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T Ohta

Publications and source records attributed to T Ohta.

At least 685 records · Page 38Linked to original sources
PubMed1994

Evidence for impaired cellular cholesterol removal mediated by apo A-I containing lipoproteins in patients with familial lecithin: cholesterol acyltransferase deficiency.

We investigated the cholesterol reducing capacity of two species of lipoproteins containing apo A-I, one containing only apo A-I (LpA-I) and the other containing apo A-I and apo A-II (LpA-I/A-II), in 7 patients (4 homozygotes and 3 heterozygotes) with familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Interaction of normal LpA-I or LpA-I/A-II with macrophage foam cells induced a mass reduction in cholesterol from these cells and the cholesterol reducing capacity of LpA-I was greater than that of LpA-I/A-II. When foam cells were incubated with these lipoproteins from homozygotes or heterozygotes, the capacity of LpA-I and LpA-I/A-II particles to reduce cellular cholesterol was decreased by approx. 50% in the homozygotes but was increased by 25-50% in the heterozygotes. These results suggest that LpA-I and LpA-I/A-II isolated from homozygotes and from heterozygotes differ in their ability to accept cellular cholesterol. The former are poor and the latter good acceptors of intracellular cholesterol. We conclude that factors other than reverse cholesterol transport via apo A-I containing lipoproteins have to be considered to explain why homozygotes for LCAT deficiency are not at high risk for premature atherosclerosis.

Adult↗
PubMed1994

A high-level and regulatable production system for recombinant glycoproteins using a human interferon-alpha promoter-based expression vector.

A novel expression vector for human cells, pIFP, which expresses a cloned gene under the control of the human interferon-alpha-encoding gene (IFN-alpha) promoter (pIFN) was constructed. As a model of glycoprotein production, a human erythropoietin-encoding cDNA (EPO) inserted downstream from pIFN in pIFP was introduced into human B-cell leukemia-derived BALL-1 cells, and EPO-producing cells were established. Upon stimulation with Sendai virus, the cells produced human EPO at high levels. The highest production level and the highest inducibility were 872 IU/ml and 67-fold, respectively. Simultaneously, the transformed cells also produced IFN-alpha and tumor necrosis factor-alpha (TNF-alpha), as the parental BALL-1 cells did. Comparing the amounts of the substances produced, activity of the exogenous pIFN introduced seemed much higher than that of the endogenous one. Further, the transformed cells could be obtained in a large quantity by being applied to the 'in vivo cell proliferation method (hamster method)'. Human EPO produced by the transformed cells had a molecular mass range of 35 to 42 kDa, similar to that of EPO produced by CHO cells. The processing of EPO seemed to occur properly. The combination of the human pIFN, BALL-1 cells and the hamster method provides us with a useful production system for bioactive glycoproteins of human origin.

Animals↗
PubMed1994

Requirement of a COOH-terminal pro-sequence for the extracellular secretion of aqualysin I (a thermophilic subtilisin-type protease) in Thermus thermophilus.

Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65 degrees C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in E. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.

Amino Acid Sequence↗
PubMed1994

Lactoferrin inhibits cholesterol accumulation in macrophages mediated by acetylated or oxidized low-density lipoproteins.

When macrophages are incubated with acetylated or oxidized low-density lipoproteins (Ac- or OxLDL), cellular cholesteryl esters (CE) increase significantly. In the present study, we investigated the effect of whey protein on Ac- or OxLDL mediated accumulation of CE in macrophages and found that lactoferrin (Lf), a minor protein component of whey, inhibits the accumulation of CE dose-dependently. In the presence of bovine Lf (1 mg/ml), CE accumulation in macrophages incubated with AcLDL (100 micrograms of protein/ml) decreased by more than 80%. Human Lf was less potent than bovine Lf, and bovine transferrin had no effect. Binding of 125I-AcLDL to macrophages was also inhibited by Lf. Agarose gel electrophoresis revealed that Lf binds to Ac- or OxLDLs and neutralizes their negative charges. These results indicate that Lf inhibits the binding of modified LDLs to macrophages by direct interaction with modified LDLs, resulting in their loss of function as ligands of the scavenger receptor. Modification of the arginine residues of Lf with 1,2-cyclohexanedione abolished its ability to bind to AcLDL, suggesting that a region rich in basic amino acid residues near the N-terminus of Lf, which resembles the ligand-binding site of the scavenger receptor, may be responsible for this binding ability. As a result, the inhibitory effect of Lf on CE accumulation in macrophages was significantly weakened by this modification. Our results suggest the possibility that Lf in the blood stream may act as an anti-atherogenic agent in vivo.

Amino Acid Sequence↗
PubMed1994

Alterations in CD45 glycosylation pattern accompanying different cell proliferation states.

CD45 is a leukocyte-specific transmembrane glycoprotein whose intracellular domain exhibits protein tyrosine phosphatase activity and plays a critical role in signal transduction. CD45 derived from stationary lymphocytes migrated faster in SDS-PAGE than that derived from exponentially growing cells. A change in N-linked saccharide structure other than the neuraminidase-sensitive terminal sialic acid portion was found to be responsible for the molecular size change in CD45. The differential glycosylation appeared to occur during late-stage posttranslational processing of CD45. We speculate that the N-glycosylation difference affects the interaction between CD45 and other factors involved in signal transduction leading to modulation of leukocyte proliferation.

Amidohydrolases↗
PubMed1994

Effects of nifedipine and Bay K 8644 on contractile activities in single skeletal muscle fibers of the frog.

The effects of dihydropyridine derivatives on contractile activity were examined in single fibers of frog skeletal muscles. Both nifedipine and Bay K 8644 enhanced twitch responses regardless of the presence or absence of Ca2+ without any effects on the resting membrane potential, evoked action potential and Ca2+ sensitivity of the contractile machinery. Twitch responses enhanced by Bay K 8644, but not by nifedipine, were partially reduced by adenine, an inhibitor of Ca(2+)-induced Ca2+ release from sarcoplasmic reticulum, suggesting the involvement of Ca(2+)-induced Ca2+ release in Bay K 8644-induced potentiation of the twitches. Both nifedipine and Bay K 8644 caused a dose-dependent potentiation of K+ contractures without any effects on the membrane potential in the presence of Ca2+, but inhibited the contractures in the absence of Ca2+. These results suggest that dihydropyridine derivatives modify contractile activities of skeletal muscles by modulating the dihydropyridine receptors on the surface of T-tubules acting as voltage sensors.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Plasma HDL levels are regulated by the catabolic rate of large particles of lipoprotein containing apo-A-I.

Lp-A-I was isolated by immunoaffinity chromatography and then separated into two fractions of large and small Lp-A-I particles by conventional ultracentrifugation with a cut-off density of 1.125 g/ml. The large and small particle-rich fractions were then radiolabeled with [125I]-Na and [131I]-Na, respectively. Both of the labeled lipoproteins were injected (20 microCi, i.v.) simultaneously into normolipidemic rabbits. The FCR of the large Lp-A-I particles was much less than that of the small Lp-A-I particles (0.801 +/- 0.026/day vs. 2.227 +/- 0.067/day, P < 0.0001). These data indicate that the two particles have distinctly different metabolic pathways and that the lower FCR of larger Lp-A-I particles can effectively raise plasma HDL levels.

Animals↗
PubMed1994

The giant extracellular hemoglobin from the polychaete Neanthes diversicolor. The cDNA-derived amino acid sequence of linker chain L2 and the exon/intron boundary conserved in linker genes.

The 4000 kDa extracellular hemoglobin from the polychaete Neanthes diversicolor consists of three types of subunits; three 15 kDa monomers (chains M1, M2 and M3), a 45 kDa disulfide-bonded trimer of chains T1, T2 and T3, and two 50-55 kDa disulfide-bonded homodimeric linkers (chains L1 and L2). The latter linker subunits are essential for the assembly of the other heme-containing subunits, monomers and a trimer. The cDNA encoding the linker chain L2 was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 235 residues has been determined. The sequence showed 22-75% identity with other linker chains. All of the linker sequences examined so far have a highly conserved cysteine-rich segment at positions 89-130: Xaa3-Cys-Xaa6-Cys-Xaa6-Cys-Xaa6-Cys-Asp-Gly-X aa2-Asp-Cys-Xaa4-Asp-Glu-Xaa4-Cys, and the motif corresponds exactly to the cysteine-rich repeats of the ligand-binding domains of vertebrate low-density lipoprotein (LDL) receptors (Suzuki, T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13548-13555). A 287 bp intron interrupts the coding sequence of Neanthes L2 gene just at the N-terminal boundary of this motif, and the position of the splice junction was exactly conserved in Neanthes and Lumbricus linker genes. This suggests that the intron has been conserved for at least 450 million years in annelid linker genes. The evolutionary origin of the remaining parts of linker chains is unclear, but it is noteworthy that the topology of the two intrachain disulfide bridges in the C-terminal segment of linker chains is homologous with that of the carbohydrate-recognition domain of animal C-type lectin.

Amino Acid Sequence↗
PubMed1994

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography.

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid-liquid extraction. The compounds were separated on a CN column using acetonitrile-0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.

Animals↗
PubMed1994

A regular 1:1 complex of two allosteric states in the single crystal of L-lactate dehydrogenase from Bifidobacterium longum.

Single crystals of the regular 1:1 complex of T and R-state tetramers of L-lactate dehydrogenase from Bifidobacterium longum have been grown from polyethylene glycol 6000 solution in the presence of NADH, fructose 1,6-bisphosphate and oxamate. The crystals belong to space group F222 with unit cell dimensions of a = 148.4 A, b = 295.9 A and c = 71.0 A. The crystals are suitable for X-ray analysis and diffract to beyond 2.5 A spacing. A crystallographic study showed that the asymmetric unit contains two subunits and that one of them belongs to the T-state tetramer and the other to the R-state tetramer.

Allosteric Site↗
PubMed1994

Clinical study of lymphatic flow to the paraaortic lymph nodes in carcinoma of the head of the pancreas.

BACKGROUND: At Kanazawa University, the authors have been developing an appropriate radical operation for the treatment of cancer of the head of the pancreas. As a result of previous research, it was believed that lymphatic metastasis of carcinoma of the head of the pancreas should be investigated more thoroughly to improve the surgical results. METHODS: Forty-two cases of carcinoma of the head of the pancreas were investigated to determine the distribution of lymphatic metastases. From among these cases, the authors injected activated carbon particles in 10 patients with pancreatic cancer and 111In colloid in seven patients with pancreatoduodenal cancer to investigate the lymphatic spread from the head of the pancreas to the paraaortic lymph nodes (area 16). RESULTS: The main lymphatic route from the head of the pancrease to lymphatic area 16 was found to pass through the nodes in the posterior part of the head of the pancrease (area 13) and around the superior mesenteric artery (area 14). Lymphatic metastases in area 16 were seen mainly in the lower segment of the middle region from the celiac artery to the inferior mesenteric artery (subarea 16b2). The carbon and 111In colloid flowed mostly to the same area 16 lymph nodes and toward the dorsal side of the renal artery rather than spreading superficially along the abdominal aorta. CONCLUSIONS: These results indicate that area 16 lymph node dissection should be extended toward the dorsal side of the renal artery rather than be performed widely along the abdominal aorta to make the radical operation for pancreatic cancer more extensive.

Abdomen↗
PubMed1994

Molecular and cellular mechanisms of mitochondrial nuclear division and mitochondriokinesis.

Our present understanding of mitochondrial division can be summarized as follows: Mitochondria contain a specific genome, synthesize their own DNA, and multiply semi-autonomously. Strands of mitochondrial DNA (mt-DNA) in the in vivo organelles of all eukaryotes are organized to form mitochondrial nuclei (nucleoids) (mt-nuclei) with specific proteins including a histone-like protein and transcription factors at the central region of the mitochondrion. We can easily observe the mt-nucleus in vivo mitochondria in various organisms such as fungi, algae, plants, and animals by using high-resolution epifluorescence microscopy. Therefore, the process of mitochondrial division can be clearly separated into two main events: division of the mt-nuclei and mitochondriokinesis analogous to cytokinesis. Mitochondria undergo binary division which is accompanied by the division of the mt-nucleus. A remarkable characteristic of mitochondrial multiplication during the mitochondrial life cycle is that mitochondria can multiply the mt-chromosome by endoduplication until 50-100 copies are present. Mitochondria can then divide without mitochondrial DNA synthesis to eventually contain 1-5 copies of the mt-chromosome. This characteristic phenomenon can be observed during cell differentiation, such as during the formation of plasmodia and sclerotia of Physarum polycephalum and during embryogenesis and the formation of meristematic tissues in plants. The mitochondrial chromosome has a mitochondrial "kinetochore (centromere)" which is A-T rich and contains specific sequences such as topoisomerase binding sites, tandem repeats, and inverted repeats. A bridge of proteins may exist between the kinetochore DNA and membrane systems. Mitochondrial chromosomes can divide according to the growth of a membrane system between the kinetochores. Mitochondriokinesis progresses steadily along with mitochondrial nuclear division. As the membrane at the equatorial region of a mitochondrion contracts, the neck of the cleavage furrow narrows, and eventually the daughter mitochondria are separated. An actin-like protein may power mitochondriokinesis by separating the daughter mitochondria. In general, mitochondriokinesis occurs by contraction rather than by partition of the inner membrane.

Animals↗
PubMed1994

Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method.

Chemical modification of glucoamylase (EC 3.2.1.3) from Clostridium sp. G0005 (CGA) with N-bromosuccinimide (NBS) was carried out in the presence or absence of an inhibitor, acarbose. CGA lost its catalytic activity through NBS oxidation in the absence of acarbose. The absorbance change at 280 nm suggested that acarbose protects about 2 Trp residues from NBS oxidation. We performed peptide mapping analysis to identify the protected Trp residues, and Trp321, Trp337, Trp433, and Trp569 were identified as candidates to be protected by acarbose. These 4 Trp residues were replaced by site-directed mutagenesis with Phe. The Trp337-->Phe mutant showed very weak catalytic activity, so Trp337 is proposed as an important residue for the catalytic activity. Further, we constructed a Glu632-->Gln mutant. Glu632 is the putative catalytic base. The presteady-state kinetics of the Trp337-->Phe and Glu632-->Gln mutants and the wild-type CGA were investigated using maltotriose as a substrate. The reaction of wild-type CGA can be explained as one involving three intermediates. On the other hand, the two mutants' reactions are explained by a two-step mechanism lacking the third intermediate. Trp337 and Glu632 appear to be crucial for the formation of the third intermediate in the wild-type reaction, which precedes the transition state.

Acarbose↗
PubMed1994

Molecular cloning of the CD45-associated 30-kDa protein.

CD45, a leukocyte-specific transmembrane protein tyrosine phosphatase, mediates signal transduction pathways critical for immune responses. However, the mechanism of CD45-mediated signal transduction and the identity of CD45-associated proteins have remained unclear. A CD45-associated 30-kDA phosphorylated protein (CD45-AP) was purified by virtue of its specific association with CD45, and its mouse cDNA was cloned by using the internal amino acid sequence information. In vitro translated CD45-AP bound specifically to CD45. CD45-AP appears to be leukocyte-specific and shares no significant homology with presently known sequences. The predicted sequence contains no consensus tyrosine phosphorylation sites or conserved sequences of GTP-binding proteins. CD45-AP may act as an adapter molecule for CD45-mediated signal transduction.

Amino Acid Sequence↗
PubMed1994

Characterization of vasoactive intestinal peptide receptors in canine liver membranes.

The binding characteristics of vasoactive intestinal peptide (VIP) in the liver membranes of the dog were examined using radioligand binding assay with 125I-VIP and unlabelled peptides and results were compared with those from the rat. The binding of VIP to canine liver membranes occurred in a reversible, saturable, specific and temperature-dependent manner. Guanine nucleotides dose-dependently inhibited VIP binding. The order of potency in competition experiments with unlabelled peptide was: VIP > pituitary adenylate cyclase activating peptide (PACAP)-27 > PACAP-38 >> peptide histidine isoleucine (PHI) = secretin in the dog, and PACAP-27 > PACAP-38 > VIP > PHI > secretin in the rat. PHI and secretin were about 5000 times less potent than VIP in the dog, but secretin was about 100 times less potent than VIP in the rat. The VIP binding sites in canine liver membranes have recognition sites for VIP which differ from those in rat liver membranes. As most of VIP in the portal vein was removed during its passage through the canine liver, the binding sites of canine liver may play a role in degradation of VIP.

Animals↗