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Biomedical subjects

H Hayashi

Publications and source records attributed to H Hayashi.

At least 865 records · Page 48Linked to original sources
PubMed1994

Molecular cloning of two new heat shock genes related to the hsp70 genes in Staphylococcus aureus.

We have identified two new heat shock protein genes, orf37 and orf35, in Staphylococcus aureus, located upstream and downstream of grpE(hsp20), dnaK(hsp70), and dnaJ(hsp40) homologous genes in the order orf37-hsp20-hsp70-hsp40-orf35. The transcripts of both orf37 and orf35 were increased by thermal upshift of the culture from 37 to 46 degrees C. The heat shock promoters were located upstream of orf37 and upstream of hsp40. The deduced peptide of orf37 showed similarity with those of orfA in Clostridium acetobutylicum and orf39 in Bacillus subtilis. orf35 was unique in S. aureus and has not yet been described in other bacteria.

Amino Acid Sequence↗
PubMed1994

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens.

The perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was cloned into an Escherichia coli-C. perfringens shuttle vector, and the pfoA gene was expressed in mutants of C. perfringens 13 which lacked the production of perfringolysin O. One group (SI117) could express the pfoA gene, and the other (SI112) could not. A mutation in the regulatory system for pfoA gene expression was suspected in SI112. A chromosomal DNA library constructed from strain 13 was transformed into strain SI112 to identify the regulatory gene(s) for the pfoA gene. Five strains of 10,000 transformants restored perfringolysin O production. All contained a 2.5-kb DNA fragment. This fragment activated the transcription of the pfoA gene and also restored the production of collagenase (kappa-toxin) and hemagglutinin in strain SI112. Deletion analysis showed that a 1.25-kb region was sufficient for the trans activity, and sequence analysis disclosed that open reading frame 2 (ORF2) was located in this region. A homology search for the deduced amino acid sequence revealed that ORF2 was homologous to a response regulator in a two-component signal transduction system. ORF2 was designated virR, and it is suggested that the virR gene plays an important role in the pathogenicity of C. perfringens.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a chromosomal gene cluster, pah, that encodes the upper pathway for phenanthrene and naphthalene utilization by Pseudomonas putida OUS82.

A 25-kb DNA SalI fragment cloned from the chromosomal DNA of Pseudomonas putida OUS82, which utilizes phenanthrene (Phn+) and naphthalene (Nah+), carried all of the genes necessary for upper naphthalene catabolism. Cosmid recombinant pIP7 complemented both the Nah- and Phn- defects of OUS8211 (Trp-Nah-Phn-Sal+[salicylate utilizing]Hna+[1-hydroxy-2-naphthoate utilizing]) and only the Phn- defect of OUS8212 (Trp-Nah-Phn-Sal-Hna+). The results indicate that strain OUS82 uses different pathways after o-hydroxycarboxylic aromatics in the catabolism of naphthalene and phenanthrene.

Cloning, Molecular↗
PubMed1994

Simultaneous measurement of intracellular Na+ and Ca2+ during K(+)-free perfusion in isolated myocytes.

To study the relationship between intracellular Na+ concentration ([Na+]i) and intracellular Ca2+ concentration ([Ca2+]i), guinea pig ventricular myocytes were loaded with both the Na(+)-sensitive probe, sodium-binding benzofuran isophthalate (SBFI), and the Ca(2+)-sensitive probe, fluo 3. [Na+]i was measured from the ratio image at 510 nm when excited at 340/380 nm. [Ca2+]i, expressed as the percent change of corrected fluo 3 fluorescence, was measured at 540 nm when excited at 500 nm. The fluorescent spectra of these probes were sufficiently different to allow for simultaneous measurement. After 30 min perfusion of K(+)-free solution, [Na+]i of rod-shaped cells increased from 6.4 +/- 0.5 to 20.6 +/- 2.6 mM, and [Ca2+]i increased to 256 +/- 36% of the control. [Ca2+]i was higher in spontaneously contracting cells and shortened cells than in rod-shaped cells at similar levels of [Na+]i. When Ca(2+)-free solution or Ni2+ (5 mM) was applied, [Ca2+]i was lower than in cells perfused with K(+)-free solution alone. It was suggested that extracellular Ca2+ and the Na(+)-Ca2+ exchange were involved in the increase in [Ca2+]i. In conclusion, we have developed a new method for the simultaneous measurement of [Na+]i and [Ca2+]i in isolated myocytes, which should be useful to study the relation between [Na+]i and [Ca2+]i.

Aniline Compounds↗
PubMed1994

Regulation of [Na+]i and [Ca2+]i in guinea pig myocytes: dual loading of fluorescent indicators SBFI and fluo 3.

To investigate the mechanisms regulating intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i, respectively), we monitored both ion concentrations simultaneously using fluorescent probes, Na(+)-binding benzofuran isophthalate (SBFI) and fluo 3, in unstimulated guinea pig ventricular myocytes. After the addition of 500 microM strophanthidin, [Na+]i increased gradually from 6.6 +/- 0.6 to 20.1 +/- 1.6 mM (mean +/- SE) at 50 min. [Ca2+]i, expressed as the percentage change of corrected fluo 3 fluorescence, was kept at the low level during the first 20 min and then began to increase to 447 +/- 81% of the control at 50 min. The addition of 1 microM hexamethylene amiloride prevented the increases in both [Na+]i and [Ca2+]i. The perfusion of Ca(2+)-free solution or 5 mM Ni(2+)-containing solution suppressed the increase in [Ca2+]i. In cells that exhibited spontaneous contractile activities, [Ca2+]i increased further than that in quiescent cells, whereas [Na+]i levels were similar. In the presence of 1 microM ryanodine, both the spontaneous contractile activities and the further increase in [Ca2+]i were eliminated. These findings indicated that 1) the pathway of Na+ entry was mainly through Na(+)-H+ exchange and that the elevated [Na+]i induced Ca2+ entry mediated by the reverse mode of Na(+)-Ca2+ exchange, and 2) the entered Ca2+ triggered the ryanodine-sensitive Ca2+ release from the sarcoplasmic reticulum, causing the dissociation in the relationship between [Na+]i and [Ca2+]i.

Amiloride↗
PubMed1994

Circadian and estrous cycle-dependent variations in blood pressure and heart rate in female rats.

To determine whether cardiovascular functions are controlled by the endogenous circadian system and whether they change with the estrous cycle in female rats, we measured mean arterial pressure (MAP), heart rate (HR), and spontaneous activity (ACT) of female rats using an implantable radiotelemetry device and a computerized data-collecting system. Under a 12:12-h light-dark (LD) cycle, these parameters exhibited daily rhythms that were entrained to the photic cycle. The patterns of the daily rhythms varied with estrous cycles, and variations were particularly marked in the proestrous stage. During the dark period of this stage, ACT levels were significantly higher, but HR was significantly lower than in other stages. Although the peak MAP occurred within 2 h after the onset of the dark phase in three of the estrous stages, it occurred around midnight in the proestrous stage. Such estrous cycle-dependent variations were eliminated by ovariectomy. The implantation of 17 beta-estradiol produced a gradual increase in MAP and an abrupt decrease in HR. During constant darkness, all three parameters were free running, maintaining the same internal phase relationships with each other as during LD cycles. These results indicate that daily variations in these parameters were controlled by the endogenous circadian oscillating system, that they vary with the estrous cycle in female rats, and that estrogen may be responsible for these estrous cycle-dependent variations.

Analysis of Variance↗
PubMed1994

Autoimmune myocarditis induced in mice by cardiac C-protein. Cloning of complementary DNA encoding murine cardiac C-protein and partial characterization of the antigenic peptides.

Autoimmune myocarditis is considered to play a major role in the pathogenesis of dilated cardiomyopathy. A new autoimmune myocarditis model was attained by repeated immunization using murine cardiac C-protein with the immunological adjuvant, Klebsiella pneumoniae O3 lipopolysaccharide. For further analysis of a pathological epitope, the cDNA encoding C-protein was isolated; a fusion protein encoded by part of this cDNA induced myocarditis in SMA mice as well as in three other strains: DBA/1J (H-2q), O20/A (H-2pz1), and SJL (H-2s). The nucleotide sequence and its deduced amino acid analysis revealed that this protein had immunoglobulin-like and fibronectin-like repeats. This study provides a new animal model of autoimmune myocarditis which may shed light on the pathogenesis of dilated cardiomyopathy.

Adult↗
PubMed1994

Immunohistochemical localization of estrogen receptors in the submandibular gland tumors of female rats.

The localization of estrogen receptors (EsR) in the tumor tissues of submandibular glands was examined in female rats, using the indirect immunoperoxidase method in combination with the in situ hybridization technique. Tumors were experimentally produced by 9,10-dimethyl-1,2-benzanthracene (DMBA), and the tumor tissues were fixed with formalin or paraformaldehyde and then embedded in paraffin. In the DMBA-induced submandibular gland tumors, immunoreactivity to EsR-peroxidase conjugate was found in nuclei of the tumor cells which occupied the peripheral rim of the tumor cell nests. In contrast, the reactivity in the normal submandibular glands without tumor was mostly confined to nuclei of the duct cells. When EsR mRNA expression was analyzed in the tumor tissue by in situ hybridization with a cDNA probe, its distribution was identical with that of immunoreactivity to EsR. These data suggest that the ductal cells of the submandibular gland are responsive to ovarial steroids, and that estrogens may play an important role in the maintenance of growth of the submandibular gland tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Effect of a benzylidene derivative, novel antirheumatic agent, on the production of tissue inhibitor of metalloproteinases.

We investigated the effects of PAL (protocatechualdehyde), a metabolite of ACP (3,4-diacetoxy benzylidene diacetate) which is a candidate as a novel antirheumatic agent, on the production of a tissue inhibitor of metalloproteinases (TIMP) using a primary chondrocyte culture from bovine nasal septum cartilage. The addition of human recombinant interleukin-1 alpha (hrIL-1 alpha) induced proteoglycan (PG) depletion from a chondrocyte matrix. PAL significantly reduced the induction of PG depletion. HrIL-1 alpha increased the casein-degrading activity, matrix metalloproteinase (MMP) activity, and the level of TIMP secretion in the culture media. PAL significantly reduced the casein-degrading activity and further increased TIMP secretion under hrIL-1 alpha stimulation. Phorbol myristate acetate (PMA) also increased the level of TIMP secretion, and staurosporine, a specific inhibitor of protein kinase C (PKC), reduced the TIMP secretion to the control level under hrIL-1 alpha or PMA stimulation. Furthermore, PAL showed a significant increase following treatment with a low concentration of PMA which alone did not increase TIMP production. These findings suggest that the activation of the PKC pathway plays an important role in TIMP production and that PAL increases the level of TIMP production through an additive or synergistic effect with the activation of the PKC pathway. In conclusion, these findings demonstrate that the inhibition of the MMP activity and the increase of TIMP production by PAL contribute to the inhibition of PG depletion in a primary chondrocyte culture from bovine nasal septum cartilage.

Alkaloids↗
PubMed1994

Confluentic acid and 2'-O-methylperlatolic acid, monoamine oxidase B inhibitors in a Brazilian plant, Himatanthus sucuuba.

Monoamine oxidase B (MAO-B) inhibitors were isolated from the bark of a Brazilian plant, Himatanthus sucuuba (SPR.). Assignments of the 1H- and 13C-NMR data using two dimensional (2D)-NMR techniques showed the active components to be known lichen depsides, confluentic acid (1) and 2'-O-methylperlatolic acid (2). The depside (1) showed selective inhibition of MAO-B with IC50 value of 0.22 microM.

Animals↗
PubMed1994

[Phosphate ion-exchange with hydrotalcite-like compound in the presence of trypsin].

A Cl(-)-intercalated hydrotalcite-like compound (HTAL) is a promising material as a better phosphate adsorbent for further clinical use than the currently marketed aluminum hydroxide gels. In the preceding study, we found that Cl- ions in the interlayer of HTAL can be exchanged with phosphate ions equivalently. In the present study, we have investigated the adsorption of porcine pancreas trypsin on HTAL and the influence of trypsin adsorption on the phosphate/Cl- ion-exchange properties of HTAL. The isotherm for trypsin adsorption at 310K and pH 8.4 showed the curve of BDDT V type and gave an adsorption capacity of 88.6 mg/g for trypsin. The zeta-potential analysis of HTAL suggested that the trypsin adsorption on HTAL at pH 8.4 was restrained by the electrostatic repulsion between HTAL surface and trypsin with positive charge. XRD and nitrogen adsorption studies showed that trypsin was adsorbed on the external surface of HTAL by occupying the area (ca. 22 nm2) per trypsin molecule. The phosphate/Cl- ion-exchange reaction with HTAL was not affected by the presence of trypsin; the phosphate ion-exchange capacity reached 73.6 mg P/g from the solution containing 3200 mg trypsin/dm3. On the other hand, the trypsin adsorption was significantly depressed by the loading of phosphate ions. The XRD analysis of the phosphate-loaded HTAL revealed that the layered structure of hydrotalcite holds after the phosphate ion-exchange, but shows a slight increase of the interlayer distance from 0.3 nm to 0.5 nm by the intercalation of phosphate ions.

Adsorption↗
PubMed1994

DNA damage induced in cultured human alveolar (L-132) cells by exposure to dimethylarsinic acid.

Gene damage in cultured human alveolar (L-132) cells induced by exposure to dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics in mammals, was studied. DNA single-strand breaks and DNA-protein cross-links were induced by the treatment of L-132 cells with 10 mM DMAA. These kinds of damage appeared at 8 hr after start of exposure to DMAA. As regards DNA-protein cross-links, the DNA was found to bind not only to core histone proteins but also linker histone (H1) and nonhistone proteins. Furthermore, the cross-links were formed by the binding to serine or threonine residues of H1 or nonhistone proteins through phosphate moieties of the DNA. The induction of the alkali-labile sites in DNA in DMAA-treated L-132 cells was observed prior to that of DNA single-strand breaks and DNA-protein cross-links. As one of the alkali-labile sites in DNA, we estimated apurinic/apyrimidinic (AP) sites in DNA. The present study suggests that the DNA single-strand breaks and DNA-protein cross-links induced by the treatment of L-132 cells with DMAA occurred via the formation of AP sites in the DNA and that the DNA-protein cross-links were produced by a Schiff-base reaction between amino groups of nuclear proteins and aldehyde groups of AP sites in the DNA and the DNA single-strand breaks, by a beta-elimination reaction on AP sites in the DNA.

Cacodylic Acid↗
PubMed1994

Ca2+ waves and intracellular Ca2+ concentration in guinea pig and rat myocytes.

We measured [Ca2+]i of guinea pig and rat myocytes with Ca2+ waves, using fura-2 fluorescence image processing. In guinea pig myocytes, Ca2+ waves were absent during the control perfusion period, but could be induced by the addition of strophanthidin (100 microM) or sodium cyanide (NaCN: 2 mM) to the perfusate. The [Ca2+]i increased from the control values of 69 +/- 5 nM and 46 +/- 2 nM, to 263 +/- 9 (p < 0.05 vs. control) nM and 225 +/- 20 (p < 0.05) nM, respectively, when cells exhibited Ca2+ waves. Although 13% (16 of 121) of the rat myocytes displayed Ca2+ waves during the control perfusion, the [Ca2+]i with Ca2+ waves (56 +/- 9 nM) did not differ from [Ca2+]i in the absence of Ca2+ waves (54 +/- 3 nM). Ca2+ waves were induced by the perfusion with a high Ca2+ solution (24.5 microM) or NaCN (2 microM), and [Ca2+]i increased from the control values of 67 +/- 11 nM and 74 +/- 5 nM, to 231 +/- 41 (p < 0.05 vs. control) nM and 266 +/- 64 nM, respectively, when cells exhibited Ca2+ waves. The Ca2+ waves were abolished by the removal of extracellular Ca2+, or by the perfusion with ryanodine (10 microM) or caffeine (20 mM). In conclusion, it was shown that Ca2+ waves were due to oscillatory Ca2+ release and that the absolute value of [Ca2+]i is important for the appearance of Ca2+ waves in guinea pig and rat myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Changes in skin disaccharide components correlate with the severity of sclerotic skin in systemic sclerosis.

The disaccharide contents of chondroitinase-digestible glycosaminoglycans extracted from a 6-mm punch biopsy of the forearm skin were determined using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labelling. In 9 patients with systemic sclerosis, the amounts of both the main disaccharide unit of dermatan sulfate and chondroitin sulfate C increased significantly, as compared with 7 site-matched controls. Furthermore, the increase in dermatan sulfate was significantly correlated with both the clinical severity and the extent of skin sclerosis, while the main disaccharide unit of hyaluronic acid tended to decrease. These results confirm that changes in skin glycosaminoglycans are closely related to fibrotic processes and suggest that the alterations of disaccharide components may play a role in the collagen deposition in systemic sclerosis.

Aged↗
PubMed1994

c-myc protein expression during cell cycle phases in differentiating HL-60 cells.

We examined c-myc protein expression in cell cycle phases during differentiation induction of HL-60 cells by flow cytometry using an indirect immunofluorescence method. In exponentially proliferating HL-60 cells, c-myc protein was expressed in a cell cycle dependent manner. During the differentiation induction of HL-60 cells with dimethylsulfoxide (DMSO), c-myc protein was rapidly down-regulated in the G1/0 specific phase prior to the appearance of differentiation associated markers. Our results indicate that c-myc protein functions in the G1/0 specific phase in cellular differentiation, and the rapid down-regulation of c-myc protein in G1/0 phase is closely associated with initial differentiation programs.

Cell Cycle↗
PubMed1994

Phenthoate metabolites in human poisoning.

Five metabolites were detected in the plasma and urine of a patient following ingestion of the organophosphate insecticide, phenthoate. Intact phenthoate was detected only in gastric lavage fluid. After methylation of acidic extracts of plasma and urine, phenthoate acid, demethyl phenthoate, demethyl phenthoate oxon acid, demethyl phenthoate S-isomer, and demethyl phenthoate acid S-isomer were identified with synthesized phenthoate analogues by gas chromatography and gas chromatograph-mass spectrometry. The main metabolites were phenthoate acid and demethyl phenthoate oxon acid. Although demethyl phenthoate oxon acid was a significant metabolite, no phenthoate oxon, phenthoate oxon acid or demethyl phenthoate oxon were detected. If the oxon was formed in the patient, it may have been rapidly degraded by carboxylesterase or glutathione transferase to demethyl phenthoate oxon acid.

Chromatography, Gas↗