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Biomedical subjects

S Okada

Publications and source records attributed to S Okada.

At least 145 records · Page 8Linked to original sources
PubMed2000

Wortmannin sensitizes human glioblastoma cell lines carrying mutant and wild type TP53 gene to radiation.

The purpose of this paper is to investigate the effect of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), on TP53 (formerly known as p53) signal transduction initiated by ionizing radiation and radiosensitization in isogenic derivatives of human glioblastoma cells differing in TP53 status. Wortmannin inhibited the accumulation of TP53 and CDKN1A (formerly known as WAF1) after 6 Gy irradiation in A-172/neo cells bearing wild-type TP53. In A-172/Trp248 cells carrying mutant TP53, X-rays induced no significant accumulation of TP53 and slight increase of CDKN1A. There were, consequently, little differences in the expression of TP53 and CDKN1A between A-172/Trp248 cells exposed to 6 Gy alone and wortmannin plus 6 Gy. However, wortmannin sensitized both A-172/neo and A-172/Trp248 cells to radiation. These studies indicate that wortmannin inhibits TP53 upregulation, but this suppression does not account for the radiosensitization by this drug. These results indicate that inhibitors of PI3K-related kinases may present a new class of radiosensitizers, regardless of the TP53 status of tumor cells.

Androstadienes↗
PubMed2000

HSP70 and ribosomal protein L2: novel 5S rRNA binding proteins in Escherichia coli.

A Northwestern analysis of Escherichia coli total proteins with radiolabeled 5S rRNA identified two novel 5S rRNA interacting proteins, a 70 kDa and a 37 kDa protein, and three ribosomal proteins reported on already. The N-terminal sequencing of the 70 kDa protein separated by SDS-PAGE from the high-salt-washed fraction of crude ribosome led to the discovery of a polypeptide identical in its first 10 amino acid residues to E. coli heat shock protein 70. The N-terminal eight amino acid sequence of the 37 kDa protein extracted from the high-salt-washed ribosome is identical to that of the ribosomal protein L2. In addition, the interaction of these proteins with 5S rRNA has been confirmed with gel mobility shift assays.

Amino Acid Sequence↗
PubMed2000

Inhibition of LPS-induced cytokines by Bcl-xL in a murine macrophage cell line.

The antiapoptotic molecule Bcl-xL has been implicated in the differentiation and survival of activated macrophages in inflammatory conditions. In this report, the role of Bcl-xL in LPS-induced cytokine gene expression and secretion was studied. Bcl-xL-transfected RAW 264 macrophages were protected from gliotoxin-induced apoptosis, indicating the presence of functional Bcl-xL. Overexpression of Bcl-xL in this macrophage cell line was also associated with a marked inhibition of LPS-induced TNF-alpha, JE/monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 secretion. Inhibition of LPS-induced cytokine secretion was paralleled by a decrease in levels of steady-state mRNA for the above cytokines and for IL-1beta. Decreased production of TNF-alpha in Bcl-xL transfectants was not due to increased mRNA degradation, as the mRNA half-lives were the same in Bcl-xL transfectants and control macrophages. Although the composition of NF-kappaB complexes detected by EMSA and supershift analysis in nuclear lysates derived from Bcl-xL transfectants and control cells was indistinguishable, LPS-induced inhibitory kappaBalpha degradation, as well as NF-kappaB binding and AP-1 activation, were slightly decreased by ectopic expression of Bcl-xL. More strikingly, LPS-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase was strongly repressed by Bcl-xL overexpression, offering a possible mechanism for the inhibition of LPS-induced cytokine production. These data provide the first evidence for a novel role for Bcl-xL as an anti-inflammatory mediator in macrophages.

Animals↗
PubMed2000

Characterization of nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated rat stomach.

We characterized nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution at a constant flow rate of 4 ml per minute. Endogenous noradrenaline released into the perfusate was electrochemically measured using high-performance liquid chromatography. Nicotinic receptor agonists were applied once into the perfusion medium for 2 min and nicotinic receptor antagonists were administered throughout the experiments. The (-)-nicotine (3x10(-5) M)-induced noradrenaline release was abolished by tetrodotoxin and hexamethonium and partially blocked by dihydro-beta-erythroidine (up to 10(-5) M) (a relatively selective antagonist of alpha4beta2 nicotinic receptors) and abolished by mecamylamine (10(-5) M) (a relatively selective antagonist of alpha3beta4 nicotinic receptors), but not influenced by alpha-bungarotoxin (3x10(-7) M) or alpha-conotoxin ImI (10(-6) M) (antagonists of alpha7 nicotinic receptors). (+/-)-Epibatidine (3x10(-7) M) (a very potent, but non-selective agonist) and (-)-cytisine (3x10(-4) M) (an agonist of beta4 nicotinic receptors) effectively evoked the release of noradrenaline, while (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (RJR-2403) (up to 10(-4) M) (an agonist of alpha4beta2 nicotinic receptors) had no effect. The potency of these agonists was as followed; (+/-)-epibatidine>>(-)-nicotine>(-)-cytisine>>>RJR -2403. These results are compatible with the published view that alpha3beta4 nicotinic receptors are predominant in other parts of the autonomic nervous system. These receptors (probably located on the gastric sympathetic ganglia) are involved in the release of noradrenaline from the rat stomach.

Animals↗
PubMed2000

p53 dependence of topoisomerase I recruitment in vivo.

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.

Antigens, Neoplasm↗
PubMed2000

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin.

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

Adjuvants, Immunologic↗
PubMed2000

Inhibition of lipoprotein lipase activity by sphingomyelin: role of membrane surface structure.

We have recently shown that sphingomyelin (SM) strongly inhibits lipoprotein lipase (LPL)-mediated lipolysis in monolayers and emulsion particles. To further evaluate how SM modulates LPL activity on the emulsion surface, the relationship between membrane surface structure and LPL activity was investigated. We measured fluorescence anisotropy of 1-palmitoyl-2-[3-(diphenylhexatrienyl)propionyl]-sn-3-phosphati dylcho line, probing surface acyl chain fluidity, and fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine in H(2)O and D(2)O buffer, assessing the degree of hydration in the head group region. The results revealed that incorporation of egg SM into triolein-egg phosphatidylcholine emulsions markedly increased acyl chain order and decreased head group hydration of the surface monolayers. In contrast, cholesterol was shown to increase head group hydration despite a strong increase in acyl chain order. The close correlation between the apparent K(m) values of LPL and the degree of head group hydration indicated that LPL interacts with the head group region rather than with the hydrophobic interior of the surface monolayers. However, apparent V(max) did not show a simple correlation with any surface structure, and the finding in which SM had no effect on apparent V(max) of medium-chain triglyceride emulsions suggested that the hydrophobic interaction between acyl chains of SM and triglyceride at the emulsion surface is important for determining the apparent V(max). These results showed conclusively that SM inhibits LPL activity mainly by changing the emulsion surface structure and not by a specific interaction between SM and LPL.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Comparison of expressed sequence tags from male and female sexual organs of Marchantia polymorpha.

A total of 935 expressed sequence tags (ESTs) from male immature sexual organ were determined, of which 600 ESTs were assembled into 110 non-redundant groups, resulting in 445 unique EST sequences. Of these, 244 sequences shared significant similarities to known nucleotide or amino acid sequences in other organisms. The remaining 201 unique sequences showed no significant matches and thus are likely to be novel transcripts. ESTs from male and female immature sexual organs of a liverwort, Marchantia polymorpha, were compared to characterize gene expression patterns during sex differentiation. Ninety-nine male ESTs turned out to be common genes found also in the female library. Interestingly, one of the ESTs found only in male shows a significant similarity to the transformer-2 gene involved in sex determination in Drosophila. In female, several unique lectin ESTs were found that are not present in the male library.

Amino Acid Sequence↗
PubMed2000

Brain phospholipase A(2)-arachidonic acid cascade is involved in the activation of central sympatho-adrenomedullary outflow in rats.

The present experiments were designed to explore the role of the brain phospholipase A(2)-arachidonic acid cascade in the activation of central sympatho-adrenomedullary outflow in rats, using melittin (an activator of phospholipase A(2)) and arachidonic acid. Intracerebro-ventricularly administered melittin (2.5, 10, and 25 microg/animal) or arachidonic acid (75, 150, 300 microg/animal) effectively and dose dependently elevated plasma levels of adrenaline and noradrenaline. The elevation of both catecholamines induced by melittin (10 microg/animal) was abolished by centrally administered mepacrine (an inhibitor of phospholipase A(2)), but not by neomycin (an inhibitor of phospholipase C). However, mepacrine had no effect on the increase induced by arachidonic acid (150 microg/animal). Indomethacin (an inhibitor of cyclooxygenase) abolished all responses induced by melittin and arachidonic acid. Furegrelate (an inhibitor of thromboxane A(2) synthase) abolished the elevation of adrenaline induced by melittin and arachidonic acid, but had no effect on the elevation of noradrenaline induced by these compounds. These results suggest that activation of the brain phospholipase A(2)-arachidonic acid cascade facilitates the central sympatho-adrenomedullary outflow in rats. Brain thromboxane A(2) is involved in the activation of central adrenomedullary outflow and an active metabolite of arachidonic acid other than thromboxane A(2) may be involved in activation of the central sympathetic outflow.

Animals↗
PubMed2000

Familial frontotemporal dementia with a P301L tau mutation in Japan.

We have reported the family line with frontotemporal dementia (FTD) in Japan. This family line has so far included four patients. Patient II-1 (man) had a 10 year history of slowly progressive personality and behavioral changes and died at the age of 56. His neuropathological examination showed severe atrophy of the bilateral frontal and temporal cortices with neuronal loss, gliosis and superficial spongiosis. Pick bodies were not found. The neuropathological diagnosis was atypical Pick's disease without Pick bodies or Pick-type in FTD. Patient III-2 is patient II-1's oldest daughter and was taken ill with personality change at the age of 52. She died at the age of 68. Patient III-4 is patient II-1's second daughter. Her onset with strange speech and behavior was at the age of 59. Patient III-5 is patient II-1's oldest son. He also had onset with personality change at the age of 54 and had the P301L mutation in tau. In all III generation cases CT/MRI revealed circumscribed frontotemporal atrophy. Patient III-5's PET/SPECT showed signs of hypoperfusion or hypometabolism in the bilateral frontotemporal areas. This is the first report of familial FTD with the P301L mutation in Japan.

DNA Mutational Analysis↗
PubMed2000

65Zn localization in rat brain after intracerebroventricular injection of 65Zn-histidine.

Previous studies have shown that 65Zn uptake in the brain expressed relative to plasma 65Zn level is enhanced by histidine infusion into the blood vessel. To study the effect of histidine on zinc uptake in the brain parenchyma via the CSF, the brains of rats injected intracerebroventricularly with 65Zn-His were subjected to autoradiography. Six days after injection, the radioactivity from 65Zn-His was distributed in the major part of the brain parenchyma higher than that from 65ZnCl(2), and relatively concentrated in the hippocampal formation, globus pallidus and hypothalamus. The radioactivity of the aqueduct was also higher in the 65Zn-His group, indicating that CSF clearance of the 65Zn-His group may be lower than that of the 65ZnCl(2) group. These results suggest an enhancement by histidine on zinc uptake in the brain parenchyma via the CSF.

Animals↗
PubMed2000

The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice.

The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR-1) irradiated with UVB at a dose of 2 kJ/m(2) twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA-untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis.

Administration, Oral↗
PubMed2000

Relationship between brain zinc and transient learning impairment of adult rats fed zinc-deficient diet.

The relationship between brain zinc and learning behavior was studied based on the data of 65Zn localization in the hippocampal formation. Learning behavior, tested by passive avoidance performance, of 6-week-old rats improved significantly compared to that of 4-week-old rats and it was maintained at 20 weeks of age. When 8-week-old rats were fed zinc-deficient diet for 4 weeks, the learning behavior was significantly impaired. However, it was recovered to almost normal level by feeding with control (zinc-adequate) diet for 5 weeks. These results demonstrate that a proper zinc supply to the brain is necessary for improvement and maintenance of learning ability. Although an appreciable decrease in brain zinc was not observed in the rats fed zinc-deficient diet for 4 weeks, significant decrease of hippocampal zinc was observed in rats fed zinc-deficient diet for 12 weeks. Moreover, synaptosomal zinc in the hippocampal formation and cerebral cortex was significantly decreased by the 12 weeks of zinc deprivation. These results suggest that the decrease of vesicular zinc in the hippocampal formation and cerebral cortex is involved in the transient learning impairment of adults rats.

Age Factors↗
PubMed2000

Histamine H(3) receptor-mediated inhibition of endogenous acetylcholine release from the isolated, vascularly perfused rat stomach.

We studied the effects of histamine H(3) receptor ligands on the release of endogenous acetylcholine from the isolated, vascularly perfused rat stomach. The stomach was perfused via the celiac artery with modified Krebs-Ringer solution containing physostigmine. Released acetylcholine from the portal vein was electrochemically measured using high-performance liquid chromatography and an enzyme system. Vagus nerves were electrically stimulated twice for 2 min (0.5 or 2.5 Hz). Acetylcholine release evoked at 2.5 Hz was slightly inhibited by histamine and effectively potentiated by thioperamide, a histamine H(3) receptor antagonist. Acetylcholine release evoked at 0.5 Hz in the presence of atropine was not influenced by thioperamide, but effectively inhibited by histamine, R-alpha-methylhistamine or imetit, histamine H(3) receptor agonists. These inhibitory effects were abolished by thioperamide or pertussis toxin. These results suggest that histamine attenuates acetylcholine release from vagus nerves through histamine H(3) receptor-mediated and pertussis toxin-sensitive mechanisms in the rat stomach.

Acetylcholine↗
PubMed2000

The effect of pentylenetetrazole-kindling on the extracellular glutamate and taurine levels in the frontal cortex of rats.

We studied the extracellular concentration of glutamate and taurine in the frontal cortex of freely-moving pentylenetetrazole (PTZ) kindled rats using an in vivo microdialysis. A significant and sustained increase in the glutamate level was observed in the kindled rats, in contrast, a slight and delayed increase was observed in the non-kindled rats when the same grade seizure was induced by PTZ. The convulsive dose of PTZ administration caused a decrease in taurine levels in the controls, however, no significant changes were found in the kindled rats.

Animals↗
PubMed2000

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g<a, Gly122-to-Asp (G122D) and His179-to-Pro (H179P), were identified. We were able to detect "ectopically" transcribed G6Pase-mRNA in Epstein-Barr virus-transformed lymphoblastoid cells and observed aberrant mRNA splicing associated with the g727t and IVS1-1g<a mutations. To our knowledge, this is the first report that ectopic expression can be utilized for the characterization of GSD-Ia mutations. Our findings suggest that a screening for the g727t, R170X, and R83H mutations by simple DNA-based diagnostic methods can detect 95% of the G6Pase mutant alleles in Japanese patients with GSD-Ia, and remaining mutations can be identified and characterized by the direct sequencing of genomic DNA and/or the analysis of ectopically expressed mRNA. The noninvasive molecular diagnosis for GSD-Ia may ultimately replace the conventional means of enzymatic diagnosis that requires liver biopsy.

Alleles↗
PubMed2000

Influence of transferrin on manganese uptake in rat brain.

To evaluate the influence of transferrin (Tf) on manganese (Mn) uptake in the brain, pH 8.6 buffer-treated (54)MnCl(2), which has a higher affinity for Tf than untreated (54)MnCl(2), and Tf-bound (54)Mn were prepared. When pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2) were incubated with apo-Tf in Tris (2-amino-2-hydroxymethylpropane-1,3-diol)-HCl buffer, the percentage of the total (54)MnCl(2) bound to Tf was approximately 85% and 10%, respectively. One hour after intravenous (iv) injection of pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2), both tracers were concentrated similarly in the choroid plexus in the ventricles and distributed in other brain regions. Six days after iv injection, both pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2) tracers were concentrated in the superior olivary complex, inferior colliculi, and red nuclei, although the former radioactivity was lower than the latter. Moreover, Tf-bound (54)Mn was prepared and injected iv into rats. The radioactivity from Tf-bound (54)Mn, which was also concentrated in the same regions, e.g., the superior olivary complex, was the lowest of all three traces. Tf-bound (54)Mn was stable during incubation with serum for 1 hr. It is likely that more Mn is transported into the brain when Mn is not bound to Tf. When Tf-bound (54)Mn and (54)MnCl(2) were unilaterally injected into the lateral ventricle, radioactivity was distributed only around the ipsilateral ventricle in the Tf-bound (54)Mn group 7 days after injection, whereas it was distributed more extensively in the (54)MnCl(2) group. It is likely that Tf-bound Mn in the CSF is less readily transported into the brain parenchymal cells than the non-Tf-bound form. These results suggest that Mn is transported into the brain efficiently via a Tf-independent uptake system.

Animals↗