Biomedical subjects

T Hamamoto

Publications and source records attributed to T Hamamoto.

At least 19 recordsLinked to original sources

PubMed2001

Activation of blood coagulation factor IX by gingipains R, arginine-specific cysteine proteinases from Porphyromonas gingivalis.

The effect of two arginine-specific cysteine proteinases (gingipains R) from Porphyromonas gingivalis, an aetiological factor of adult periodontitis, on the activation of human factor IX was investigated in the presence of ethylene glycol, an activity enhancer of activated factor IX (factor IXa), with the use of a fluorogenic oligopeptide substrate. Each gingipain R rapidly activated factor IX but the 95 kDa proteinase complex (HRgpA) that contains both haemagglutinin/adhesion and catalytic domains was 2.4-fold more efficient than the single-chain 50 kDa gingipain R (RgpB), which has only a catalytic domain. SDS/PAGE and N-terminal sequence analysis of factor IX digestion fragments indicated that, like all endogenous activators, gingipains R also produce factor IXabeta via an IXa intermediate. Significantly, phospholipids augmented the activation of factor IX by HRgpA but not by RgpB in a Ca(2+)-dependent manner. In the presence of both cofactors the kinetic efficiency of HRgpA to activate factor IX (k(cat)/K(m)=1.9x10(6) M(-1).s(-1)) was 8.5-fold higher than that of RgpB (k(cat)/K(m)=2.3x10(5) M(-1).s(-1)) and double that of the factor VIIa-tissue factor complex, but 8-fold lower than that for factor XIa. A comparison of the relative activation rates of factor IX, factor X and prothrombin directly in plasma by HRgpA suggests a significant contribution for factor IX conversion in blood coagulation induced by gingipains R. Taken together, gingipains R are the first-reported activators of factor IX of bacterial origin. By this effect they could be involved in the production of thrombin as well as the subsequent generation of prostaglandins and interleukin 1, all of which have been found to be associated with the development and progression of periodontitis.

Adhesins, Bacterial↗

PubMed2001

Effect of isosorbide dinitrate on nitric oxide synthase under hypoxia.

Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) formation from L-arginine in the presence of molecular oxygen and NADPH. NO is involved in the regulation of microvasculature. Isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) have been widely used as vasodilators to treat acute myocardial ischemia, their biological effects being due to the release of NO. In this investigation, the effects of ISDN and GTN on NOS activity in the presence or absence of oxyhemoglobin under hypoxia and normoxia were studied. The apparent K(m) values for molecular oxygen were 21.6 +/- 1.5 and 9.4 +/- 1.3 micromol/l for nNOS and eNOS, respectively. ISDN liberated NO in a concentration- and pH-dependent manner, but no differences between hypoxia and normoxia were observed. The NO release from ISDN was also measured directly by an electron spin resonance spectral method with N-(dithiocarboxy)sarcosine-Fe complex as a NO-trapping agent. ISDN increased nNOS and eNOS activities in the presence of 30 micromol/l oxyhemoglobin under hypoxia, while it did not affect nNOS and eNOS activities under normoxia. In the absence of oxyhemoglobin, ISDN inhibited nNOS and eNOS activities under both hypoxic and normoxic experimental conditions. The rate of oxygen release from oxyhemoglobin under hypoxia was increased 3 times in the presence of 1 mmol/l ISDN. In contrast to ISDN, GTN could not release NO spontaneously, and it also did not affect nNOS and eNOS activities in the absence or presence of 30 micromol/l oxyhemoglobin under both hypoxic and normoxic conditions. These results indicated that the NO release from ISDN is different from that of GTN, and the increase of NOS activity by ISDN in the presence of oxyhemoglobin under hypoxia is ascribed to the increase in molecular oxygen concentration.

Animals↗

PubMed2000

Proteoliposomes colocalized with endogenous mitochondria in mouse fertilized egg.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Animals↗

PubMed2000

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

Animals↗

PubMed2000

Redefined substrate specificity of ST6GalNAc II: a second candidate sialyl-Tn synthase.

The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.

Animals↗

PubMed2000

Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis.

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

ATP Synthetase Complexes↗

PubMed2000

BMP type II receptor is required for gastrulation and early development of mouse embryos.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, play a variety of roles during mouse development. BMP type II receptor (BMPR-II) is a type II serine/threonine kinase receptor, which transduces signals for BMPs through heteromeric complexes with type I receptors, including activin receptor-like kinase 2 (ALK2), ALK3/BMPR-IA, and ALK6/BMPR-IB. To elucidate the function of BMPR-II in mammalian development, we generated BMPR-II mutant mice by gene targeting. Homozygous mutant embryos were arrested at the egg cylinder stage and could not be recovered at 9.5 days postcoitum. Histological analysis revealed that homozygous mutant embryos failed to form organized structure and lacked mesoderm. The BMPR-II mutant embryos are morphologically very similar to the ALK3/BMPR-IA mutant embryos, suggesting that BMPR-II is important for transducing BMP signals during early mouse development. Moreover, the epiblast of the BMPR-II mutant embryo exhibited an undifferentiated character, although the expression of tissue-specific genes for the visceral endoderm was essentially normal. Our results suggest that the function of BMPR-II is essential for epiblast differentiation and mesoderm induction during early mouse development.

Animals↗

PubMed2000

Smad6 is a Smad1/5-induced smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter.

Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. We have isolated the mouse Smad6 promoter and identified the regions responsible for transcriptional activation by BMPs. The proximal BMP-responsive element (PBE) in the Smad6 promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for Drosophila Mad and Medea. We generated a luciferase reporter construct (3GC2-Lux) containing three repeats of the GC-rich sequence derived from the PBE. BMPs and BMP receptors induced transcriptional activation of 3GC2-Lux in various cell types, and this activation was enhanced by cotransfection of BMP-responsive Smads, i.e. Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE was observed. These results indicate that the expression of Smad6 is regulated by the effects of BMP-activated Smad1/5 on the Smad6 promoter.

Animals↗

PubMed2000

Novel functions of human alpha(1)-protease inhibitor after S-nitrosylation: inhibition of cysteine protease and antibacterial activity.

alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)). We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase. S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity. Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione. NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap. Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase. As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated.

Animals↗

PubMed2000

An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine.

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc.

Amino Acid Sequence↗

PubMed2000

[A case of duodenal adenoma consists of multiple nodular lesions].

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Adenoma↗

PubMed1999

Regulation of energy metabolism in human cells in aging and diabetes: FoF(1), mtDNA, UCP, and ROS.

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.

Aging↗

PubMed1999

Quantitative analysis of expression of mouse sialyltransferase genes by competitive PCR.

The present paper describes a rapid and systematic method for semi-quantitative analysis of the expression of sialyltransferase genes. So far, fifteen sialyltransferase cDNAs have been cloned from mice. Most of these genes are expressed in developmental stage-dependent and/or tissue-specific manners, and the expression levels of some of them are too low to detect on Northern blot analysis. To resolve how each sialyltransferase contribute to synthesize sialylglycoconjugates, it is necessary to establish the method for quantification of gene expression levels of these fifteen sialyltransferases. Therefore, we developed a competitive PCR-based method for analyzing the quantitative relationship of the gene expression of fifteen sialyltransferases. Using this method, we can investigate the levels of gene expression of sialyltransferases in various cell lines and various tissues of mice, and can accurately determine their expression levels.

Animals↗

PubMed1999

Alternatively spliced variant of Smad2 lacking exon 3. Comparison with wild-type Smad2 and Smad3.

An alternatively spliced variant of Smad2 with a deletion of exon 3 (Smad2Deltaexon3) is found in various cell types. Here, we studied the function of Smad2Deltaexon3 and compared it with those of wild-type Smad2 containing exon 3 (Smad2(wt)) and Smad3. When transcriptional activity was measured using the p3TP-lux construct, Smad2Deltaexon3 was more potent than Smad2(wt), and had activity similar to Smad3. Transcriptional activation of the activin-responsive element (ARE) of Mix.2 gene promoter by Smad2Deltaexon3 was also similar to that by Smad3, and slightly less potent than that by Smad2(wt). Phosphorylation by the activated transforming growth factor-beta type I receptor and heteromer formation with Smad4 occurred to similar extents in Smad2Deltaexon3, Smad2(wt), and Smad3. However, DNA binding to the activating protein-1 sites of p3TP-lux was observed in Smad2Deltaexon3 as well as in Smad3, but not in Smad2(wt). In contrast, Smad2(wt), Smad2Deltaexon3, and Smad3 efficiently formed ARE-binding complexes with Smad4 and FAST1, although Smad2(wt) did not directly bind to ARE. These results suggest that exon 3 of Smad2 interferes with the direct DNA binding of Smad2, and modifies the function of Smad2 in transcription of certain target genes.

Alternative Splicing↗

PubMed1999