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T Hamamoto

Publications and source records attributed to T Hamamoto.

At least 37 records · Page 2Linked to original sources
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

[Candida].

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Antibodies, Fungal↗
PubMed1999

[beta-D-glucan].

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Glucans↗
PubMed1999

[Cryptococcus].

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Antibodies, Fungal↗
PubMed1999

[Aspergillus].

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Antibodies, Fungal↗
PubMed1998

Molecular cloning and functional expression of a fifth-type alpha 2,3-sialyltransferase (mST3Gal V: GM3 synthase).

The cDNA encoding a new type of alpha 2,3-sialyltransferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV. The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively. The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosylceramide (LacCer), and synthesized GM3. The apparent Km value for LacCer was 9.3 microM. mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides. The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3. The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside. The gene expression seemed to be coincident with ganglioside expression in mouse. Thus, we conclude that mST3Gal V is the fifth-type alpha 2,3-sialyltransferase carrying GM3 synthetic activity.

Amino Acid Sequence↗
PubMed1998

Acidic stimulation induces a negative regulatory factor that affects alternative exon selection in vitro in human ATP synthase gamma-subunit pre-mRNA.

Tissue-specific alternative RNA splicing in human F1gamma pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1gamma gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1gamma minigene containing parts of exon 8, intron 8, and exon 9 of the human F1gamma gene and then analyzed a negative factor that inhibited inclusion of exon 9 via an in vitro splicing assay using acid-stimulated HT1080 cell nuclear extract. In vitro splicing of the F1gamma minigene, similarly to the beta-globin minigene used as a control, was observed in HeLa cell nuclear extract. Next, we performed supplemental experiments using HeLa and HT1080 cell nuclear extracts. The splicing reaction of the F1gamma minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human beta-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1gamma minigene in vitro, and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1gamma pre-mRNA.

Alternative Splicing↗
PubMed1998

Large-scale preparation of human thrombin: polyethylene glycol potentiates the factor Xa-mediated activation of prothrombin.

We investigated the ability of polyethylene glycol 4000 to accelerate thrombin generation in a mixture of prothrombin and factor X at concentrations of 1-30%. In the presence of 5 mM of CaCl2, polyethylene glycol 4000 promoted prothrombin activation at concentrations above 1%. The peak of activation was seen at levels of 14 and 20% of polyethylene glycol 4000. The effect of the polyethylene glycol was remarkably dependent on its molecular weight; molecular weights greater than 2000 were required for accelerating thrombin generation. Under optimal conditions, polyethylene glycol 4000, in the presence of CaCl2, promoted conversion of all of the prothrombin into thrombin and its derivatives. We conclude that polyethylene glycol 4000, at concentrations ranging from 14 to 20%, effectively accelerates thrombin generation in the presence of 5 mM of CaCl2. This new method for preparing thrombin is based on the use of polyethylene glycol 4000 and CaCl2 and is applicable to the manufacture of thrombin.

Calcium Chloride↗
PubMed1998

The effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa--tissue factor activity by antithrombin III.

We investigated the effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa-tissue factor activity by antithrombin III (ATIII) on a human bladder carcinoma (J82) cell line and an ovarian carcinoma (OC-2008) cell line, two tumor cell lines which constitutively synthesize and express high levels of cell surface tissue factor. We observed that ATIII inactivated factor VIIa-tissue factor more readily on OC-2008 cells than on J82 cells in the absence of added heparin. Likewise, factor Xa was more effectively inactivated on OC-2008 cells than on J82 cells. The ability of ATIII to inactivate factor VIIa-tissue factor activity on the OC-2008 cell was reduced following treatment of the cells with heparinase. This indicated that heparin-like GAGs were expressed on the OC-2008 cell surface, and that these GAGs were important for the inhibition of factor VIIa-tissue factor activity by ATIII. In addition, we demonstrated that the ability of ATIII to inactivate factor VIIa-tissue factor activity was markedly reduced following treatment of cells with calcium ionophore (A23187). However, the effect of cell surface GAGs on the inhibition of factor Xa by ATIII remained even after treatment of OC-2008 cells with A23187. In contrast to the manner of inhibition by ATIII/heparin, TFPI effectively inactivated factor VIIa-tissue factor activity on the cell surfaces even after induced physical damage or disruption of the cell by treatment with A23187. Our collective findings suggest that GAGs on cell surfaces play an important role in regulating factor VIIa-tissue factor activity by ATIII under normal conditions, or in the early phases of physical damage or destruction of the cell. However, TFPI may play a more important role than ATIII in regulating the activity of factor VIIa-tissue factor in a vascular trauma site following extensive cell injury.

Antithrombin III↗
PubMed1998

Phosphopentomutase of Bacillus stearothermophilus TH6-2: the enzyme and its gene ppm.

Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis. We identified a sequence 5'-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene. The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735. The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide. The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.

Amino Acid Sequence↗
PubMed1997

Intramolecular rotation in ATP synthase: dynamic and crystallographic studies on thermophilic F1.

A single molecule of ATP synthase (F0F1) is by itself a rotary motor, the smallest ever found, and this biomotor is driven by an electrochemical potential of H+ (delta microH+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The major breakthroughs in studies on the mechanochemical coupling have been the direct observation of the rotation of a stable alpha 3 beta 3 gamma complex of thermophilic F1 (TF1), and X-ray crystallography of the alpha 3 beta 3 gamma portion of mitochondrial F1 (MF1) and the alpha 3 beta 3 oligomer of TF1. This review focuses on the dynamics of TF1, demonstrated by a crucial experiment. The torque of the rotation was estimated to be 42 pN.nm from the delta microH+ and frictional force. Important unsolved problems are the crystallography of F0, elastic energy conversion, and the stator and rotor of this biomotor.

Adenosine Triphosphate↗
PubMed1997

Introduction of DNA into rat liver with a hand-held gene gun: distribution of the expressed enzyme, [32P]DNA, and Ca2+ flux.

DNA-coated Au particles were accelerated by pressurized He gas to supersonic velocities for introduction of a gene into cells. Experimental and theoretical analyses both revealed a heterogeneous distribution of the particles per shot (1 mg Au = 2.4 x 10(7) particles with 2 microg [32P] DNA = 2.5 x 10(11) moles). For introduction of genes into the liver of living rats, the best results were obtained with a newly developed hand-held gene delivery system. The beta-galactosidase gene introduced into rat liver with Au particles by He at 250 psi was expressed (1.2 microunits/microg protein) in a limited area of the liver surface (8 x 8 mm, depth 0.5 mm). When the same gene gun was used on a monolayer of cultured COS7 cells (about 5 microm thick), cells were lost in the central area of heavy bombardment. Cell death caused by influx of Ca2+ was prevented by the use of the cytosol-type culture medium.

Animals↗
PubMed1997

The stabilizing residues and the functional domains in the hyperthermophilic V-ATPase of Desulfurococcus.

To clarify a universal mechanism of the intramolecular rotation of ATP-synthase, an operon encoding a stable, ancestral ATPase was cloned from a heterotrophic archaeum Desulfurococcus strain SY. The operon of about 7 kbp contained genes E, C, G, A, B and D encoding subunits with predicted molecular weights of 23,217, 41,659, 11,499, 65,476, 52,295, and 24,897, respectively. The sequence was compared with that of Na-ATPase of Enterococcus hirae, A-ATPase of Halobacterium salinarium, V-ATPase of Methanosarcina mazei, and ATP synthase of Methanococcus jannaschii, which are homologous. (1) The cause of hyperthermostability: The main exchanges in the amino acid residues of hyperthermophilic proteins included Asp --> Glu (11 residues of A subunit of E.h.) and, Ser --> Ala. (2) The domains needed for the intramolecular rotation: The domains similar to those established in F-type ATPases were also found in the V-type ATPases of species with a different energy metabolism.

Adenosine Triphosphatases↗
PubMed1997

Genes of human ATP synthase: their roles in physiology and aging.

The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (delta mu H+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the delta mu H+ and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle, C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with rho degree cells (cells without mtDNA).

Aging↗
PubMed1997

Mouse beta-galactoside alpha 2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression.

Four types of beta-galactoside alpha 2,3-sialyltransferase (ST3Gal I-IV) have been cloned from several animals, but some contradictory observations regarding their substrate specificities and expression have been reported. Therefore, it is necessary to concurrently analyze the substrate specificities of the four enzymes, of which the source should be one animal. Accordingly, the acceptor substrate specificities and gene expression of mST3Gal I-IV were analyzed. Since we had already cloned ST3Gal I and II, as previously reported (Lee, Y.-C. et al., Eur. J. Biochem., 216, 377-385 (1993); J. Biol. Chem., 269, 10028-10033 (1994)), the cDNAs of ST3Gal III and IV were cloned from mouse cDNA libraries. Each of the four enzymes was expressed in COS-7 cells as a recombinant enzyme fused with protein A, and applied on an IgG-Sepharose gel to eliminate endogenous sialyltransferase activity. ST3Gal I and II showed the highest activity toward Gal beta 1, 3 GalNAc (type III), very low activity toward Gal beta 1,3GlcNAc (type I), but none toward Gal beta 1,4GlcNAc (type II). ST3Gal III and IV exhibited high activity toward the type I and II disaccharides, but very low activity toward the type III one. On the other hand, asialo-GM1 (Gg4Cer) was as good a substrate for ST3Gal I and II as the type III disaccharide, though ST3Gal III and IV hardly utilized glycolipids as substrates, as indicated by in vitro experiments. Northern blot analysis revealed that enzymes of the ST3Gal-family are expressed mainly in a tissue-specific manner. The ST3Gal I gene was strongly expressed in spleen and salivary gland, and weakly in brain, liver, heart, kidney, and thymus. The ST3Gal II gene was strongly expressed in brain, and weakly in colon, thymus, salivary gland, and testis, and developmentally expressed in liver, heart, kidney, and spleen. The ST3Gal III and IV genes were expressed in a wide variety of tissues. These differences in tissue specific expression suggest the expression of each ST3Gal influences the distribution of sialyl-glycoconjugates in vivo.

Amino Acid Sequence↗