Search PubMed⌕ Search

Biomedical subjects

S Harada

Publications and source records attributed to S Harada.

At least 307 records · Page 17Linked to original sources
PubMed1994

1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels.

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Structure of azurin from Achromobacter xylosoxidans NCIB11015 at 2.5 A resolution.

The crystal structure of azurin from a denitrifying bacterium, Achromobacter xylosoxidans NCIB11015, has been refined at 2.5 A resolution using diffraction data obtained by means of synchrotron radiation at KEK. Crystals suitable for X-ray experiment were obtained by the macro-seeding method and an intensity data were obtained on imaging plates mounted on a Weissenberg camera (Rmerge = 0.09). The initial model was obtained by the molecular replacement method using the structure of azurin from Alcaligenes denitrificans NCTC8582 as a starting model. The structure was refined by molecular dynamics optimization and the restrained least-squares method to a crystallographic R-value of 0.205. However, the current model gave an electron-density of the side-chain regions of several residues close to the N-terminus quite different from those expected from the amino acid sequences reported. Very recently, two kinds of azurins (Az-I and Az-II) were isolated from this bacterium by a slightly modified purification method and have been characterized and found to have different CD spectra. On analysis of amino acid sequences around the N-terminus, the second azurin (Az-II) was proved to be a new type of azurin in this bacterium. It was consequently revealed that the current model corresponds to a new type of azurin because of the complete agreement between the electron-density and the amino acid sequence of the newly determined 20 residues from the N-terminus. Determination of the whole amino acid sequence of this azurin and further refinement are in progress.

Alcaligenes↗
PubMed1994

Interferon-alpha (INF-alpha) and etretinate in the treatment of mycosis fungoides.

A combination therapy of mycosis fungoides (MF) with interferon-alpha (INF-alpha) and etretinate was reported. A 53-year-old patient suffering from MF was treated with intramuscular administration of INF-alpha at a dosage of 9 x 10(6) IU daily; skin lesions poorly responded to the treatment. After oral etretinate with a dosage of 50 mg daily was combined with INF-alpha, marked clinical improvement was seen in skin lesions with minor side effects. INF-alpha in combination with etretinate can have beneficial effects in the treatment of MF.

Administration, Oral↗
PubMed1994

Anti-human IgE monoclonal antibodies recognizing epitopes related to the binding sites of high and low affinity IgE receptors.

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the C epsilon 1, C epsilon 2, C epsilon 2/C epsilon 3 junction and C epsilon 3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (Fc epsilon RI and Fc epsilon RII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-Fc epsilon RII binding was clearly inhibited by the mAb recognizing the C epsilon 2/C epsilon 3 junction, suggesting that Fc epsilon RII binds to a rather limited area around the C epsilon 2/C epsilon 3 junction. The IgE-Fc epsilon RI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in C epsilon 2 was blocked simultaneously with that at the C epsilon 2/C epsilon 3 junction or with that in C epsilon 3, indicating that these three distinct epitopes are related to the Fc epsilon RI binding sites. When these three epitopes were shown in the stereograph of human IgE, the Fc epsilon RI binding area was spread largely on the groove side between C epsilon 2 and C epsilon 3 domains. These results suggest that Fc epsilon RI acquires the high affinity through multiple bindings.

Amino Acid Sequence↗
PubMed1994

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.

Amino Acid Sequence↗
PubMed1994

The EBNA-2 arginine-glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.

Amino Acid Sequence↗
PubMed1994

Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices.

Nitric oxide (NO) is shown to be synthesized in the central nervous system as well as in vascular endothelial cells. However, the physiological role of NO in cardiovascular regulation in the central nervous system remains unclear. The present study examines whether NO plays a role in the regulation of neuronal activity in the nucleus tractus solitarius (NTS). Single-unit extracellular recordings were obtained from NTS neurons in slices (400 microns) of the rat brainstem, which had spontaneous discharges at a frequency of 0.5 to 3 spikes per second. Eighty-one neurons were tested for sensitivity to L-arginine, which is the physiological precursor of NO. L-Arginine (10(-7) to 10(-4) mol/L) increased neuronal activity dose dependently in 33 (40.7%) of 81 neurons tested, but D-arginine (10(-5) mol/L) did not. The neurons that responded to L-arginine responded to glutamate as well. NG-Monomethyl-L-arginine (10(-5) to 3 x 10(-5) mol/L), an inhibitor of the formation of NO, dose-dependently blocked increases in the neuronal activity evoked with L-arginine (10(-5) mol/L). Hemoglobin (1.5 mg/L), a trapper of NO, and methylene blue (10(-5) mol/L), an inhibitor of guanylate cyclase, also blocked increases in the neuronal activity evoked with L-arginine (10(-5) mol/L). Sodium nitroprusside (SNP, 10(-5) to 10(-4) mol/L), which spontaneously produces NO, increased the neuronal activity in the neurons that responded to L-arginine. SNP did not alter the neuronal activity of the neurons that did not respond to L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts.

PGE1 and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEFG), a secreted endothelial cell-specific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and E1 increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation of VEGF expression by PGF2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism.

Alprostadil↗
PubMed1994

Characterization of retinoic acid- and cell-dependent sequences which regulate zif268 gene expression in osteoblastic cells.

We have previously shown that retinoic acid (RA) induces differentiation in an osteoblastic cell line derived from embryonic rat calvaria and that RA has selective effects on zif268 gene expression in these preosteoblastic cells,distinct from those in more mature osteoblasts. In this study we demonstrate that the RA-dependent transcriptional increase in zif268 gene expression is mediated by the interaction of RA receptors (RARs) with a 17 base pair sequence in the zif268 promoter containing a single half-site motif (GTTCA), identical to each of the direct repeats seen in the RAR beta 2 gene. The sequence appears relatively RA-specific, since the zif268 RA-responsive element is not activated by 1,25-dihydroxyvitamin D3 or thyroid hormone (T3). However, cotransfection of RAR expression vectors and an SV-40 promoter chloramphenicol acetyltransferase (CAT) construct containing the single zif268 RA-responsive motif into CV-1 cells demonstrates that the alpha-, beta-, and gamma-RARs transactivate through this element. Extensive mutagenesis of the zif268 promoter region containing the RA response element (RARE) motif confirms that the transactivation and nuclear protein binding activity of this region requires only the half-site motif. The direct involvement of RAR in this DNA-protein interaction has been demonstrated by competitive gel retardation analysis using consensus RAREs and super-shifting of the DNA-protein complex with mouse alpha- or gamma-RAR monoclonal antibodies. In addition, we found that cell-specific suppression of RA-stimulated zif268 gene expression can be attributed to a 29 base pair nucleotide sequence, located downstream of the RA-responsive region in the zif268 gene. This sequence appears to be bound specifically by nuclear protein(s) from several cell types, including osteoblasts. The presence of this sequence in cis to the zif268 RARE or the consensus beta RARE completely blocks the RA-responsiveness of the zif268 gene in differentiated osteoblasts. These data extend the broad spectrum of RA-responsive sequences necessary for DNA binding and transactivation to include regulation via single RARE half-site motifs and suggest that the lack of RA responsiveness in differentiated osteoblasts may be mediated by cell-specific suppression of gene expression.

Animals↗
PubMed1994

Age-dependent decreases in fibrinolytic enzyme activities in serum of healthy subjects.

We investigated the age-dependency of serum levels of 9 kinds of proteases. The results showed that the enzymatic activities corresponding to urokinase, plasmin, and thrombin, all involved in blood clotting and fibrinolysis, were inversely correlated with age. This suggests that there is some similarity between the normal process of aging and the pathologic process of Alzheimer's disease. Compared with our previous data on Alzheimer patients, the present results indicate that some derangement in the aging process is involved in the pathogenetic mechanisms of Alzheimer's disease.

Aging↗
PubMed1994

Influence of arotinolol hydrochloride on heart rate spectrum in hypertensive subjects.

Influence of arotinolol hydrochloride and atenolol on the balance between the sympathetic and parasympathetic nervous systems was evaluated in 8 hypertensive subjects by spectral analysis of heart rate (HR) and systemic blood pressure (BP). Before and after administration of either arotinolol (n = 7) or atenolol (n = 7) for 2 weeks, BP was continuously and non-invasively monitored by a finger-cuff manometry (Finapres). A time series of instantaneous HR was constructed from the BP signal. A time series of mean BP was also constructed. Spectral analysis was performed by the use of an autoregressive algorithm on these time series (approximately 180 sec). Each spectrum was subdivided into low-(0.05-0.15 Hz, LF) and high-frequency (0.15-0.4 Hz, HF) components, and each component was divided by the sum of the two for normalization. As a measure of the balance between the sympathetic and parasympathetic nervous systems, the ratio of LF to HF (LF/HF) was evaluated. Arotinolol increased fractional HF in the HR spectrum from 0.45 +/- 0.12 to 0.73 +/- 0.08 (p < 0.01) and decreased fractional LF from 0.55 +/- 0.12 to 0.27 +/- 0.08 (p < 0.01); consequently, it decreased LF/HF from 1.4 +/- 0.5 to 0.4 +/- 0.2 (p < 0.01). Atenolol had similar effects on these parameters. Neither of these beta-adrenergic blockades produced a discernible decrease in LF/HF in the BP spectrum. In conclusion, these beta-adrenergic blockades decreased LF/HF in the HR spectrum in hypertensive subjects, which suggests that they improved the balance between the sympathetic and parasympathetic nervous systems.

Adrenergic beta-Antagonists↗
PubMed1994

Relationship between antigens and IgG subclasses in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized in part by the presence of tissue-bound and circulating antibodies (mostly of IgG) to the basement membrane zone (BMZ). We previously reported that IgG subclasses of BP antibodies were IgG1, IgG2 and IgG4, and that only BP IgG1 fixed complements. In this study, we examined whether BP IgG sub-classes bound to the same epitope of BP antigen or a different epitope. In an inhibition immunofluorescence studies, the complement fixing capability of IgG1 was inhibited by the pretreatment with IgG4 and partially inhibited by IgG2. On immunoblot analysis, IgG1 and IgG4 were bound to the same MW of BP antigen. In enzyme-linked immunosorbent assay (ELISA), the binding capability of IgG subclass fractions from patients with BP to synthetic peptide P1-2, exceeding normal IgG subclass fractions was seen in five IgG1, one IgG2 and two IgG4, from eight BP patients. The binding capability of IgG subclass fractions from the patients with BP to P1-1, exceeding the normal IgG fractions was seen in two IgG1, three IgG2 and one IgG4 from ten BP patients. On inhibition ELISA, the binding activity to P1-2 of IgG4 was partially inhibited by the pretreatment of IgG1 and IgG2. These findings suggest that BP IgG1, IgG2 and IgG4 could bind to the same epitope though considerable variation occurred between patients.

Adult↗
PubMed1994

TAN-1496 A, C and E, diketopiperazine antibiotics with inhibitory activity against mammalian DNA topoisomerase I.

Fungal metabolites with an epi-oligothiadiketopiperazine structure, TAN-1496 A, C and E, were isolated from the culture broth of Microsphaeropsis sp. FL-16144. Their molecular formulas were determined to be C22H28N2O9S2, C22H28N2O9S3 and C22H28N2O9S4, respectively. Structures were determined by comparing the NMR data with those of known diketopiperazine antibiotics, sirodesmins. These metabolites inhibited the relaxation of supercoiled pBR322 DNA by calf thymus topoisomerase I but did not affect the decatenation of kinetoplast DNA by calf thymus topoisomerase II at concentration up to 500 microM. They strongly suppressed the growth of various murine and human tumor cells and induced apoptosis. Moreover, various derivatives were synthesized to investigate the relationship of their functional groups and biological activities.

Animals↗
PubMed1994

New naphthacenecarboxamide antibiotics, TAN-1518 A and B, have inhibitory activity against mammalian DNA topoisomerase I.

New naphthacenecarboxamide antibiotics, TAN-1518 A and B, were isolated from a culture broth of Streptomyces sp. AL-16012. Their structures were elucidated from their reactions and from spectroscopic analyses. The relaxation of supercoiled pBR322 DNA by calf thymus DNA topoisomerase I was inhibited by these metabolites as potently as by camptothecin. However, the decatenation of kinetoplast DNA by calf thymus DNA topoisomerase II was little affected by these agents. The major metabolite, TAN-1518 A, strongly suppressed the growth of various murine and human tumor cells, inducing apoptosis. Unlike camptothecin, TAN-1518 A did not stimulate cleavable complex formation in the nuclei of CHO-K1 cells and had weak activity in intercalating into DNA strands. This metabolite arrested the growth of human tumor cell lines in G1 phase of the cell cycle. These results suggest that TAN-1518 A and B are novel antitumor agents targeting topoisomerase I.

Animals↗
PubMed1994

Structures of dnacin A1 and B1, new naphthyridinomycin-type antitumor antibiotics.

Dnacin A1 and B1 were revealed to be new naphthyridinomycin-type antitumor antibiotics with formulae of C20H23N5O4 and C19H24N4O5, respectively. The gross structure of dnacin A1 was elucidated by the spectroscopic analyses. Conversion of dnacin B1 into A1 by treatment with potassium cyanide indicated the presence of an alpha-carbinolamine moiety in dnacin B1. The relative stereochemistry of dnacins was clarified by analysis of the NOESY spectra.

Antibiotics, Antineoplastic↗
PubMed1994

Family characteristics and offspring growth in various countries. I. Frequency of various family types in Bulgaria, Japan, Korea, Mexico and Poland.

9936 families were analyzed from Bulgaria, Japan, Korea, Mexico, and Poland. A factor analysis revealed four factors: culture (F1), income (F2), genetics (F3), and family and apartment size (F4). Family types we coded as 1 if below the median and 2 if above the median. The most frequent types were represented by 1122, 2221, and 2211 (frequency 8.7-8.0%), and the least frequent by 1221 and 2111 (frequency 4.2-4.3%). Some similarity with respect to family types were found between populations: from Polish regions with heavy industry and seaside regions; from Polish towns under industrialization and from Mexico; from the Polish medium-size town of Lublin and Bulgarian towns; from Polish villages; from Polish mountain areas and north-eastern towns; from Korea; from Japan; from Polish cities.

Body Height↗
PubMed1994

[Association among genotypes of aldehyde dehydrogenase 2 and alcohol consumption].

A large population survey was made for the determination of aldehyde dehydrogenase 2 (ALDH2) genotypes among 630 unrelated Japanese adults by PCR using allele specific primers. The frequencies of three genotypes were 60% ALDH2*1/ALDH2*1 (NN) type, 35% ALDH2*1/ALDH2*2 (ND) type and 5% ALDH2*2/ALDH2*2 (dd) type. Average amounts of alcohol consumption per month for three genotypes were 965.8 g/month for NN type, 629.5 g/month for ND type and 179 g/month for DD type. The differences in average alcohol consumption were significant (p < 0.01) among the three genetic different groups. When male and female date were analyzed separately for these three groups, significant differences among three groups were found in both genders. These results indicated that ALDH2 mutant is an important genetic factor for limiting the alcohol consumption. The frequencies of ALDH2 genotypes were not significantly different between male and female population groups, however, their average alcohol consumption were significantly different between the two groups. Therefore, various social factors and life styles, in addition to genetic factors, might concern in the difference of alcohol consumption.

Adult↗