported.

Aged↗

PubMed2003

Xylosidases associated with the cell surface of Penicillium herquei IFO 4674.

Penicillium herquei IFO 4674 is a filamentous fungus that produces a large amount of hydrolases for fibrous polysaccharides. We purified two beta-xylosidases, S1 and S2. The molecular masses of S1 and S2 determined by MALDI-TOF-MS were 103,700 and 37,460 Da. The optimum pHs of S1 and S2 were 4.0 and 6.5, respectively. By several kinds of alcohols, especially glycerol, S1 was activated while S2 was unaffected or inhibited. S1 had a transxylosylation activity, while S2 did not. The s2 gene encoding xylosidase S2 was cloned by PCR with primers designed on the basis of partial amino acid sequences of S2. The s2 consisted of 1005 by encoding 335 amino acids (37,433 Da) and had no secretion signal sequence. The deduced amino acid sequence shows a high identity to that of Bacteroides ovatus xylosidase/arabinosidase (56%), which is a member of the family 43 glycoside hydrolase.

Journal Article↗

PubMed2003

Carcinoid tumor in the female urethral orifice: rare case report and a review of the literature.

We experienced an extremely rare tumor in the female urethral orifice in a 57-year-old Japanese woman. To our knowledge, only two cases of primary urethral carcinoid tumor have been reported. The previous reports of urethral carcinoid tumor were recognized in the male middle urethra and penile urethra. The present case was resected, and diagnosed as a carcinoid tumor by histological, immunohistochemical and ultrastructural findings. The tumor cells were stained by chromogranin A, synaptophsin and neuron-specific enolase, and neurosecretory granules were confirmed with electron microscopy. The patient did not complain of any symptoms until 5 years after the resection of the tumor. Therefore, the case we describe here is the first known report of carcinoid tumor in the Japanese female urethra.

Biomarkers, Tumor↗

PubMed2003

Glycated high-density lipoprotein regulates reactive oxygen species and reactive nitrogen species in endothelial cells.

Nonenzymatic glycosylation of plasma proteins may contribute to the excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelial dysfunction in diabetes. To clarify whether glucose-modified HDL affects the function of endothelial cells, we first examined herein the level of H(2)O(2) generation from cultured human aortic endothelial cells (HAECs) exposed to a glycated oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation for 48 hours with 100 microg/mL of gly-ox-HDL induced significant release of H(2)O(2) from cells and gly-ox-HDL-induced H(2)O(2) formation was inhibited in the presence of diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, stimulation of HAECs with gly-ox-HDL for 48 hours elicited a marked downregulation of catalase and Cu(2+), Zn(2+)-superoxide dismutase (CuZn-SOD), suggesting H(2)O(2) formation by gly-ox-HDL to be due to a disturbance involving oxidant and antioxidant enzymes in the cells. Treatment of HAECs with gly-ox-HDL attenuated the expression of endothelial nitric oxide synthase (eNOS), but not inducible nitric oxide synthase (iNOS), and this was followed by decreased production of nitric oxide (NO) by the cells. Furthermore, in vitro experiments with glycated HDL (gly-HDL) in the presence of 2 mmol/L EDTA and Cu(2+)-oxidized HDL suggested the effect of gly-HDL on endothelial function to be possibly potentiated by additional oxidative modification. Taking all of the above findings together, gly-ox-HDL may lead to the deterioration of vascular function through altered production of reactive oxygen species and reactive nitrogen species in endothelial cells.

Blotting, Western↗

PubMed2003

beta-Catenin mutation is selected during malignant transformation in colon carcinogenesis.

Activating mutations in the beta-catenin gene is thought to be responsible for the excessive beta-catenin signaling involved in the majority of colon carcinomas in rodent models. Our recent study which indicated that beta-catenin mutations are present frequently in early dysplastic lesions of rat colon induced by a colon-specific carcinogen, azoxymethane led us to perform more specifically a comparative study regarding types of the beta-catenin mutation as well as K-ras mutations between such early appearing lesions and colon tumors. Male F344 rats, 6 weeks old, received s.c. injections of azoxymethane (15 mg/kg body weight) once a week for 3 weeks, and were killed at 16 and 46 weeks of age. Colons of animals killed at 16 weeks of age were processed for early altered lesions. Colon tumors from animals killed at 46 weeks of age were evaluated histopathologically. Laser capture microdissection system was used to obtain DNA of epithelial cells in both intramucosal lesions and colon tumors. After amplification of exon 3 of the beta-catenin gene and exon 1 of the K-ras gene, the products were then sequenced directly in both directions. Mutations in the exon 3 of beta-catenin gene were detected in 22 of 56 early lesions (39.3%) and 21 of 37 colon cancers (56.8%). Remarkably, all beta-catenin mutations detected in the colon tumors converged at codons encoding functionally important residues that may directly mediate beta-catenin degradation, whereas mutations in the early appearing lesions were found to be scattered in the exon 3 of the gene. K-ras mutations were also detected in 24 of 56 early lesions (42.9%) and 11 of 37 colon cancers (29.7%). All K-ras mutations converged at codon 12 and codon 13, even in the early lesions. The results of this study provide evidence for the first time that beta-catenin mutation is selected during the colon carcinogenesis. Our results also suggest that the activation of beta-catenin signaling pathway is not only an initiating event, but also plays a pivotal role in the promotion stage of colorectal carcinogenesis.

Animals↗

PubMed2003

Structures of the mating-type loci of Cordyceps takaomontana.

Nucleotide sequences of the mating-type loci MAT1-1 and MAT1-2 of Cordyceps takaomontana were determined, which is the first such report for the clavicipitaceous fungi. MAT1-1 contains two mating-type genes, MAT1-1-1 and MAT1-1-2, but MAT1-1-3 could not be found. On the other hand, MAT1-2 has MAT1-2-1. A pseudogene of MAT1-1-1 is located next to MAT1-2.

Base Sequence↗

PubMed2003

Carbohydrate binding specificity of the recombinant chitin-binding domain of human macrophage chitinase.

The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.

Base Sequence↗

PubMed2003

A case of normoreninemic aldosterone-producing adenoma associated with chronic renal failure: case report and literature review.

The diagnosis of aldosterone-producing adenoma (APA) is challenging for endocrinologists, as APA does not always present with the typical constellation of clinical and laboratory features, such as hypertension, hypokalemia, suppressed plasma renin activity (PRA), and high plasma aldosterone concentration (PAC). Very recently, several studies have indicated that APA can be discovered even in normokalemic subjects with normal PRA more frequently than previously considered. Here we report a case of APA associated with chronic renal failure, which showed normokalemia and normal PRA. The patient was referred to our clinic for evaluation of an incidentally discovered adrenal mass with abnormally high PAC. After 6 yr, it was found that the right adrenal tumor showed a marked increase in size. Endocrinological examinations indicated normal PRA and markedly high PAC. Aldosterone showed a better response to the upright posture test than that to ACTH stimulation test. The diagnosis of APA was made based on the markedly high PAC to PRA ratio and the adrenocortical scintigraphy, which showed unequivocal uptake into the tumor. Right laparoscopic adrenalectomy was performed, revealing a right adrenocortical adenoma with massive hemorrhage. Histopathological examinations revealed the presence of two independent adrenocortical adenomas, one APA with predominant clear tumor cells and few c17 (17alpha-hydroxylase) immunoreactivity and the other, cortisol producing adenoma with compact cytoplasm and abundant C17 immunoreactivity. This case indicates a difficulty of diagnosis of "normoreninemic APA" with renal failure. This case is in line with the recent concept that APA is a continuous condition in which only a minority of patients have the classical clinical picture of primary aldosteronism such as hypokalemia. It is possible that normokalemic APA constitutes the most common presentation of the disease.

Adrenal Cortex Neoplasms↗

PubMed2003

Involvement of poly(ADP-ribose) synthetase in acoustic trauma of the cochlea.

We investigated effects of poly(ADP-ribose) synthetase (PARS) inhibitors on acoustic trauma. Albino guinea pigs were intravenously given 3-aminobenzamide, nicotinamide or 3-aminobenzoic acid (an inactive analog of 3-aminobenzamide) just prior to exposure to a 2 kHz pure tone of 120 dB sound pressure level (SPL) for 10 minutes. The threshold of the compound action potential (CAP) and the amplitude of distortion-product otoacoustic emissions (DPOAEs) were measured before and 4 hours after the acoustic overexposure. Statistically significant decreases in the CAP threshold shifts and significant increases in the DPOAE amplitudes were observed 4 hours after the acoustic overexposure in the animals treated with 3-aminobenzamide or nicotinamide, whereas 3-aminobenzoic acid did not exert any protective effect. These results strongly suggest that excessive activation of PARS is involved in generation of the acoustic trauma.

Acoustic Stimulation↗

PubMed2002

Microadenomatous lesions involving loss of Apc heterozygosity in the colon of adult Apc(Min/+) mice.

Mutations in the human adenomatous polyposis coli (APC) gene are causative for familial adenomatous polyposis (FAP), a rare condition in which numerous colonic polyps arise during puberty and, if left untreated, lead to colon cancer. Mouse model for human FAP, Apc(Min/+) mouse, contains a truncating mutation in the Apc gene and spontaneously develops intestinal adenomas. However, the distribution of tumors along the intestine found in Apc(Min/+) mice is different from that in human FAP. A majority of intestinal polyps in the Apc(Min/+) mice is known to be located in the small intestine but rarely detected in the colon. We report here that adult Apc(Min/+) mice develop dozens of microadenomatous lesions in the colon (>20 lesions/colon). Surprisingly, the vast majority of such adenomatous lesions consisting of colonic crypts were <300 microm in their greatest dimension, whereas lesions in the small intestine showed various sizes. The allelic loss analysis revealed that these adenomatous crypts in the colon have already lost the remaining allele of Apc. Such findings suggest that, in contrast to tumorigenesis in the small intestine, loss of heterozygosity of the Apc gene is not sufficient for tumor development in the colon of Apc(Min/+) mice. Our results may give an account for the low incidence of colonic tumors in Apc(Min/+) mice.

Adenomatous Polyposis Coli↗

PubMed2002

Lack of alpha2-antiplasmin promotes pulmonary heart failure via overrelease of VEGF after acute myocardial infarction.

Identification of a novel therapy for prevention of sudden death by ischemic cardiac infarction is an area of intensive investigation. We here report that the mortality due to an experimental acute myocardial infarction (AMI) was markedly increased in mice deficient in alpha2-antiplasmin (alpha2-AP(-/-) mice) but not in mice deficient in other components acting in fibrinolysis (tissue-type PA, urokinase type PA, or plasminogen activator inhibitor-1) even if the infarct area in alpha2-AP(-/-) mice was not different from those in the other mice. Echocardiography showed in alpha2-AP(-/-) mice after AMI an overload of the right ventricle and that pulmonary permeability was increased. According to the experiments using explanted myocytes and vascular smooth muscle cells, it was found that the amount of secreted vascular endothelial cell growth factor (VEGF) in alpha2-AP(-/-) mice was markedly increased compared with that in wild-type mice. Finally, an injection of an anti-VEGF antibody decreased the mortality after AMI in alpha2-AP(-/-) mice. Plasmin cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment and is inactivated mainly by alpha2-AP. Therefore, lack of alpha2-AP could markedly result in overrelease of VEGF by the continuous activation of plasmin because of AMI and could result in an acute cor pulmonale. Our results provide new aspects on the role of alpha2-AP and VEGF in the pathogenesis of cardiac events.

Animals↗

PubMed2002

Crystallization and preliminary crystallographic analysis of human L-xylulose reductase.

Human L-xylulose reductase was crystallized from buffered polyethylene glycol solutions using the hanging-drop vapour-diffusion method. The crystals diffract to 2.1 A resolution and belong to the orthorhombic P222 space group, with unit-cell parameters a = 72.9, b = 74.1, c = 87.9 A. This is the first crystallization report of a xylulose reductase that is identical to diacetyl reductase.

Crystallization↗

PubMed2002

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney.

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.

Acetoin Dehydrogenase↗

PubMed2002

Determination of prednisolone in the cochlear tissue.

Although glucocorticoids are widely used to treat inner ear diseases, glucocorticoid concentration has never been determined in the cochlear tissue. The aim of the present study was to measure the prednisolone concentration in the cochlear tissue after intravenous administration. At 0.5, 1, 2, 4 or 8 h after the injection (100 mg/kg), cochlea, hepatic and brain tissue and serum were removed, and prednisolone extracted from these samples was measured using high-performance liquid chromatography. Although prednisolone was not detected in the brain tissue, it was detected in the hepatic tissue and serum, demonstrating the peak value at 30 min after administration and a rather rapid decline with time thereafter. Prednisolone was also detected in the cochlear tissue, but the uptake and elimination patterns were entirely different from other samples. The prednisolone level in the cochlea reached the peak value 1 h after administration and gradually declined. The present study shows that the prednisolone administered is gradually transported to the cochlear tissue from blood and remains at higher concentrations than in the hepatic tissue or serum over several hours. It is highly likely that this slow elimination is closely related to the therapeutic effect of steroids in inner ear diseases.

Animals↗

PubMed2002

Long-term observations on the reversibility of cochlear dysfunction after transient ischemia.

To examine the reversibility of functional damage to the cochlea after transient ischemia, cochlear ischemia of 0-60 min was induced in 34 albino guinea pigs. Thresholds of auditory brainstem response (ABR) were then followed for 5 days after ischemia. Although the ABR threshold returned to almost the pre-ischemic value after 15 min ischemia, ischemia of 30 and 60 min duration induced irreversible dysfunction. Aminoguanidine, an inducible NO synthase (iNOS) inhibitor, significantly ameliorated the post-ischemic cochlear dysfunction induced by 60 min ischemia. Morphological findings of the hair cells were consistent with these functional results. These results indicate that ischemia of 30 min or longer induces irreversible damage to the cochlea and that iNOS plays injury-producing roles in this type of injury.

Animals↗