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Biomedical subjects

K Ohta

Publications and source records attributed to K Ohta.

At least 19 recordsLinked to original sources
PubMed1992

Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes. In three unrelated Japanese children with combined pituitary hormone deficiency, we identified three point mutations in the Pit-1 gene, Pro24Leu, Arg143Gln, and Arg271Trp, located on the major transactivation region, POU-specific domain, and POU-homeodomain, respectively.

Amino Acid Sequence↗
PubMed1992

Cellular mechanism of thrombin on endothelin-1 biosynthesis and release in bovine endothelial cell.

We have studied the cellular mechanism responsible for induction of preproendothelin (preproET)-1 mRNA and release of ET-1 by thrombin in cultured bovine endothelial cells (ECs). Thrombin induced an immediate and dose-dependent formation of inositol-1,4,5-trisphosphate (IP3) with a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). The thrombin-induced ET-1 release was abolished either by a phospholipase C inhibitor, a protein kinase C (PKC) inhibitor, or an intracellular Ca(2+)-chelator, whereas a Ca(2+)-channel antagonist was ineffective. A selective thrombin inhibitor (argatroban) decreased IP3 formation and the increase in [Ca2+]i and ET-1 release stimulated by thrombin. Northern blot analysis revealed that thrombin-induced expression of preproET-1 mRNA was inhibited completely by a PKC inhibitor and partially by argatroban. These data suggest that thrombin is involved in the mechanism of preproET-1 mRNA expression and subsequent ET-1 release, possibly through activation PKC and mobilization of intracellular Ca2+ resulting from the receptor-mediated phosphoinositide breakdown in ECs.

Animals↗
PubMed1992

Characterization of the gene encoding human pituitary-specific transcription factor, Pit-1.

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone- and prolactin-encoding genes. A chromosomal gene related to human Pit-1 isolated from human gene libraries was over 14 kb long and split into six exons. All of the splice donor and acceptor sites conformed to the GT/AG rule. The gene was mapped to human chromosome region 3p11.

Base Sequence↗
PubMed1992

Interleukin 6/B cell stimulatory factor-II is expressed and released by normal and transformed human bronchial epithelial cells.

Airway epithelial cells have a potential to participate in local immune and inflammatory responses via releasing biologically active compounds. We studied the expression and release of interleukin 6 (IL-6), a multifunctional cytokine possibly involved in tissue immune responses. Primary culture of normal human bronchial epithelial cells and its transformed cell line BEAS-2B released significant amount of biologically and immunologically intact IL-6 into media. A protein synthesis inhibitor cycloheximide abolished the IL-6 release, suggesting a de novo synthesis. Northern blot analysis demonstrated the expression of the specific IL-6 mRNA. Human bronchial epithelial cells can produce IL-6 and contribute to the local activity of IL-6, suggesting that these cells may play a role in the regulation of airway immune responses.

Antibodies, Monoclonal↗
PubMed1992

Glutathione-binding proteins identified by monoclonal antibodies which depress the behavioral response evoked by glutathione in Hydra.

Hydra shows at least 5 components of the behavioral response (R1-R5) which are evoked at different concentrations of S-methylglutathione (GSM). We have prepared several monoclonal antibodies (mAbs), each of which depressed specific responses and visualized specific structures. In this paper, we analyzed molecules participating in the signaling pathway of R5 response with use of three mAbs (J245, J5 and J5/1), all of which depressed the response. A behavioral analysis of the response in the presence of mixtures of these mAbs suggested that J245 and J5 both acted on a component of the receptor-effector system which was not affected by J5/1. Correspondingly, an immunoblotting analysis showed two 220 kDa proteins which reacted with both J245 and J5 but not with J5/1. ELISA analyses also showed that the J245 antigens formed only a portion of the J5 antigens. A labeled major peak was found in the 220 kDa protein fraction by gel permeation HPLC after immunoprecipitation of the J245 antigen photolabeled with [35S]-S-(p-azidophenacyl)-glutathione. Competition of the photolabeling by the ligands GSM and L-glutamate (a competitive inhibitor of the R5 response) indicated dissociation constants for their binding to the protein of 55 and 90 microM, respectively. These values were consistent with those expected from behavioral experiments. The 220 kDa proteins therefore appear to be candidates for the receptor molecules mediating R5.

Affinity Labels↗
PubMed1992

Apoptotic cell death of primed CD45RO+ T lymphocytes in Epstein-Barr virus-induced infectious mononucleosis.

The expansion of activated T cells, characterized by the expression of CD45RO as well as HLA-DR antigens, is a central feature in acute infectious mononucleosis (IM) induced by primary infection of Epstein-Barr virus (EBV). However, the fate of these activated T cells in this disease is not clearly understood. We found that, on simple culture, a large proportion of T cells isolated from acute IM patients died rapidly, but only a few T cells from normal individuals did. Morphologic observations and DNA fragmentation analysis showed that the loss of viability of IM T cells after incubation was mediated by apoptosis. IM T cells undergoing apoptosis resided exclusively in the CD45RO+ populations of both CD4+ and CD8+ T cells, most of which were shown to coexpress apoptosis-related Fas antigen. Some cytokines such as interleukin-2 (IL-2), IL-5, and IL-6 could rescue IM T cells from apoptotic cell death. The results seemed to imply that most of primed (CD45RO+) T cells in acute IM might be subject to apoptotic cell death, possibly when leaving from the local sites actively producing certain soluble factors required for their survival. Our studies suggest the programmed cell death of peripheral mature T cells as a mechanism of antigen-driven selection.

Antigens, CD↗
PubMed1992

Efficient in vitro folding of the three-disulfide derivatives of hen lysozyme in the presence of glycerol.

Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In the reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and 'correct' disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS-derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for 'correct' in vitro folding.

Animals↗
PubMed1992

Intestinal active absorption of sugar-conjugated compounds by glucose transport system: implication of improvement of poorly absorbable drugs.

The intestinal absorption of glucose- and galactose-conjugated compounds was studied in the everted sac of the rat small intestine. The absorption clearance of p-nitrophenyl beta-D-glucopyranoside (p-NPglc) at 250 microM in the mucosal side (4.45 +/- 0.34 microL/min/cm, mean +/- SE, N = 4), calculated by dividing the absorption rate by the drug concentration, was significantly decreased (0.476 +/- 0.036 microL/min/cm) in the presence of 1 mM phloridzin, an inhibitor of glucose transport, and in the absence of Na+, a cosubstrate of the glucose transport carrier (0.424 +/- 0.018 microL/min/cm). The absorption clearance of p-NPglc was decreased as its concentration increased. In the same experiment, the absorption clearance of p-nitrophenyl beta-D-galactopyranoside (1.99 +/- 0.23 microL/min/cm) was also significantly decreased in the presence of phloridzin and in the absence of Na+. However, the absorption clearance of p-nitrophenyl beta-D-mannopyranoside (0.811 +/- 0.013 microL/min/cm) was low and not significantly decreased in the presence of phloridzin (P greater than 0.1). Furthermore, the absorption clearance of beta-naphthyl beta-D-glucopyranoside and beta-naphthyl beta-D-galactopyranoside was also significantly decreased in the presence of phloridzin (P less than 0.001). These results indicated that the glucose and galactose moieties provided these compounds with a new route by way of the glucose transport carrier for intestinal absorption.

Animals↗
PubMed1992

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.

Animals↗
PubMed1992

Concomitant expression of receptor subtype and isopeptide of endothelin by human adrenal gland.

We studied whether specific receptors for endothelin (ET) isopeptide exist in human aldosterone-producing adenoma and normal adrenal cortex, and whether ET isopeptides are produced by human adrenal gland. Competitive binding studies using [125I]ET-1 as a radioligand revealed the presence of a single class of high-affinity binding sites for ET-1 with the apparent KD of 70 +/- 31 pM and Bmax of 226 +/- 139 fmol/mg protein in adenoma membranes almost comparable to those in adjacent normal cortex. The apparent Ki for ET-2 and ET-3 were 89 +/- 33 pM and 82 +/- 16 pM, respectively. Northern blot analysis of poly(A)+ RNA of adenoma and adjacent normal cortex using cDNAs for ET receptor subtype (ETA, ETB) and ET isopeptide (ET-1, ET-3) as probes revealed that ETA and ETB receptors as well as ET isopeptides (preproET-1, preproET-3) are concomitantly expressed in both tissues. Our data demonstrate for the first time that ET receptor subtype (ETA and ETB) and ET isopeptide (ET-1 and ET-3) are concomitantly expressed by human adrenal cortex, suggesting the potential role of ETs as a local mediator in human adrenal gland.

Adenoma↗
PubMed1992

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.

Animals↗
PubMed1992

Effects of three distinct natriuretic peptides on receptor binding and guanylate cyclase activities in rat glioma cells.

Receptor binding and cyclic GMP generation by three distinct natriuretic peptides (ANP, BNP, CNP) were studied in a cultured rat glioma cell line (C6). Binding studies revealed the presence of high-affinity binding sites for three natriuretic peptides with almost comparable affinities. In contrast, CNP and BNP were almost equipotent in stimulating intracellular cyclic GMP generation over the low concentration range, but CNP caused further elevation in the high concentration range, whereas ANP was minimally effective. Our data suggest that the glioma cells possess receptors more responsive to CNP than ANP and BNP despite no apparent correlation between receptor binding affinities and cyclic GMP responses.

Animals↗
PubMed1992

Regulation of a major microtubule-associated protein by MPF and MAP kinase.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.

Animals↗
PubMed1992

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1992

Cerebral microcirculatory changes during and following transient ventricular tachycardia in cats.

Although reduced cerebral perfusion is believed to be the cause of syncope due to cardiac arrhythmias, investigations on the cerebral microcirculation during cardiac arrhythmias have been rare. We therefore studied the effects of transient ventricular tachycardia on the local cerebral blood volume and blood flow. Experimental ventricular tachycardia was induced in cats by electrically stimulating the ventricle of the heart at a rate of 300/min for 1 min. Using our photoelectric method, the local cerebral blood volume, mean transit time of blood, and cerebral blood flow in the parieto-temporal region were measured during and for 3 h after ventricular tachycardia. Transient ventricular tachycardia of as short as 1 min caused cerebral ischemia with a blood flow reduction of approximately 30%. This was considered to be due to reduced blood pressure plus transient autonomic dysfunction, or dysautoregulation, during the ventricular tachycardia. Mild and transient reactive hyperemia occurred immediately after termination of the dysrhythmia, but continuous reductions of cerebral blood flow were observed thereafter for 3 h. This delayed hypoperfusion is attributable to either vasoconstriction of the large resistance vessels or changes in the hemorheological properties of the blood caused by cerebral ischemia. Ventricular tachycardia of the type reported has significant and long-lasting effects on the cerebral microcirculation.

Animals↗
PubMed1992

Local and systemic influenza haemagglutinin-specific antibody responses following aerosol and subcutaneous administration of inactivated split influenza vaccine.

An easily administered and safe vaccine is required to produce the herd immunity necessary to control influenza epidemics worldwide. A commercial quadrivalent inactivated split influenza vaccine was administered intranasally in aerosol form to a group of 46 volunteers; other groups were given the same vaccine subcutaneously and saline intranasally. The results show that mucosal stimulation via intranasal vaccination resulted in a marked increase in local HA-specific IgA antibodies, and that this stimulation was necessary for serum HA-specific IgA responses. Serum HA-specific IgA antibody levels can be used as indicators of local antigenic stimulation, providing a method for evaluating potency and antigenicity in humans of intranasal influenza vaccine. This vaccination route shows much promise for the future.

Adult↗
PubMed1992

A simple and sensitive ELISA of antibodies to pertussis antigens.

A precise method for quantifying serum antibody levels to antigens of Bordetella pertussis is required to evaluate pertussis vaccines, to diagnose pertussis infections, and to detect transplacentally transmitted maternal antibodies. The purpose of this paper is to report a rapid, sensitive, easy and standardized ELISA method using polystyrene balls for the assay of serum antibodies against pertussis toxin (PT) and filamentous haemagglutinin (FHA), which are protective antigens of B. pertussis. By the polystyrene ball method, a clear-cut difference was observed between values of pre- and postvaccination serum samples while by the conventional plate method those values overlapped. It was concluded that the PS ball method is one of the most sensitive and simple methods for detecting antibodies against PT and FHA.

Adhesins, Bacterial↗