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Biomedical subjects

Y Ohba

Publications and source records attributed to Y Ohba.

At least 127 records · Page 7Linked to original sources
PubMed1994

Sterol mediated regulation of SREBP-1a,1b,1c and SREBP-2 in cultured human cells.

Under conditions of cholesterol depletion and SREBP-1 accumulation, changes in the levels of sterol regulatory element binding protein(s) (SREBPs) and sterol regulated gene mRNA were studied in Hep G2 cells by RNase protection assay. Cholesterol depletion increased the expression of mRNAs for cholesterol biosynthetic enzymes and low density lipoprotein (LDL) receptor. mRNAs levels for SREBP-1c and SREBP-2 were also increased by the cholesterol depletion. In contrast, levels for SREBP-1a and 1b (1a/b) mRNA increased transiently and then decreased. To examine the effect of SREBP-1 accumulation, Hep G2 cells were incubated with a SREBP-1 degradation inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN). The ALLN treatment increased the LDL receptor mRNA significantly, and also increased mRNA levels for HMG-CoA reductase, SREBP-1a/b and SREBP-2. The mRNA level for squalene synthase was not changed, and for SREBP-1c was decreased by the treatment. In conclusion, the regulation of differential expression of SREBP mRNA may be involved in sterol mediated regulation of gene expression. Moreover, the regulation of the SREBP-1 level may be a critical step in the regulation of sterol mediated LDL receptor expression.

Amino Acid Sequence↗
PubMed1994

Oxidation-induced aggregation of rabbit low-density lipoprotein by azo initiator.

Oxidation of low-density lipoprotein (LDL) has been considered as an important step in the early pathogenesis of atherosclerosis. We investigated the oxidative modification of LDL by a water-soluble azo-initiator AAPH (2,2,-azo-bis(2-amidinopropane).2HCl) and analyzed the uptake of AAPH-oxidized LDL with mouse peritoneal macrophages. Oxidative modification of LDL by AAPH was similar to the modification induced by copper in regard to the degree of oxidation and formation of aggregated LDL. The aggregated oxidized (AO-) LDL was fractionated by gel permeation chromatography and compared with the monomeric oxidized (MO-) LDL to make clear their characterization. The results of binding, cell association, and degradation with macrophages indicated that both AO- and MO-LDL were bound and endocytosed by macrophages. The cross competition experiment showed that nonreciprocal competition existed among MO-LDL, AO-LDL, and monomeric acetylated (MAc-) LDL. By the sterol accumulation experiment in macrophages with the various types of modified LDL, the cellular sterol accumulation was shown as the following order, AO-LDL > MAc-LDL > MO-LDL. These results indicated that the oxidation by AAPH can induce the aggregation of LDL and that the AO-LDL contribute to lipid accumulation into macrophages more than the MO-LDL.

Acetylation↗
PubMed1994

[Retrospective analysis of deep-seated candidiasis among cases autopsied between 1982 to 1991].

Candida is present in the flora of the oral cavity, skin, intestinal tract and vagina, and is also known to be an opportunistic pathogen. Infection with this fungus has been increasing annually along with wide spread use of broad-spectrum antimicrobial agents. The subjects included 95 patients (48 males and 47 females) who had been diagnosed as having had deep-seated candidiasis, among patients autopsied between 1982 to 1991. In regard to annual changes in deep-seated candidiasis, the incidence reached a peak in the 1985 to 1988 period, and thereafter decreased. The number of cases with leukemia as the underlying disease was the largest, 36 (37.9%), followed by malignant lymphoma in 10, and aplastic anemia 5. The number of cases with infection of the stomach was largest, 42 (44.2%), followed by the esophagus in 33 (34.7%), the lung and kidney. The cases with deep-seated candidiasis showed low values of or level of lymphocyte, hemoglobin, CRP, total protein and cholesterol and high values or levels of LDH, urea N, creatinine and total bilirubin. Cases with marked decrease in neutrophils showed no regional infiltration of inflammatory cells in any of the organs infected with Candida. Cases with disseminated candidiasis showed vascular invasion by Candida. The laboratory findings also showed that most of the cases had been undernourished and had high values of CRP which supports the presence of inflammation. Common sites of infection are the esophagus, stomach, and intestinal tract. In the presence of granulocytopenia and immunodeficiency, tissue invasion become severe and associated with vascular invasion.

Aged↗
PubMed1994

Changes in the distribution and intensity of alkaline phosphatase activity in rat lymph node and spleen cells after antigen stimulation.

We investigated the reaction of the popliteal lymph nodes (PLN) to the injection of two antigens, keyhole limpet hemocyanin (KLH) and lipopolysaccharide (LPS), into the footpads of rats, as well as the changes occurring in the PLN after allogeneic cell stimulation. Changes in alkaline phosphatase (ALP) activity of the lymph nodes were examined enzyme histochemically. Paralleling with PLN weight gain, increased ALP activity was observed in the medullary regions of the lymph nodes of stimulated rats. ALP reactivity in the stimulated lymph nodes was observed to be weak in the germinal centers and strong in the medullary regions. The spleens of rats subjected to systemic graft-vs.-host (GVH) reaction were examined in a similar fashion. The ALP-positive areas of the GVH spleens increased in size as compared with normal spleens. These positive areas of lymph node and spleen appear to correspond mainly to areas containing OX12-positive cells. These results suggest that enzyme-histochemical analysis of ALP activity together with immunohistochemical analysis of lymphocyte phenotypes may be a useful method for examining lymph node and spleen reactions to soluble and cellular antigens in rats.

Alkaline Phosphatase↗
PubMed1994

Characterization of aggregated low density lipoproteins induced by copper-catalyzed oxidation.

Oxidation of low density lipoproteins (LDL) has been shown to lead to enhanced uptake by macrophages mediated by the scavenger receptor. In the present study, changes in LDL induced by copper-catalyzed oxidation were investigated using gel permeation chromatography (GPC), and the results were compared with several parameters of oxidized LDL (ox-LDL). When LDL at 200 micrograms/ml was oxidized with 10 microM Cu2+ at 37 degrees C for up to 24 hours, increases in thiobarbituric acid-reactive substances and electrophoretic mobility were first observed within 3 hours. An increase in fluorescence and a decrease in intact apolipoprotein B (apoB) were than observed in parallel with an increase in 125I-LDL degradation by macrophages after 6 hours. Finally, LDL aggregation separated by liquid chromatography was observed after 24 hours. The aggregated and monomeric fractions of ox-LDL were analyzed and the results compared with the monomeric fraction of native LDL. Both fractions of ox-LDL contained hardly any intact apoB and showed an intense fluorescence. The electrophoretic mobility increment of aggregated ox-LDL was almost half that of monomeric ox-LDL, yet the lysine residues of aggregated ox-LDL were more extensively decreased than those of monomeric ox-LDL. Degradation of aggregated ox-LDL by macrophages showed a slightly greater increase than that of monomeric ox-LDL. GPC analysis is a useful method to estimate the LDL aggregation, and these results provide a basis to investigate the formation of aggregated LDL.

Animals↗
PubMed1994

[Determination of globin synthesis studies using high performance liquid chromatography technique].

In vitro globin chain synthesis ratios (beta/alpha) were determined using high performance liquid chromatography (HPLC) in twenty-five alpha or beta-thalassemiaes as well as six normal controls. Among thalassemiaes nineteen were Japanese and six Korean. Two kinds of cation exchange columns.; Mono S, HR 5/5 by Pharmacia and TSK gel by Tohso were compared. Hemolysates (10%), in stead of purified globin peptides, were directly subjected to the both columns, and 3H-labeled globin chain in vitro synthesized were separated on them. The Tohso column gave better resolution of globin peptides, resulting in sharp elution profiles. The HPLC system took about two hours for an analysis of a sample. In contrast, the conventional carboxymethyl cellulose (CMC) chromatography by Clegg need larger sample amounts for globin extraction and twenty-four hours to be completed. The HPLC is evidently simple and rapid. The globin chain ratios thus obtained by HPLC were 1.04-1.08, 0.23-0.89, 1.22-1.62 and 2.05-4.29 for normals, thirteen beta-thalassemia minors, seven alpha-thalassemia traits and three Hb H diseases respectively.

Chromatography, High Pressure Liquid↗
PubMed1993

CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3.

To elucidate the differential roles of N- and C-terminal halves of hirudins in thrombin inhibition, we produced novel recombinant hirudin analogs, CX-397 and CX-397R, having a hybrid amino acid sequence of hirudin variants-1 (HV-1) and -3 (HV-3). CX-397 is composed of the N-terminal half of HV-1 (HV-11-36) combined with the C-terminal half of HV-3 (HV-337-66). CX-397R is the opposite combination. Their anti-thrombin activity was determined by a fluorogenic enzyme assay and compared with that of recombinant HV-1 (rHV-1) and rHV-3. The order of the magnitude of dissociation constants (Ki) of these four hirudin analogs in thrombin inhibition was as follows: CX-397R (0.294 pM) > rHV-1 (0.148 pM) > rHV-3 (0.0593 pM) > CX-397 (0.0433 pM), indicating that CX-397 is the strongest inhibitor among them.

Amino Acid Sequence↗
PubMed1993

Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human cdc25B phosphatase.

In mammalian cells, p34cdc2 kinase undergoes phosphorylation at threonine-14, tyrosine-15 and threonine-161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine-14 and tyrosine-15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine-15 in vitro, but whether it also does at threonine-14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine-14 and tyrosine-15 but not threonine-161.

CDC2 Protein Kinase↗
PubMed1993

[The characterization of human cdc2 kinase and CDK2].

p34cdc2 kinase plays a key role in the initiation of mitosis. The activity of this kinase requires the binding of a protein, named cyclin, to it. The kinase forms a heterodimer with cyclin. Cyclin A or B is the counterpart of this complex. The differences in the activity between cyclin A/cdc2 kinase and cyclin B/cdc2 kinase have not been cleared. In recent years, the other cdc2-like kinases were identified. One of them was CDK2 (cyclin dependent kinase 2). CDK2 could rescue the defect of the budding yeast CDC28 mutation, which arrested the cells at a point named START, in G1 phase. Then, CDK2 was thought to be worked at G1 through S phase in a cell cycle, but the details on the role of this kinase has not been cleared so far. In this study, we separated the human cyclin A/cdc2 kinase, cyclin B/cdc2 kinase and CDK2, each other by use of column chromatography, and characterized the each kinase. These kinases had the same substrate specificities when the synthesized peptides were tested. They phosphorylated the threonine residue in the sequence -Thr-Pro-Lys-Lys-Ala- but hardly phosphorylated threonine residue the sequence -Thr-Pro-Lys-Ala-Lys-. They had some differences in the substrate-preference when the native proteins were tested. In a cell cycle of human cells, the activity of cdc2 kinase increased at G2/M phase and the activity of CDK2 was high from S through M phase. These data suggested that cdc2 kinase works at the transition from G2 to M phase and that CDK2 works from G1 through G2/M phase. They could phosphorylate different protein-substrates having the common phosphorylated sequence -Thr-Pro-X-Lys-.

Amino Acid Sequence↗
PubMed1993

[Turning point in quality control in Japan--current status & problems and future issues].

Some authorities suggest that quality control in Japan is now at a turning point. During the 30 years since its introduction in the 1960s, quality control has improved dramatically, and a number of new quality control techniques have been designed and applied to routine examination. In actual practice, however, people tend to become insensitive to abnormalities or to ignore them, causing an increase in the number of abnormal results. This article will review a survey of quality control and describe the current status, problems and future issues in quality assurance.

Forecasting↗
PubMed1993

[Intestinal flora of inpatients and isolation frequency of methicillin-resistant Staphylococcus aureus].

The authors compared intestinal flora from 30 healthy volunteers and 128 inpatients. E. coli, B. fragilis, and Bifidobacterium were each detected in the stools of healthy subjects at a frequency of more than 90%, while the incidences of such flora were low in the stools of inpatients: A significant difference was observed between the two groups. E. faecium, P. aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), C. difficile, and Candida were detected at high frequencies in the stools of inpatients, as compared with healthy subjects. This finding is attributed to the administration of antimicrobial agents. It is also considered that microbial interaction in maintaining the balance among normal intestinal flora had been lost in patients from whom MRSA and/or C. difficile was isolated. The decrease or elimination of bacterial species antagonistic to such resistant strains must be guarded against, because this can lead to weakening of the defence mechanism against intestinal infection.

Enterococcus faecium↗
PubMed1992

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.

Amino Acid Sequence↗
PubMed1992

The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase.

The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.

Amino Acid Sequence↗
PubMed1992

M-phase-specific histone H1 kinase in fish oocytes. Purification, components and biochemical properties.

We demonstrate, for the first time in fish, that a Ca(2+)-independent and cyclic-nucleotide-independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them. The kinase, M-phase-specific histone H1 kinase (M-H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine-specific protein kinase encoded by the fission yeast cdc2+ gene (cdc 2 kinase). The M-H1K and maturation-promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M-H1K and MPF. The final preparation was purified 5000-fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP peptide was used. The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33, 34, 46 and 48 kDa. Anti-PSTAIR antibody recognizing cdc2 kinase cross-reacted with the 33-kDa and 34-kDa proteins, while the 46-kDa and 48-kDa bands cross-reacted with monoclonal antibodies raised against cyclin B. The 33-kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2-related kinase which has the PSTAIR sequence and binds to p13suc1 but does not form a complex with cyclin B. M-H1K activity corresponded well to the 34-kDa, 46-kDa and 48-kDa proteins but not to the 33-kDa protein. These results strongly suggest that M-H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M-H1K, although it is found in the highly purified M-H1K. The purified M-H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0-10.5. The kinase was thermolabile and sensitive to freezing/thawing.

Amino Acid Sequence↗
PubMed1992

The role of transcription and messenger RNA stability in the regulation of epidermal growth factor receptor gene expression in regenerating mouse liver.

The influence of partial hepatectomy on epidermal growth factor receptor gene expression was studied in mouse liver. Epidermal growth factor receptor binding and epidermal growth factor receptor messenger RNA levels in the liver showed a rapid peak 8 hr after partial hepatectomy, whereas the sham operation had no effects on these levels. The peak epidermal growth factor receptor messenger RNA level was approximately threefold higher than preoperative values. The increase in epidermal growth factor receptor messenger RNA levels occurred primarily as a consequence of an increase in the rate of transcription. Partial hepatectomy slightly increased the half-life of epidermal growth factor receptor messenger RNA in the liver from 2.8 to 3.6 hr. Treatment of partially hepatectomized mice with cycloheximide increased hepatic epidermal growth factor receptor messenger RNA levels about fivefold by prolonging the half-life of the messenger RNA to 11.2 hr, although this treatment inhibited the increase in transcription induced by partial hepatectomy. Cycloheximide also increased epidermal growth factor receptor messenger RNA levels in the liver or kidney of sham-operated mice about threefold, primarily through stabilizing epidermal growth factor receptor messenger RNA. In contrast, cycloheximide had no effects on beta-actin messenger RNA levels in the liver and kidney. These results suggest that transcription induced by partial hepatectomy requires protein synthesis and that labile proteins are involved in the regulation of the stability of epidermal growth factor receptor messenger RNA.

Actins↗