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J Aikawa

Publications and source records attributed to J Aikawa.

At least 19 recordsLinked to original sources
PubMed2001

Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo.

The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase/N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker alpha-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-O-sulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.

Animals↗
PubMed2001

Replacements of amino acid residues at subsites and their effects on the catalytic properties of Rhizomucor pusillus pepsin, an aspartic proteinase from Rhizomucor pusillus.

Site-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-binding and catalysis. Mutations of two amino acid residues, E13 in the S3 subsite and N219 in the S3/S4 subsites, caused marked changes in kinetic parameters for two substrate peptides with different sequences. Further site-directed mutagenesis at E13 suggested that E13 plays a critical role in forming the correct hydrogen bond network around the active center. In the crystal structure of Rhizomucor miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the Oepsilon atom of N219 forms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhibitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolytic activity without significant loss of clotting activity. This mutant may present a promising candidate for a useful milk coagulant.

Amino Acid Substitution↗
PubMed2000

Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4.

We report the cloning and partial characterization of the fourth member of the vertebrate heparan sulfate/heparin: GlcNAc N-deacetylase/GlcN N-sulfotransferase family, which we designate NDST4. Full-length cDNA clones containing the entire coding region of 872 amino acids were obtained from human and mouse cDNA libraries. The deduced amino acid sequence of NDST4 showed high sequence identity to NDST1, NDST2, and NDST3 in both species. NDST4 maps to human chromosome 4q25-26, very close to NDST3, located at 4q26-27. These observations, taken together with phylogenetic data, suggest that the four NDSTs evolved from a common ancestral gene, which diverged to give rise to two subtypes, NDST3/4 and NDST1/2. Reverse transcription-polymerase chain reaction analysis of various mouse tissues revealed a restricted pattern of NDST4 mRNA expression when compared with NDST1 and NDST2, which are abundantly and ubiquitously expressed. Comparison of the enzymatic properties of the four murine NDSTs revealed striking differences in N-deacetylation and N-sulfation activities; NDST4 had weak deacetylase activity but high sulfotransferase, whereas NDST3 had the opposite properties. Molecular modeling of the sulfotransferase domains of the murine and human NDSTs showed varying surface charge distributions within the substrate binding cleft, suggesting that the differences in activity may reflect preferences for different substrates. An iterative model of heparan sulfate biosynthesis is suggested in which some NDST isozymes initiate the N-deacetylation and N-sulfation of the chains, whereas others bind to previously modified segments to fill in or extend the section of modified residues.

Amidohydrolases↗
PubMed1999

Molecular cloning and expression of a third member of the heparan sulfate/heparin GlcNAc N-deacetylase/ N-sulfotransferase family.

N-Deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin initiate a series of chemical modifications that ultimately lead to oligosaccharide sequences with specific ligand binding properties. These reactions are catalyzed by GlcNAc N-deacetylase/N-sulfotransferase (NDST), a monomeric enzyme with two catalytic activities. Two genes encoding NDST isozymes have been described, one from rat liver (NDST1) and another from murine mastocytoma (NDST2). Both isozymes are expressed in tissues in varying amounts, but their relative contribution to heparan sulfate formation in any one tissue is unknown. We now report the identification of a third member of the NDST family, designated NDST3. A full-length cDNA clone (3.2 kilobase pairs) encoding a 873-amino acid protein was obtained from a human fetal/infant brain cDNA library. Human NDST3 (hNDST3) has a nucleotide sequence homologous but not identical to hNDST1 and NDST2. The deduced amino acid sequence shows 70% and 65% amino acid identity to that of hNDST1 and NDST2, respectively. A soluble chimera of hNDST3 and protein A exhibited both N-deacetylase and N-sulfotransferase activity, confirming its enzymatic identity. Northern blot analysis of human fetal brain poly(A)+ RNA showed a single transcript of 6.4 kilobase pairs. Reverse transcription polymerase chain reaction analysis revealed more restricted tissue expression of hNDST3 than hNDST1 and NDST2, and high levels in brain, liver, and kidney. Analysis of Chinese hamster ovary cells revealed expression of NDST1 and NDST2, but not NDST3. In a Chinese hamster ovary cell mutant exhibiting reduced N-sulfotransferase activity and reduced sulfation of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065), expression of NDST1 was greatly reduced, but NDST2 was expressed normally, suggesting that both enzymes are involved in heparan sulfate assembly. The discovery of multiple NDST isozymes suggests that the assembly of heparan sulfate is much complicated than previously appreciated.

Acetyltransferases↗
PubMed1998

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning.

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells.

Animals↗
PubMed1998

Neonatal intrahepatic cholestasis with hepatic siderosis and steatosis.

Neonatal intrahepatic cholestasis is a heterogeneous disease of undetermined cause. There is an unreported subset of idiopathic neonatal intrahepatic cholestasis with an unusual histological combination of hepatic siderosis and macrovesicular steatosis. The patients were a 34-day-old female and a 39-day-old male with normal birth weights. Their mothers had received oral iron supplement 4-6 weeks before delivery. The patients had obstructive jaundice noticed at the well-baby clinic at 1 month of life. They had high levels of serum galactose and tyrosine, hyperferritinemia. Urinary organic acid and bile acid analyses were negative, and galactose-1-phosphate uridyltransferase activity in red cells was normal. Liver biopsies showed diffuse iron deposits and macrovesicular fat. By substituting formula milk with lactose-free milk, the patients responded, and had normal biochemical tests within 5 months of life. Follow-up biopsies, at the age of 12 months, showed mild residual fibrosis without iron or fat deposits. They are both well at 3 and 6 years of age, respectively, without biochemical liver dysfunction and neurologic impairment. Prenatal iron-overload might contribute to the pathogenesis of the disease, but further studies are needed to confirm the assumption.

Cholestasis, Intrahepatic↗
PubMed1998

Asparagine-linked glycosylation of the rat leukemia inhibitory factor expressed by simian COS7 cells.

The leukemia inhibitory factor (LIF) is a secretory glycoprotein and a pluripotent growth factor which acts in diverse cell systems. LIF has been reported to be heavily glycosylated. In this paper, we examine the transient expression of rat LIF (rLIF) in COS7 cells and its glycosylation by a PNGaseF treatment and lectin blot. rLIF expression in COS7 cells resulted in seven molecular species being produced with zero to six N-glycosyl moieties. Mutated rLIF proteins with substitutions at the seven possible N-glycosylation sites were also expressed. An analysis of the molecular weight of the mutated rLIF confirmed the six N-glycosylation sites. Bioassays of mouse leukemia cell lines were performed to analyze the contribution of the glycosyl moieties to their functions. We found that the glycosyl moieties at each of the N-glycosylation sites were not essential to their function of the protein, but the reduced functions to promote the proliferation of DA-1a cells that had been observed for some mutants suggests a biochemical role for the in vitro function.

Amidohydrolases↗
PubMed1998

A micromethod for protein assay using a light microscope.

A micromethod which utilizes a protein-dye binding reaction on an acetate membrane and a light microscope with an automatic exposure instrument has been developed for measuring small amounts of protein from limited biological materials. After discoidal cellulose acetate membranes (2.0 mm diameter), which absorbed 0.5 microliter of protein solution containing 0.2-2.0 micrograms of protein, were stained with Coomassie Brilliant Blue G-250, they were examined by a light microscope with an automatic exposure instrument. A linear relation was confirmed between the intensity of dye binding to protein and exposure time under specific optical conditions.

Animals↗
PubMed1998

Peripheral blood stem cell transplantation for hepatoblastoma with microscopical residue: a therapeutic approach for incompletely resected tumor.

We report a nine-month-old boy with stage III B hepatoblastoma of caudate lobe origin. Surgical resection was attempted following six courses of chemotherapy, but viable tumor cells remained microscopically at resection margins. Subsequently, he received peripheral blood stem cell transplantation (PBSCT), whose preparative regimen being consisted of carboplatin, etoposide, tetrahydropyranyl adriamycin, and melphalan. Since then, the patient shows no relevance of local relapse or distant metastasis without any chemotherapy. PBSCT for patients with post-operative residue may improve the outcome of advanced hepatoblastoma and worth of a further clinical investigation.

Disease-Free Survival↗
PubMed1997

Molecular cloning of rat leukemia inhibitory factor receptor alpha-chain gene and its expression during pregnancy.

Leukemia inhibitory factor (LIF) is a secreted glycoprotein and a pluripotent growth factor that acts on diverse cell systems. LIF transmits its effects via binding to transmembrane receptors, of which both high- and low-affinity forms have been identified. In this study, we analyzed the structure and expression of rat LIF receptor alpha-chain (rLIFR alpha) cDNA. A full-length clone of the cDNA encoding the membrane-bound form of rLIFR alpha protein was prepared by a combination of LA-PCR and 5' RACE using DNA reverse-transcribed from total RNA isolated from the livers of day-12 and day-14 pregnant rats as templates. The nucleotide sequence of a full-length clone was determined and further confirmed by analysis of shorter DNA fragment prepared by PCR using pfu polymerase. The gene for rLIFR alpha encodes a 1093 amino acid residue protein. The rLIFR alpha protein shows a high degree of similarity to mouse and human LIF receptor alpha-chain protein (89% and 76% amino acid sequence identities, respectively). Only one molecular species of mRNA for the rLIFR alpha gene was detected in the liver and placenta. rLIFR alpha was expressed in liver of both non-pregnant and pregnant rats. The level of mRNA for the rLIFR alpha gene in placenta was maximum on day 16 of pregnancy.

Amino Acid Sequence↗
PubMed1997

Stimulation of angiotensin subtype 2 receptor reduces basal cGMP levels in the neointima of rat aorta after balloon injury.

1. The association between the stimulation of the angiotensin subtype 2 receptor (AT2-R) and the change in tissue levels of cyclic nucleotide was assessed on neointima formation in rat aorta following aortic balloon injury. 2. Tissue levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate levels (cAMP) in the injured and uninjured aorta was determined by enzyme immunoassay at baseline and again 30 s after administration of 10(-7) M angiotension II. 3. Injured and uninjured aorta showed no difference in basal levels of cGMP. Angiotension II reduced the basal level of cGMP in the injured aorta only. 4. This decrease was blocked by a selective AT2-R antagonist (PD123319) and by a nonselective angiotensin II antagonist (angiotensin II antipeptide), but not by a selective angiotensin subtype 1 antagonist (CV-11974). 5. Stimulation with a selective AT2-R caused no change in the level of cAMP in the injured or uninjured aorta. 6. Results suggest that stimulation of AT2-R in proliferative neointima leads to a decreased tissue level of cGMP.

Angiotensin II↗
PubMed1997

Site-directed mutagenesis of conserved Trp39 in Rhizomucor pusillus pepsin: possible role of Trp39 in maintaining Tyr75 in the correct orientation for maximizing catalytic activity.

Replacement of Trp39 of Rhizomucor pusillus pepsin (RMPP) by Asn or Cys resulted in a marked decrease in the milk-clotting and proteolytic activities. Kinetic analysis with chromogenic synthetic oligopeptides as substrates revealed that the mutations caused marked changes in the kcat value, but only slight changes in the Km value. Similar enzymatic properties were observed in mutants of Tyr75, which was shown to have a role in enhancing the catalytic activity. Both Tyr75Asn and Trp39Asn mutants rapidly lost the activity at high temperatures due to autocatalytic digestion at two sites. The structures of several aspartic proteinases including RMPP, as revealed by X-ray crystallographic studies, showed that Trp39 occupies a position close to Tyr75 and the N delta atom of Trp39 within hydrogen-bonding distance of the hydroxyl side chain of Tyr75. These observations suggest that Trp39 plays a role in maintaining Tyr75 in the correct orientation in aspartic proteinases, including RMPP.

Amino Acid Sequence↗
PubMed1996

Effect of endothelin on angiotensin converting enzyme activity in cultured vascular smooth muscle cells.

We evaluated the effect of endothelin-1 (ET) on the angiotensin converting enzyme (ACE) activity in rat aortic smooth muscle cells (VSMCs). ACE activity was determined by radioimmunoassay of the amount of angiotensin II generated after the addition of angiotensin I (500 pg/ml) to cultured VSMCs. The antibody used had less than 0.1% cross-reactivity with angiotensin I. ACE activity increased 1.9-fold 5 hr after the addition of 10(-6) M ET under serum-free conditions. This stimulatory effect of ET on ACE activity in VSMCs was completely inhibited by 10(-7) M captopril. Results suggested that the ACE present in SMCs is stimulated by ET.

Angiotensin II↗
PubMed1996

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusillus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency.

Residue 75 on the flap, a beta hairpin loop that partially covers the active site cleft, is tyrosine in most members of the aspartic proteinase family. Site-directed mutagenesis was carried out to investigate the functional role of this residue in Rhizomucor pusillus pepsin, an aspartic proteinase with high milk-clotting activity produced by the fungus Rhizomucor pusillus. A set of mutated enzymes with replacement of the amino acid at position 75 by 17 other amino acid residues except for His and Gly was constructed and their enzymatic properties were examined. Strong activity, higher than that of the wild-type enzyme, was found in the mutant with asparagine (Tyr75Asn), while weak but distinct activity was observed in Tyr75Phe. All the other mutants showed markedly decreased or negligible activity, less than 1/1000 of that of the wild-type enzyme. Kinetic analysis of Tyr75Asn using a chromogenic synthetic oligopeptide as a substrate revealed a marked increase in kcat with slight change in K(m), resulting in a 5.6-fold increase in kcat/K(m). When differential absorption spectra upon addition of pepstatin, a specific inhibitor for aspartic proteinase, were compared between the wild-type and mutant enzymes, the wild-type enzyme and Tyr75Asn, showing strong activity, had spectra with absorption maxima at 280, 287 and 293 nm, whereas the others, showing decreased or negligible activity, had spectra with only two maxima at 282 and 288 nm. This suggests a different mode of the inhibitor binding in the latter mutants. These observations suggest a crucial role of the residue at position 75 in enhancing the catalytic efficiency through affecting the mode of substrate-binding in the aspartic proteinases.

Amino Acid Sequence↗
PubMed1996

Vascular smooth muscle relaxation by alpha-adrenoceptor blocking action of dobutamine in isolated rabbit aorta.

We investigated the mechanism of vascular relaxation produced by dobutamine, a positive inotropic agent with beta 1-adrenergic action. Dobutamine concentration-dependently (10 nM-10 microM) relaxed ring segments of rabbit aorta partially precontracted with 1 microM phenylephrine (PE) but did not relax those precontracted with 40 mM K+ or 5 microM prostaglandin F2 alpha (PGF2 alpha). The relaxation was not completely inhibited by pretreatment with 10 microM propranolol. Dobutamine did not significantly increase tissue cyclic AMP levels concomitantly with relaxation as does isoproterenol (ISO) in rabbit aorta. Dobutamine produced a parallel rightward shift in concentration-response curves to PE. The Schild plot analysis resulted in a linear regression of a slope of 1.077 +/- 0.077, which was not significantly different from unity. The pA2 value of dobutamine as compared with PE in rabbit aorta was 6.81 +/- 0.03. Dobutamine causes arterial dilatation mediated not only through a beta-adrenergic action but also through an alpha-adrenergic blocking action in rabbit aorta.

Abortifacient Agents↗
PubMed1996

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities.

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.

Animals↗