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Biomedical subjects

A Hara

Publications and source records attributed to A Hara.

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

Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis.

A hamster sperm 26 kDa protein (P26h) is strikingly homologous with mouse lung carbonyl reductase (MLCR) and is highly expressed in the testis, but its physiological functions in the testis are unknown. We show that recombinant P26h resembles NADP(H)-dependent MLCR in the tetrameric structure, broad substrate specificity, inhibitor sensitivity, and activation by arachidonic acid, but differs in a preference for NAD(H) and high efficiency for the oxidoreduction between 5alpha-androstane-3alpha,17beta-diol (k(cat)/K(M) = 243 s(-1) mM(-1)) and 5alpha-dihydrotestosterone (k(cat)/K(M) = 377 s(-1) mM(-1)). The replacement of Ser38-Leu39-Ile40 in P26h with the corresponding sequence (Thr38-Arg39-Thr40) of MLCR led to a switch in favor of NADP(H) specificity, suggesting the key role of the residues in the coenzyme specificity. While the P26h mRNA was detected only in the testis of the mature hamster tissues, its enzyme activity was found mainly in the mitochondrial fraction of the testis and in the nuclear fraction of the epididymis on subcellular fractionation, in which a mitochondrial enzyme, isocitrate dehydrogenase, exhibited a similar distribution pattern. The enzyme activity of P26h in the two tissue subcellular fractions was effectively solubilized by mixing with 1% Triton X-100 and 0.2 M KCl, and enhanced more than 10-fold. The enzymes purified from the two tissue fractions exhibited almost the same structural and catalytic properties as those of the recombinant P26h. These results suggest that P26h mainly exists as a tetrameric dehydrogenase in mitochondria of testicular cells and plays a role in controlling the intracellular concentration of a potent androgen, 5alpha-dihydrotestosterone, during spermatogenesis, in which it may be incorporated in mitochondrial sheaths of spermatozoa.

Alcohol Oxidoreductases↗
PubMed2001

Expression of hepatocyte growth factor and matrix metalloproteinase-2 in human glioma.

Hepatocyte growth factor (HGF) has a stimulatory effect on the synthesis of matrix metalloproteinase-2 (MMP-2), which is involved in glioma invasion. In this study, to clarify the correlation between the expression of HGF and MMP-2 in glioma tissues, immunohistochemical analysis of HGF and MMP-2 was performed in 11 cases of astrocytoma, 10 cases of anaplastic astrocytoma, and 9 cases of glioblastoma. As a result, expression of HGF and MMP-2 was correlated with the grade of malignancy (P = 0.0181 and 0.0001, respectively), and a significant correlation between the immunoreactivity of HGF and that of MMP-2 was observed (P < 0.05). Immunofluorescence study revealed the concomitant expression of HGF and MMP-2 in glioma tissue. In cultured glioma cell lines (SNB-19, U87MG, and U373MG), exogenous recombinant HGF effectively acted on the production of the active and latent forms of MMP-2 protein in a dose-dependent manner. Active MMP-2 increased more effectively than the latent form. Taken together, these results suggest that HGF may promote glioma invasion in vivo by production of MMP-2.

Astrocytoma↗
PubMed2001

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene.

We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.

Anti-Bacterial Agents↗
PubMed2001

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis.

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.

Acyl-CoA Oxidase↗
PubMed2001

Assessment of leg oedema by dynamic lymphoscintigraphy with intradermal injection of technetium-99m human serum albumin and load produced by standing.

This study was a preliminary evaluation of the utility of dynamic lymphoscintigraphy with technetium-99m human serum albumin (HSA) and a load produced by standing in the assessment of lymphatic dysfunction in patients with leg oedema. The 71 subjects investigated included 53 patients with lymphoedema, six with venous occlusion alone and five with lymphovenous occlusion, as well as seven normal subjects. After intradermal injection of 99mTc-HSA into an interdigital space in each foot, dynamic scintigrams were recorded with the patient supine for 15 min. The subjects then stood in place and images were recorded for an additional 15 min. Relative changes in lymphatic tracer transport before and after standing were analysed on time-activity curves (TACs). This test was compared with a conventional test in a supine position in six patients with lymphoedema, and was repeated in five other patients with lymphoedema. It was found that in the normal limbs, a standing load activated tracer transport to the draining lymphatic vessels, resulting in a rapid stepwise increase in tracer activity, large spiking waves and a decreasing phase following a peak in tracer activity on TACs. In 59 lymphoedematous limbs, including some with a mild form of oedema without morphological abnormalities on scintigrams, this load failed to induce a sufficient activation of tracer transport, and the frequencies of each of the three normally appearing changes described above significantly decreased compared with those in the 14 normal limbs (P < 0.0001, P < 0.01 and P < 0.0001, respectively). In addition, there were significant reductions in the relative increases in maximum activity and clearance times after standing (both P < 0.0001). These abnormalities significantly correlated with the grade of severity of oedema. Six limbs with lymphovenous occlusion showed significant reductions in tracer transport compared to six limbs with venous occlusion. Lymphatic dysfunction was accentuated more by this test than by the conventional test, and repeated tests showed consistent results in the same individuals. It is concluded that under a standardized load, this quick test seems of value in providing a sensitive and objective assessment of lymphatic dysfunction in the lower limbs, and is also advantageous for image interpretation since accelerated tracer transport clearly visualizes compromised lymphatics. This test may also be helpful in distinguishing purely venous oedema from mixed lymphovenous disease.

Adult↗
PubMed2001

Relationship between thallium-201 kinetics and proliferative activity assessed by monoclonal antibody MIB-1 in brain tumours.

To clarify the relationship between thallium-201 chloride kinetics and proliferative activity in brain tumours. a single-photon emission tomographic (SPET) study was performed and the results correlated with monoclonal antibody MIB-1 staining of the tumour tissue. 201T1 SPET was performed 10 min (early scan) and 2 h (delayed scan) after intravenous administration of 201TI (111 MBq) in 34 intra-axial tumours including 24 malignant tumours, and in 27 extra-axial tumours including one malignant tumour. Tumour 201T1 kinetic parameters [early and delayed uptake ratios (ER and DR, respectively), retention index (RI), and the ratio of tumour delayed activity to early activity (Td/Te)] were compared with tumour tissue MIB-1 labelling indices (MIB-1 LI) representative of tumour cell proliferative activity. In the intra-axial tumours, ER and DR and MIB-1 LI were significantly higher in the malignant tumours than in the benign tumours. ER and DR were significantly correlated with MIB-1 LI (P<0.01 and P<0.05, respectively), but RI and Td/Te were not. In the extra-axial benign tumours, ER was as high as that in the intra-axial malignant tumours, while MIB-1 LI was equal to that in the intra-axial benign tumours. There were no significant correlations between any 201T1 kinetic parameters and MIB-1 LI. This study indicates that 201T1 ER may be the most reliable parameter for predicting the proliferative activity of intra-axial tumours.

Adolescent↗
PubMed2001

Computational analysis of full-length mouse cDNAs compared with human genome sequences.

Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes.

Algorithms↗
PubMed2001

Genetic evidence of the neoplastic nature of gemistocytes in astrocytomas.

Gemistocytic astrocytoma is characterized by a predominance of large astrocytes with plump processes and massive accumulation of glial fibrillary acidic protein (gemistocytes). This histological variant of low-grade diffuse astrocytoma (WHO grade II) is prone to more rapid progression to anaplastic astrocytoma and glioblastoma than the ordinary fibrillary astrocytoma. The biological basis of this unfavorable prognosis is unclear, since gemistocytes themselves have low proliferative activity, even if present in anaplastic astrocytomas or glioblastomas. This has raised the question of whether gemistocytes are neoplastic cells or dysplastic reactive astrocytes. In this study, gemistocytes and non-gemistocytic neoplastic cells were separated by laser-assisted microdissection from six gemistocytic astrocytomas carrying TP53 mutations. In all cases, identical TP53 mutations were identified in both cell types, indicating that gemistocytes are indeed neoplastic cells. Their lack of proliferative activity may indicate terminal differentiation.

Adult↗
PubMed2001

Effect of acoustic overstimulation on regulation of glucocorticoid receptor mRNA in the cochlea of the guinea pig.

Semi-quantitative reverse transcription polymerase chain reaction was performed to determine the distribution of mRNA levels of glucocorticoid receptor (GR) within the guinea pig cochlea and to examine the change in their expression after acoustic overstimulation. Using an original PCR primer for the guinea pig, the highest GR mRNA level was revealed in the modiolus and lowest in the medial portion including the organ of Corti. Total RNA was extracted from the whole cochlea of the guinea pig 0, 2, 6 and 24 h after exposure to a 2 kHz pure tone of 110, 120 or 130 dB SPL for 10 min. The level of GR mRNA significantly decreased immediately and 2 h after exposure to the sound of 120 dB SPL, and 2 and 6 h after exposure to that of 130 dB SPL. These results suggest the presence of a down-regulation of GR mRNA induced by acoustic overstimulation, although the exact mechanism of this phenomenon remains unsolved.

Acoustic Stimulation↗
PubMed2001

Does xanthine oxidase contribute to the hydroxyl radical generation in ischemia and reperfusion of the cochlea?

We investigated the effect of a hydroxyl radical scavenger, 1,3-dimethyl-2-thiourea (dimethylthiourea), and two xanthine oxidase inhibitors, oxypurinol and allopurinol, on the threshold shift of the compound action potential (CAP) after transient ischemia of the cochlea. Transient ischemia of 30 min duration was induced in albino guinea pigs via a skull base approach. The animals were treated with perilymphatic perfusion of dimethylthiourea, oxypurinol or allopurinol from 10 min before the onset of ischemia to 4 h after the termination of ischemia. Dimethylthiourea ameliorated the CAP threshold shifts at 4 h after the onset of reperfusion in a dose-dependent manner. However, oxypurinol and allopurinol did not affect the post-ischemic cochlear dysfunction. These results imply that the hydroxyl radical plays an important role in generation of cochlear dysfunction induced by ischemia-reperfusion and that xanthine oxidase may not be the primary source of this radical.

Action Potentials↗
PubMed2001

Ischemia-reperfusion injury of the cochlea: effects of an iron chelator and nitric oxide synthase inhibitors.

Release of free iron from cellular stores and activation of nitric oxide synthase (NOS) has been implicated in a wide variety of cochlear injuries. In order to evaluate the effects of deferoxamine (a iron chelator), 3-bromo-7-nitroindazole (a relatively selective neuronal NOS (nNOS) inhibitor) or aminoguanidine (a relatively selective inducible NOS (iNOS) inhibitor) on the post-ischemic cochlear dysfunction, albino guinea pigs were subjected to 30 min ischemia, and the threshold shifts of the compound action potential (CAP) from pre-ischemic values were compared with those of control animals 4 h after the onset of reperfusion. A statistically significant reduction in the post-ischemic CAP threshold shift was observed in the animals treated with deferoxamine or 3-bromo-7-nitroindazole. However, aminoguanidine did not affect the post-ischemic CAP threshold shift. These results suggest that free iron and nNOS play deleterious roles in the cochlear injury induced by transient ischemia.

Action Potentials↗
PubMed2001

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.

Acridines↗
PubMed2001

Roles of galactose and sulfate residues in sulfatides for their antagonistic functions in the blood coagulation system.

We previously reported that the sulfatide (galactosylceramide I3-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I6-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.

Animals↗
PubMed2001

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA.

To improve adenovirus-mediated gene delivery to skeletal muscle, we have used a recombinant adenovirus vector encoding the human Coxsackievirus and adenovirus receptor (hCAR). Because CAR is expressed at a lower level in rodent myoblasts and muscle fibers than in other tissues, we expected that elevated expression of CAR in skeletal muscle would improve the efficacy of adenovirus-mediated gene transfer. Since the mouse myoblasts, C2C12 cells, showed low sensitivity to infection by recombinant adenovirus 5, we initially infected these cells at a high multiplicity of infection (MOI) of 250 with the recombinant adenovirus containing hCAR cDNA and LacZ gene. Subsequent infection by recombinant adenovirus containing the marker gene, green fluorescence protein, became efficient even at a low MOI of 25. Thus, elevated hCAR expression in mouse muscle fibers made a second virus inoculation at low doses possible. We also demonstrated that the elevated hCAR expression did not influence muscle membrane integrity. Our results suggest that co-expression of CAR and a therapeutic gene by adenovirus vector constitutes a novel strategy to advance gene therapy for hereditary muscle diseases.

Adenoviridae↗
PubMed2001

Gene therapy for preventing neuronal death using hepatocyte growth factor: in vivo gene transfer of HGF to subarachnoid space prevents delayed neuronal death in gerbil hippocampal CA1 neurons.

To develop a novel strategy to prevent delayed neuronal death (DND) following transient occlusion of arteries, the gene of hepatocyte growth factor (HGF), a novel neurotrophic factor, was transfected into the subarachnoid space of gerbils after transient forebrain ischemia. Importantly, transfection of HGF gene into the subarachnoid space prevented DND, accompanied by a significant increase in HGF in the cerebrospinal fluid. Prevention of DND by HGF is due to the inhibition of apoptosis through the blockade of bax translocation from the cytoplasm to the nucleus. HGF gene transfer into the subarachnoid space may provide a new therapeutic strategy for cerebrovascular disease.

Animals↗
PubMed2001

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.

Alcohol Oxidoreductases↗
PubMed2001

Peroxynitrite affects Ca2+ influx through voltage-dependent calcium channels.

The effect of peroxynitrite (OONO-) on voltage-dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO- time- and dose-dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4-benzoic acid) porphyrin, a scavenger for OONO-. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO(-)-induced [45Ca2+] influx. OONO-, as well as 30 mm KCl, significantly increased fluorescence intensity of cell-associated bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose-dependently suppressed OONO(-)-induced [45Ca2+] influx. Although each of 1 microM nifedipine and 1 microM omega-agatoxin VIA (omega-ATX) significantly inhibited the OONO(-)-induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 microM omega-conotoxin GVIA (omega-CTX) showed no effect on the influx. On the other hand, OONO- itself reduced 30 mM KCl-induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO- alone, and the magnitude of this reduction was as same as that of KCl-induced [45Ca2+] influx by omega-CTX. These results indicate that OONO- increases [45Ca2+] influx into the neurones through opening P/Q- and L-type VDCCs subsequent to depolarization, and inhibits the influx through N-type VDCCs.

Anesthetics, Local↗