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Biomedical subjects

T Nakaoka

Publications and source records attributed to T Nakaoka.

At least 37 records · Page 2Linked to original sources
PubMed1994

Adenosine as an endogenous mediator of hypoxia for induction of vascular endothelial growth factor mRNA in U-937 cells.

Adenosine induced by hypoxia exerts various effects via different types of receptors. Recently, hypoxia was shown to be a strong inducer of vascular endothelial growth factor, a secreted endothelial cell specific mitogen. In this report, we studied on effects of adenosine on inducibility of VEGF and possible mediation of hypoxia for its induction in U-937 cells. Hypoxia induced expression of VEGF mRNA with an early peak at 1 hour. 5'-N-ethylcarboxamidoadenosine, an adenosine analog, strongly induced VEGF mRNA, which was inhibited by 3,7-dimethyl-1-propargylxanthine (DMPX), an A2-antagonist. The hypoxic induction was inhibited by adenosine deaminase, 7-(beta-hydroxyethyl)theophylline, a non-selective adenosine receptor antagonist and DMPX. These results suggest that the hypoxic induction of VEGF mRNA is mediated by adenosine via A2-receptor in U-937 cells.

Adenosine↗
PubMed1994

Cloning and expression of cDNA for a new type of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase.

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding a new type of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3GalA.2) has been isolated from both mouse and rat brain cDNA libraries. The cDNA sequences included an open reading frame coding for 350 amino acids, and the primary structure of this enzyme suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases. The deduced amino acid sequence of ST3GalA.2 (mouse) showed 76% identity in the active domain with that of the previously cloned mouse Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3GalA.1 (Lee, Y.-C., Kurosawa, N., Hamamoto, T., Nakaoka, T., and Tsuji, S. (1993) Eur. J. Biochem. 216, 377-385)). Northern blotting indicated that the expression of ST3GalA.2 mRNA is tissue-specific, it being prominent in brain and liver, while that in the other tissues is very low. This enzyme expressed in COS-7 cells exhibited transferase activity only toward the disaccharide moiety of Gal beta 1,3GalNAc of glycolipids as well as glycoproteins and oligosaccharides like ST3GalA.1, but showed a difference in acceptor substrate preference, i.e. asialo-GM1 and GM1 were much more suitable substrates for ST3GalA.2 than for ST3GalA.1.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of chick embryo Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Comparison with the mammalian enzyme.

DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open-reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase.

cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.

Acetylgalactosamine↗
PubMed1994

Effect of long-term cyclic mechanical load on protein synthesis and morphological changes in cultured myocardial cells from neonatal rat.

We studied the effect of prolonged cyclic mechanical stimulation on protein synthesis and morphological changes in cultured cardiac myocytes isolated from 3-day-old neonatal rats to develop an in vitro model for cardiac hypertrophy induced by a mechanical load. Myocardial cells were cultured on deformative culture plates with M-199 culture medium in the presence or absence of fetal calf serum (FCS), and a cyclic mechanical load was applied for 2 hours up to 15 days. Mechanical stimulation for 2 hours increased the 14C-phenylalanine incorporation rate of myocardial cells, both in the absence and presence of FCS or when the myocardial cells were either beating or arrested with tetrodotoxin. The incorporation rate always increased by mechanical stimulation during 15 days of cell culture as compared to cells that sustained no mechanical stress. The cell size and protein content, which increased gradually with mechanical stimulation and reached a maximum at 10 days, were even greater in the presence of FCS. Within 10 days myocardial cells had aligned in the direction of the maximal cyclic mechanical load. In these cells electron microscopy revealed an increase in the number of myofilaments associated with the development of mitochondria as recognized in the adult myocardial cells. These results indicate that long-term cyclic mechanical loading of cultured myocardial cells may be a good in vitro model for the study of cardiac hypertrophy.

Animals↗
PubMed1994

Expression of mouse Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase in an insoluble form in Escherichia coli and partial renaturation.

Mouse Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase was produced in an insoluble form in Escherichia coli cells harboring expression plasmids. The insoluble protein was solubilized with 8 M urea and diluted for renaturation of the enzyme. The substrate specificity and kinetic parameters, except for the specific activity, of the renatured enzyme were similar to those of the enzyme obtained from rat liver. These results suggest that a bacterial expression system is a potentially powerful tool for the large scale production of sialyltransferases and for elucidating the molecular mechanisms of sialyltransferases.

Animals↗
PubMed1994

Rapid induction of vascular endothelial growth factor expression by transient ischemia in rat heart.

Vascular endothelial growth factor (VEGF or vascular permeability factor), a direct-acting, endothelial cell-specific mitogen, has been suggested to be involved in development and maintenance of vasculatures in tumor neovascularization and in normal tissues. To investigate possible roles of VEGF in ischemic hearts, we studied induction of VEGF mRNA by ischemia and hypoxia using coronary artery-ligated hearts in vivo and perfused hearts and cultured myocardial cells in vitro. VEGF mRNA was potently induced by ischemia in the heart in vivo. In perfused hearts, maximum expression was rapidly induced (within 30 min) by transient reversible ischemia (5-10 min of ischemia) and lasted at least 3 h. Induction was also caused by hypoxia, which was confirmed in perfused hearts and cultured myocardial cells. These results suggest that induction of VEGF mRNA is upregulated by oxygen deprivation in the heart and that not only infarction but also chronic ischemia in the clinical setting could induce VEGF as a potent angiogenesis factor to stimulate coronary collateral formation.

Animals↗
PubMed1994

Antigen stimulation alters the expression of S-100 protein in the giant macrophages and follicular dendritic cells of the guinea pig lymph nodes.

Injections of chick erythrocytes were employed to examine the relationship between expression of S-100 protein and antigen stimulation in the giant macrophages (GM) and follicular dendritic cells (FDC) of guinea pig lymph nodes by an immunocytochemical method. Popliteal lymph nodes were observed 1, 3, 6 and 12 h, then 1, 2 and 3 days, and finally 1, 2 and 4 weeks after injection of chick erythrocytes into hind foot pads. All GM in control lymph nodes were dendritic in shape at the cortical surface and strongly immunoreactive to S-100 protein. In lymph nodes of injected animals, three types of GM were observed at the cortical surface: cells were S-100-protein-positive/phagocytosis-positive, S-100-protein-positive/phagocytosis-negative and S-100-protein-negative/phagocytosis-positive. GM that were S-100-protein-negative/phagocytosis-positive appeared from 1 h onwards, reaching a maximum number at 6 h and then decreasing in number with time at the cortical surface. In the medulla, such cells were observed after 1 h, increasing to a maximal level at 6 h and, thereafter, gradually decreasing in number. These results indicate that expression of S-100 protein in GM is altered by antigen stimulation and that GM move in the lymph nodes. The same pattern of expression of S-100 protein was demonstrated in the FDC 1 week after injection.

Animals↗
PubMed1993

Molecular cloning and expression of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain.

DNA clones encoding beta-galactoside alpha 2,3-sialyltransferase have been isolated from mouse brain cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amino acid sequence showed 80% identity with that of porcine submaxillary gland Gal beta 1,3GalNAc alpha 2,3-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2-terminal part including the cytoplasmic tail, signal-anchor domain and stem region was replaced with an immuno-globulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. This enzyme exhibited the transferase activity toward only the disaccharide moiety of Gal beta 1,3GalNAc of glycoproteins and glycolipids, no significant activity being detected for the other substrates tested.

Amino Acid Sequence↗
PubMed1993

Two step single primer mediated polymerase chain reaction. Application to cloning of putative mouse, beta-galactoside alpha 2,6-sialyltransferase cDNA.

Using the 2 step single primer mediated polymerase chain reaction(PCR), mouse beta-galactoside alpha 2,6-sialyltransferase cDNA was cloned. Single primer mediated PCR is a method to amplify a particular DNA fragment beyond its known sequence region. It employs only one primer for the reaction. Compared to other PCR methods to amplify an adjacent sequence of known DNA fragment, this method requires no enzymatic manipulation on template DNA and is applicable to a template on long DNA fragment. First, a short DNA fragment of the enzyme was obtained from mouse cDNA by the usual PCR method using degenerate primers synthesized according to a relatively conserved region in rat and human beta-galactoside alpha 2,6-sialyltransferase. Four primers were synthesized based on this sequence, then 2 step single primer mediated PCR were performed to obtain 5' and 3' flanking sequences of this short fragment resulting in 1.0 kb and 1.3 kb fragments being amplified respectively. The integrity of the two fragments was confirmed by an additional PCR using primers synthesized according to the joined sequence, which contained 1.2kb complete putative mouse beta-galactoside alpha 2,6-sialyltransferase coding region. The result showed that the specificity and consequently applicability of the single primer mediated PCR for amplifying a particular DNA fragment beyond known sequence region was remarkably improved by the successive 2nd reaction.

Amino Acid Sequence↗
PubMed1993

Phosphatidylserine specific binding protein in rat brain: purification and characterization.

A calcium-independent phosphatidylserine specific binding protein detected on liposome blotting analysis was purified from rat brain and revealed to be identical to myristoylated, alanine-rich C kinase substrate (MARCKS). MARCKS specifically binds to phosphatidylserine but not phosphatidylcholine. The binding of MARCKS to phosphatidylserine was abolished on protein kinase C-dependent phosphorylation. Since bacterially expressed MARCKS also specifically binds to phosphatidylserine, myristoylation of the N-terminal glycine seems not to be essential for the binding of MARCKS to phosphatidylserine. These data suggest that phosphatidylserine is a membranous target molecule of MARCKS.

Amino Acid Sequence↗
PubMed1993

High incidence of mitral and tricuspid regurgitation in patients with Graves' disease detected by two-dimensional color Doppler echocardiography.

With two-dimensional (2D) color Doppler echocardiography, the cardiac and valvular function of 24 consecutive patients with a history of Graves' disease (17 were hyperthyroid and 7 were euthyroid at the time of the examination) were evaluated. The incidences of mitral regurgitation (MR), tricuspid regurgitation (TR) and MR plus TR were significantly higher in the patients with Graves' disease than in the age-matched control group of patients without this disease. In the patients who had signs of congestive heart failure (CHF) while they were hyperthyroid, a significantly higher incidence of severe TR was observed. This is the first report of a 2D color Doppler echocardiography study on the incidences of TR and/or MR in patients with Graves' disease. Our data indicate that in Graves' disease valvular dysfunction can be caused by systemic disorders and that severe TR is a possible risk factor for CHF.

Adult↗
PubMed1992

Bimodal regulation of protein phosphorylation by a ganglioside in rat brain membrane.

Whether or not a ganglioside influences the protein phosphorylation in the rat brain membrane fraction was investigated. Phosphorylation of the 72 kDa protein was significantly affected by the addition of 80 nM GQ1b in vitro, which is far below the reported concentration of gangliosides that affects protein phosphorylation in the neuronal membrane fraction. This action of GQ1b was bimodal: it being not only stimulatory as to the incorporation of phosphate into the 72 kDa protein on incubation of 20 sec, but also as to the release of phosphate from or breakdown of the 72 kDa protein on incubation for more than 5 min. Eighty nM GQ1b did not noticeably affect ATPase in the same fraction. These results suggest that the transphosphorylation of the 72 kDa protein is affected by the interaction of GQ1b with either the responsible enzymes or the 72 kDa protein as a substrate.

Adenosine Triphosphatases↗
PubMed1991

S-100 immunoreactive cells in the spleen and bursa of Fabricius of broiler chickens.

The distribution of S-100 protein in spleens and bursae of Fabricius of broiler chickens with various diseases was investigated by an immunohistochemical method with antiserum to bovine S-100 protein. S-100 protein-positive cells were found in six cases of 34 chickens. In spleens, S-100 protein was detected in reticular cells in the ellipsoids of the sheathed arteries and eosinophilic granulocytes. Follicular dendritic cells in germinal centres were also S-100 protein-positive. In the bursa, reticular cells of the follicles and eosinophilic granulocytes in the follicular cortex and surface epithelium were positive for S-100 protein. The evidence suggests that S-100 protein may be involved in diseases of chickens.

Animals↗
PubMed1989

Risk factors for the complication of cerebral infarction in Japanese patients with acute myocardial infarction.

The incidence and risk factors for cerebral infarction in Japanese patients with acute myocardial infarction were evaluated. Seven (5.0%) of 140 patients with acute myocardial infarction suffered from cerebral infarction during their initial hospitalization. The incidence was slightly higher than those reported in Western countries. Anterior wall myocardial infarction and a past history of cerebrovascular disease were considered to be probable risk factors for the complication. A beneficial effect of anticoagulant therapy in preventing cerebral infarction in cases of acute myocardial infarction with those risk factors is suggested.

Aged↗
PubMed1987

Effect of ascorbic acid on the metabolism of N-nitrosopiperidine.

Guinea-pigs were fed diets containing 48, 200 or 2000 mg/kg ascorbic acid (AsA) for 20-30 days and given a single oral dose of 50 mg N-nitrosopiperidine (NPIP)/kg body weight. The levels of glutaric acid, 3-hydroxy-NPIP and 4-hydroxy-NPIP, but not of 1,5-pentanediol, in urine increased with the dose of AsA in the diet.

Animals↗