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N Kojima

Publications and source records attributed to N Kojima.

At least 109 records · Page 6Linked to original sources
PubMed1996

[Currarino triad : a case report].

The authors report a case of Currarino triad comprising anorectal malformations, sacral bony anomaly and presacral mass. A 1-year-old boy was presented with constipation as his chief complaint. No neurological deficit was found on admission. There was no cutaneous evidence of underlying spinal dysraphism. Lumbar X-ray films showed bony defect caudal to the third sacral vertebra. A barium-enema examination revealed an anterior displacement of the rectum. A myelography showed a presacral cavity filled with contrast medium. MRI demonstrated a thick filum terminale, and a round hypointense mass in the pelvis on T1 weighted images and hyperintense on T2 weighted images. Surgically we released the thick filum terminale, and obliterated the anterior sacral meningocele, because total removal would have been hazardous. Postoperatively transient dysuria was observed for a month, and the difficulty in defecation persisted. Recognition of this rare condition will lead to correct diagnosis and proper treatment.

Abnormalities, Multiple↗
PubMed1995

A developmentally regulated member of the sialyltransferase family (ST8Sia II, STX) is a polysialic acid synthase.

We found polysialic acid synthase activity of ST8Sia II (STX) in vitro and in vivo. Previously, we showed that mouse ST8Sia II exhibits alpha 2,3-sialylated N-glycan alpha 2,8-sialyltransferase activity, but the polysialic acid synthase activity of ST8Sia II was not detected at that time [Kojima, N. et al. (1995) FEBS Lett. 360, 1-4]. When fetuin was [14C]sialylated with ST8Sia II and then its N-linked oligosaccharides were analyzed, a part of the N-linked oligosaccharides was eluted in the void volume from a Sephadex G-50 column, and was eluted in the void volume from a Sephadex G-50 column, and was eluted from the DEAE-Toyopearl column at almost the same salt concentration as that where colomic acid was eluted. In addition, a series of 14C-labeled oligo-sialic acids were obtained from the oligosaccharides on partial mild acid hydrolysis. These results indicated that a part of N-linked oligosaccharides of fetuin were polysialylated with ST8Sia II. Transfection of ST8Sia II gene into several cell lines including NIH3T3 led to the expression of polysialic acids on the cell surface. Thus, ST8Sia II can directly synthesize polysialic acid chains on alpha 2,3-sialylated N-linked oligosaccharides of glycoproteins without any initiator sialytransferase.

3T3 Cells↗
PubMed1995

Molecular cloning of Sia alpha 2,3Gal beta 1,4GlcNAc alpha 2,8-sialyltransferase from mouse brain.

A cDNA encoding a new alpha 2,8-sialyltransferase (ST8Sia III), which exhibits activity toward the Sia alpha 2,3Gal beta 1, 4GlcNAc sequences of N-linked oligosaccharides, was cloned from mouse brain by means of the polymerase chain reaction-based approach. The predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4% identity with those of so far cloned mouse alpha 2,8-sialyltransferases, i.e. GD3 synthase (ST8Sia I) and STX (ST8Sia II), respectively. Transfection of the protein A-fused ST8Sia III gene into COS-7 cells led to alpha 2,8-sialyltransferase activity toward sialylated glycoproteins and alpha 2,3-sialylated glycosphingolipids, such as alpha 2,3-sialylparagloboside and GM3. However, the kinetic properties of ST8Sia III revealed that it is much more specific to N-linked oligosaccharides of glycoproteins than glycosphingolipids. The expression pattern of the ST8Sia III gene was clearly different from those of other alpha 2,8-sialyltransferase genes. The expression of the ST8Sia III gene was tissue and stage specific. The ST8Sia III gene was expressed only in brain and testis, and it appeared first in 20 postcoitum embryonal brain and then decreased. Therefore, the new alpha 2,8-sialyltransferase is closely involved in brain development.

Amino Acid Sequence↗
PubMed1995

Characterization of the phosphatidylserine-binding region of rat MARCKS (myristoylated, alanine-rich protein kinase C substrate). Its regulation through phosphorylation of serine 152.

We reported previously that recombinant myristoylated, alanine-rich protein kinase C substrate (MARCKS) expressed in Escherichia coli as well as MARCKS purified from rat brain specifically bound to phosphatidylserine (PS) in a calcium-independent manner and that the binding was regulated through phosphorylation of MARCKS (Nakaoka, T., Kojima, N., Hamamoto, T., Kurosawa, N., Lee, Y. C., Kawasaki, H., Suzuki, K., and Tsuji, S. (1993) J. Biochem. (Tokyo) 114, 449-452). In this study, to identify the minimum PS-binding region of MARCKS and the regulatory phosphorylation site, the binding of MARCKS to PS was examined in deletion mutants producing glutathione S-transferase (GST) fusion proteins. The mutant proteins GST-6-180 and GST-127-160 had almost the same ability to bind to immobilized PS as MARCKS purified from rat brain, whereas GST-127-152 did not bind to it. In addition, the binding of GST-6-156 to immobilized PS was 62% of that of GST-6-180, but that of GST-6-152 was only 8% and that of GST-6-135 was not detected. The effect of phosphorylation by protein kinase C was examined in several mutants of GST-6-180 whose serine residues were substituted with alanine. After phosphorylation, the mutants GST-6-180[S156A and S163A], GST-6-180]S156A], and GST-6-180[S163A] did not bind to immobilized PS like native MARCKS and GST-6-180. However, even after phosphorylation, GST-6-180-[S152A] and GST-6-180[S152A and S156A] could bind to immobilized PS. These results strongly suggest that MARCKS binds to PS molecules in the inner leaflet of the plasma membrane through residues 127-156, with residues 153-156 (FKKS) being particularly important in the binding of MARCKS to PS, and that the binding is regulated through the protein kinase C-catalyzed phosphorylation of the serine at residue 152.

Amino Acid Sequence↗
PubMed1995

Enzymatic activity of a developmentally regulated member of the sialyltransferase family (STX): evidence for alpha 2,8-sialyltransferase activity toward N-linked oligosaccharides.

We have detected sialyltransferase activity of recombinant mouse STX, which was cloned from rat brain as a new member of the sialyltransferase family, but sialyltransferase activity of which had not been detected previously [Livingston and Paulson, J. Biol. Chem. (1993) 268, 11504-11507]. The activity of mouse STX was specific toward sialylated glycoproteins. N-Glycanase treatment and linkage-specific sialidase treatment of glycoproteins revealed that STX transfers sialic acids through alpha 2,8-linkages to only N-linked oligosaccharides of glycoproteins. However, polymerase activity for polysialic acid synthesis was not detected for this sialyltransferase. Since this alpha 2,8-sialyltransferase gene is highly restricted in fetal and newborn brain, it may be involved in the polysialylation of glycoproteins, especially of N-CAM.

Animals↗
PubMed1995

Enzymic method for the amperometric determination of nicotinic acid in meat products.

An enzymic method for the determination of nicotinic acid is described, based on the indirect electrochemical monitoring of nicotinic acid via its reaction with oxygen in the presence of nicotinic acid hydroxylase. Derivative amperometric signals due to oxygen depletion enable a one-point kinetic analysis to be carried out. Nicotinic acid hydroxylase catalyses the hydroxylation of nicotinic acid to produce 6-hydroxynicotinic acid, which is accompanied by a stoichiometric consumption of oxygen. The method was applied successfully to the analysis of real samples.

Catalysis↗
PubMed1995

Cloning and sequence analysis of cDNA for a possible DNA-binding protein 5E5 in the nervous system.

Monoclonal antibody 5E5 recognized an intranuclear antigen of neurons in the rat. We isolated 5E5cDNA and determined its nucleotide and deduced amino acid sequences. The 5E5cDNA had an open reading frame of 825 amino acids and its amino acid sequence showed no significant homology to any protein or to any DNA binding motif so far known. 5E5 protein had an abundance of basic amino acids, especially arginine, and included a glycine-rich region and a proline cluster. Monoclonal antibody 12H raised against 5E5cDNA fusion protein recognized an intranuclear substance in rat brain sections and a single protein band of about 98 kDa in the brain nuclear extract fraction on immunoblotting. DNA-cellulose column chromatography indicated that 5E5 protein might have DNA-binding ability. Transfection studies indicated that 5E5 protein expressed in COS-1 is localized in cell nuclei. These results suggest that 5E5 protein is a possible DNA-binding protein which is expressed especially in neurons.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a third type of N-glycan alpha 2,8-sialyltransferase from mouse lung.

AcDNA encoding a new alpha2,8-sialyltransferase (ST8Sia IV), which exhibits activity toward the alpha,2,3-linked sialic acids of N-linked oligosaccharides, was cloned from a mouse lung cDNA library by means of the PCR-based approach. The predicted amino acid sequence of ST8Sia IV showed 15.2, 56.0, and 26% identity with those of so far cloned mouse alpha2,8-sialytransferases, i.e. GD3 synthase (ST8Sia I), STX(ST8Sia II), and Sia(alpha)2,3Galbeta1,4GlcNAc(alpha)2,8-sialyl-transferase (ST8Sia III). ST8Sia IV exhibits high amino acid sequence identity (99.2%) with recently cloned hamster polysialyltransferase-1 gene, which is necessary to polysialic acid expression, but no enzymatic activity of the gene product was reported [Eckhardt, M. et al. (1995) Nature 373, 715-718]. The ST8Sia IV gene was strongly expressed in lung, heart, and spleen, but only weak expression of the gene was observed in brain, without remarkable developmental regulation. The activity of mouse ST8Sia IV was specific toward sialylated glycoproteins. The linage-specific sialidase treatment of glycoproteins as well as N-linked oligosaccharides from the glycoproteins revealed that ST8Sia IV exhibits an alpha2,8-sialytransferase activity toward alpha2,3-linked sialic acids of N-linked oligosaccharides. ST8Sia IV can synthesize polysialic acid chain in vitro without any initiator sialytransferase.

Amino Acid Sequence↗
PubMed1995

Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates.

A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, alpha-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and alpha-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km = 2.8 x 10(4) M-1.S-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase. An active site-directed inhibitor of metalloendopeptidase-24.15, N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala- Asn-Ser-2,4-dinitroanilinoethylamide (kcat/Km = 9.3 x 10(5) M-1.S-1). An angiotensin antagonist, [Sar1, Ala8]-angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Kl = 7.6 microM). MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.

Amino Acid Sequence↗
PubMed1995

Monoclonal antibody directed to Le(y) oligosaccharide inhibits implantation in the mouse.

We investigated the role of carbohydrates in blastocyst attachment to the uterine epithelium. Le(y) (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc) was localized by indirect immunofluorescence to the surface of the mouse blastocyst and uterine epithelium. Western blot analysis showed that Le(y) is carried on many uterine glycoproteins in both pregnant and nonpregnant females; however, new species were detected on Day 4 postcoitum (p.c.) coincident with the onset of uterine receptivity. The function of Le(y) in implantation was tested by injecting monoclonal antibody (mAb) directly into the uterine lumen on Days 3-5 p.c. The effects of intrauterine injections on implantation were scored by comparing the number of viable embryos to the number of CL on Day 10 p.c. Injection of purified anti-Le(y) IgM into the uterine lumen on the afternoon of Day 4 significantly inhibited implantation. This effect was dose-dependent and was obtained during a narrow time window, from 87 to 93 h p.c. Inhibition of implantation was not observed in contralateral uterine horns injected with saline, nor was it observed in uterine horns injected with other anti-carbohydrate mAbs. We conclude that binding of anti-Le(y) to the blastocyst or luminal epithelium masks a ligand involved in implantation. Although the mechanism of inhibition is unknown, we show that Le(y) can interact with another oligosaccharide (H) that has been described as a possible uterine ligand for blastocyst attachment. We hypothesize that Le(y) and H form carbohydrate-carbohydrate interactions that promote close apposition of cell surface membranes during an early step in implantation.

Animals↗
PubMed1995

Changes of bone mineral density in pregnant and postpartum women.

OBJECTIVE: To explore the effects of pregnancy and postpartal lactation on bone mineral density (BMD). METHODS: In this study, the BMD of 22 pregnant women in a longitudinal study, and of 75 pregnant and 111 puerperant women in a cross-sectional study was estimated at the distal radius of the forearm by dual energy X-ray absorptiometry. BMD was measured on 8 separate occasions from the first trimester of pregnancy to 24 months' postpartum. RESULTS: In none of 22 pregnant women was there any noticeable change in BMD during pregnancy. Whereas no significant change in BMD occurred during the 12-month postpartum period in 11 non-lactating women, 11 women who breastfed had a significant decrease in BMD at 1, 3, and 6 months' postpartum, with all of them showing a further decrease in BMD at 12 months' postpartum. The BMD of the radius was significantly lower in the breast-feeders than in the formula-feeders at all postpartal times of evaluation except at 24 months' postpartum. CONCLUSION: It can be recommended that lactating women receive appropriate treatments for saving BMD during lactation.

Adult↗
PubMed1994

Induction of cholinergic differentiation with neurite sprouting by de novo biosynthesis and expression of GD3 and b-series gangliosides in Neuro2a cells.

The expression of a single glycosyltransferase, GD3 synthase, caused cholinergic differentiation with neurite sprouting. The cells that expressed GD3 were established from Neuro2a cells by transfection of a mammalian expression vector into which were carried a cDNA encoding GD3 synthase and the blasticidin-S-deaminase gene with a SV40 promoter, followed by selection with blasticidin-S-hydrochloride. The blasticidin-S-hydrochloride-resistant colonies derived from the cells transfected with the cDNA encoding GD3 synthase and the clonal cells (N2a-GD3) were spontaneously sprouting neurites but not those derived from cells transfected with only the vector without the cDNA encoding GD3 synthase (N2a-bsr). GD3 expression by N2a-GD3 was confirmed by immunostaining of the cells using the anti-GD3 monoclonal antibody, KM643. N2a-GD3 expressed not only GD3 but also GQ1b, one of the b-series gangliosides, whereas N2a-bsr did not express these gangliosides. Cell proliferation of N2a-GD3 was greatly reduced, as compared with that of N2a-bsr, and, after several passages, it completely stopped. In addition, N2a-GD3 expressed acetylcholine esterase, indicating that the differentiation of Neuro2a cells was induced by expression of GD3 synthase and subsequent modification of the biosynthesis and expression of gangliosides. These results strongly suggest that the de novo synthesis and expression of GD3 and/or b-series gangliosides induce neurite outgrowth and differentiation of Neuro2a cells. Exogenous GM1 stimulated the neuritogenesis of N2a-bsr but not differentiated N2a-GD3, indicating that the mechanism of neurite sprouting in this system may be overlapped en route with that of exogenous GM1.

Acetylcholinesterase↗
PubMed1994

Cloning and expression of Gal beta 1,3GalNAc-specific GalNAc alpha 2,6-sialyltransferase.

A cDNA clone encoding a new type of GalNAc alpha 2,6-sialyltransferase (ST6GalNAc II) with a structure similar to that of a previously cloned GalNAc alpha 2,6-sialyltransferase (ST6GalNAc I; Kurosawa, N., Hamamoto, T., Lee, Y.-C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409) was obtained from chicken testes. The predicted amino acid sequence of ST6GalNAc II encodes a protein with type II transmembrane topology, as found for other glycosyltransferases, and showed 32% identity with that of ST6GalNAc I. Transfection of the full length ST6GalNAc II gene into COS cells led to GalNAc alpha 2,6-sialyltransferase activity with a different substrate specificity from that of ST6GalNAc I. Moreover, asialofetuin after treatment with beta-galactosidase did not serve as an acceptor for this enzyme. 14C-Sialylated oligosaccharides obtained from resialylated asialobovine submaxillary mucin with this enzyme were identical to Gal beta 1,3([14C]NeuAc alpha 2,6)GalNAc-ol but not [14C]NeuAc alpha 2,6GalNAc-ol. These results clearly show that the expressed enzyme is a novel type of sialyltransferase that requires beta-galactoside residues linked to GalNAc residues, whereas sialic acid residues linked to galactose residues are not essential for the activity.

Amino Acid Sequence↗
PubMed1994

Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

Amino Acid Sequence↗
PubMed1994

Kinetic properties and acceptor substrate preferences of two kinds of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain.

The cDNAs encoding two kinds of Gal beta 1,3GalNAc alpha 2,3-sialytransferases (ST3GalA.1 and ST3GalA.2) have been cloned from mouse brain, both of which could synthesize the NeuAc alpha 2,3Gal beta 1,-3GalNAc sequence of gangliosides as well as O-glycosidically linked oligosaccharides of glycoproteins [Lee et al. (1993) Eur. J. Biochem. 216, 377-385; Lee et al. (1994) J. Biol. Chem. (in press)]. Kinetic analysis of the two sialyltransferases using Gal beta 1,3GalNAc, asialoGM1, or asialofetuin revealed that ST3GalA.1 exhibits the highest Km value for asialoGM1 (Km = 1.25 mM) and the lowest one for asialofetuin (Km = 0.10 mM), whereas the Km values of ST3GalA.2 for the substrates are very similar (Km approximately 0.5 mM). The synthesis of GM1b from asialoGM1 by ST3GalA.1 was clearly inhibited in the presence of Gal beta 1,3GalNAc or asialofetuin, but that by ST3GalA.2 was not at all. On the other hand, the activity of ST3GalA.2 toward Gal beta 1,3GalNAc or asialofetuin was inhibited by asialoGM1 or GM1. The results of acceptor competition experiments involving asialoGM1, Gal beta 1,3GalNAc, and asialofetuin indicated that ST3GalA.2 exhibits noncompetitive inhibition between asialoGM1 and Gal beta 1,3GalNAc or between asialoGM1 and asialofetuin, whereas ST3GalA.1 exhibits competitive inhibition between all kinds of acceptors. These results strongly indicate that acceptor preference of ST3GalA.1 is different from that of ST3GalA.2, although their acceptor substrate specificities are the same; i.e., gangliosides serve as predominant acceptors for the latter over O-glycosidically linked oligosaccharides of glycoproteins, which are much better acceptors for the former.

Animals↗
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 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↗