Biomedical subjects

T Obata

Publications and source records attributed to T Obata.

At least 73 records · Page 4Linked to original sources

PubMed1999

Reserpine prevents hydroxyl radical formation by MPP+ in rat striatum.

The present study investigated the blockage of dopamine (DA) oxidation by reserpine. 1-Methyl-4 phenylpyridinium ion (MPP+) increased the release of DA and the formation of hydroxyl radical ( r22. OH). The r22. OH generated by DA when captured as the hydroxylated derivative of salicylic acid was measured by the high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. MPP+ concentration for half-maximal effect of DA producing release (EC50) was 5.2 mM. The maximum attainable concentration of dialysate DA (Emax) by MPP+ was 7.7 microM. However, the EC50 and Emax values with reserpinized animal were 5.2 mM and 1.2 microM, respectively. When high concentration of pargyline (10 mM) were infused in MPP+ (5 mM)-pretreated animals, a marked elevation of DA and r22. OH formation was observed. The level of DA and 2, 3-DHBA formations was drastically reduced, as compared with the MPP+-only treated group. Although the levels of MPP+-induced DA and 2,3-DHBA formation after pargyline treatment increased, pargyline failed to increase either the level of MPP+-induced DA or 2,3-DHBA in the reserpinized group. When DA was administered to the MPP+-pretreatment group, a marked elevation was observed, showing a positive linear correlation DA and r22. OH formation trapped as 2,3-DHBA (R2=0.978) in the dialysate. When corresponding experiment were performed with iron (II), the same results were obtained: a positive linear correlation between the release of DA and 2,3-DHBA (R2=0.989) in the dialysate. These results indicated that reserpine-induced DA depletion may reduce MPP+-induced r22. OH formation.

1-Methyl-4-phenylpyridinium↗

PubMed1999

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure.

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.

Amino Acid Substitution↗

PubMed1999

Analysis of the consensus binding sequence and the DNA-binding domain of ZF5.

Murine ZF5 is a transcription factor with five zinc finger motifs that represses the c-myc gene by binding to two GC-rich elements at the promoter region. Because of its ubiquitous expression in a variety of tissues, elucidation of biological functions and cellular target genes of ZF5 is of great interest. As the first step of identifying cellular target genes, we have attempted to determine the consensus binding motif for ZF5. We succeeded in isolating 19 oligonucleotide duplex DNAs to which ZF5 binds and determined the binding sequences with DNase I footprinting analysis. From these sequences, we deduced the consensus binding motif for ZF5 to be GSGCGCGR. In addition, we have analyzed the DNA-binding domain of ZF5 by testing a series of deletion mutants. It turned out that the zinc fingers 3 and 4 of the five finger motifs play a critical role in DNA binding.

Animals↗

PubMed1999

Protective effect of histidine on MPP+-induced hydroxyl radical generation in rat striatum.

We investigated the efficacy of histidine on MPP+-induced hydroxyl radical (.OH) formation in extracellular fluid of rat striatum. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol microl-1 min-1) was infused through a microdialysis probe to detect the generation of.OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. MPP+ (5 mM) clearly produced an increase in.OH formation. However, histidine (25 mM) reduced the.OH formation by the action of MPP+. These results indicate that histidine protects MPP+-induced.OH formation in rat striatum.

1-Methyl-4-phenylpyridinium↗

PubMed1999

Early detection of radiation-induced liver injury in rat by superparamagnetic iron oxide-enhanced MR imaging.

The detectability of early liver injury induced by irradiation was studied using magnetic resonance (MR) imaging enhanced with superparamagnetic iron oxide (SPIO), a tissue-specific contrast agent against the reticuloendothelial system (RES). In rat, 3 days after focal irradiation (0-10 Gy), MR imaging was performed and specimens were obtained to observe the phagocytic function of RES. The irradiated portion of the liver was visualized with a clear demarcation from the nonirradiated part by SPIO-enhanced MR images as a decrease in negative enhancement reflecting the function of RES (P < 0.05), whereas this was impossible with nonenhanced MR images. Significant regression was observed as a dose-related change of the signal intensity in the irradiated portion on SPIO-enhanced MR images (R = 0.867, P < 0.0001). SPIO-enhanced MR imaging was reliable for detecting the range and extent of liver injury a few days after low-dose irradiation, and it may be a useful procedure for verifying the target area in clinical cases of radiation therapy.

Animals↗

PubMed1999

A new antidiabetic agent (JTT-501) rapidly stimulates glucose disposal rates by enhancing insulin signal transduction in skeletal muscle.

A newly synthesized antidiabetic agent, JTT-501 is an isoxazolidinedione rather than a thiazolidinedione. An oral dose of JTT-501 (100 mg x kg(-1) x day(-1)) given to 12-week-old male Zucker fatty rats for 7 days led to the amelioration of both hyperinsulinaemia (40% of non-treated) and hypertriglyceridaemia (23% of non-treated) as well as a 2.4-fold increased insulin sensitivity as determined by a euglycaemic insulin clamp. In our study, we further evaluated the acute effect of JTT-501 on both the glucose infusion rates (GIR) and insulin signalling in skeletal muscle. Male Sprague-Dawley (SD) rats aged 10 weeks were injected intravenously with JTT-501 (5 mg/kg) and then a euglycaemic insulin clamp was initiated and glucose infusion rates monitored for 150 min. We found that this treatment increased the glucose infusion rate by 33% during the last 30 min in SD rats. After the clamp had been initiated for 30 min, the insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activities co-immunoprecipitated with insulin receptor substrate 1 (IRS-1) were also enhanced, resulting in increased glycogen synthase activities in the soleus muscles. Treatment with JTT-501 also enhanced the phosphorylation of insulin receptors and insulin receptor-substrate 1 rapidly as well as the phosphatidylinositol 3-kinase activities, which were stimulated by a bolus injection of insulin. Similarly, JTT-501 stimulated the glucose infusion rate by 30% and enhanced insulin signalling in Zucker fatty rats. In conclusion, a newly developed isoxazolidinedione, JTT-501, rapidly potentiates the insulin sensitivity of skeletal muscle by enhancing insulin signalling and could be useful for the treatment of insulin-resistant diabetic subjects.

Animals↗

PubMed1999

Eicosapentaenoic acid inhibits prostaglandin D2 generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells.

BACKGROUND: Eicosapentaenoic acid (EPA) is catalysed by cyclo-oxygenase (COX), as is arachidonic acid, and is a competitive inhibitor of arachidonate metabolism. OBJECTIVES: We examined the effect of EPA on prostaglandin (PG) D2 generation in the cultured human mast cells with IgE-anti-IgE challenge incubation. METHODS: Cultured human mast cells were incubated with EPA (1 micromol/L) for 20 h, then challenged with anti-IgE incubation after treatment with IgE. At the same time, COX inhibitors were tested to identify COX-1 and COX-2 activity. PGD2 synthetic activity was also assayed in a cell-free homogenate of cultured mast cells with COX inhibitors and EPA. Histamine in the culture medium and in cells was assayed with the HPLC-fluorescent method. PGD2 and PGD3 were assayed with gas chromatography-mass spectrometry and the stable isotope dilution method. RESULTS: Although EPA incubation did not affect histamine release by cultured human mast cells in response to IgE-anti-IgE challenge incubation, it did decrease PGD2 generation by inhibiting the COX-2 pathway. In contrast, in the cell-free homogenate of cultured human mast cells, EPA inhibited both COX-1 and COX-2 activities. CONCLUSION: Pre-incubation with EPA primarily affects the COX-2 pathway in cultured human mast cells and reduces PGD2 generation in response to IgE-anti-IgE challenge incubation. These findings suggest that COX-1 and COX-2 have different substrate flow systems in mast cells. They also suggest that endogenous EPA diet supplementation would reduce PGD2 production and could serve as an anti-inflammatory substrate in human mast cells.

Antibodies↗

PubMed1999

Isolation of deoxycytidine kinase from Ehrlich carcinoma cells by affinity chromatography based on a substrate analog, 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosyl-N4-palmitoylcytosine++ +.

Deoxycytidine kinase from Ehrlich carcinoma cells was purified 10400-fold by ammonium sulfate fractionation and affinity chromatography using Sepharose 4B coupled to 2'-C-cyano-2'-deoxy-1-beta-D-arabinofuranosyl-N4-palmitoylcytosine , with a yield of 45%. The purified enzyme preparation showed a single major band with a molecular weight of 32000 on SDS-PAGE. The enzyme phosphorylated deoxyadenosine, deoxyguanosine, cytidine, and several deoxycytidine analogues as well as deoxycytidine. Also, the kinetic parameters of the enzyme for the substrates were estimated.

Animals↗

PubMed1999

Protective effect of histidine on hydroxyl radical generation induced by potassium-depolarization in rat myocardium.

We investigated the efficacy of histidine on potassium-depolarization induced hydroxyl radical (*OH) generation in the extracellular fluid of rat myocardium by a flexibly mounted microdialysis technique (O system). After the rat was anesthetized, a microdialysis probe was implanted in the left ventricular myocardium, and then sodium salicylate in Ringer's solution (0.5 nmol/microl per minute) was infused to detect the generation of *OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Infusion of KCl (70 mM) clearly produced an increase in *OH formation. However, when KCl in the presence of a high concentration of histidine (25 mM) was infused through the microdialysis probe, KCl failed to increase the 2,3-DHBA formation. To examine the effect of histidine on ischemia-reperfusion of the myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. However, when corresponding experiments were performed with histidine (25 mM)-pretreated animals, histidine prevented the ischemia-reperfusion induced *OH formation trapped as 2,3-DHBA. These results indicate that histidine may protect against K+-depolarization-evoked *OH generation in rat myocardium.

Animals↗

PubMed1999

A preliminary study for clinical pharmacokinetics of oral fluorine anticancer medicines using the commercial MRI system 19F-MRS.

These preliminary studies of dynamic natural abundance 19F-magnetic resonance spectroscopy (19F-MRS) on 5-FU based medicines were performed in the human liver using commercial 1.5 T MRI equipment. A single tuned, custom-made circular shape surface coil with a diameter of 15 cm operating at 60 MHz was used for the 19F-MRS study. Localized proton shimming with a whole body coil was performed with adequate volume to include the observing area of the surface coil, and the line width of the water signal was less than 40 Hz. Very different spectroscopic appearance patterns of 5-FU were observed. We examined whether the pharmacokinetics in the liver of orally administered 1-hexylcarbamoyl-5-fluorouracil (HCFU) differ from those of orally administered 5'-deoxy-5-fluorouridine (5'-DFUR). This preliminary study suggested the 19F-MRS technique could be a useful method of evaluating in vivo the metabolism of 5-FU based medicines.

Administration, Oral↗

PubMed1998

Glibenclamide, an antagonist of ATP sensitive K+ channels, blocks free radical generation in the rat myocardium.

The present study examined the effect of glibenclamide, an ATP-sensitive K+ (K(ATP)) channels antagonist, on the potassium chloride (KCl)-induced hydroxyl free radical (.OH) generation. Sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused directly through a microdialysis probe to detect the generation of .OH as reflected by the formation of dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rat. The high concentration of KCl (70 mM) significantly increased the level of 2,3- and 2,5-DHBA by the action of depolarization by KCl. However, in the presence of glibenclamide (10 microM), KCl failed to increase the 2,3- and 2,5-DHBA formation. Moreover, when allopurinol (10 mg/kg), a xanthine oxidase inhibitor, was administered by i.v. injection, the elevation of DHBA was not observed. These results suggest that openings of cardiac K(ATP) channel by depolarization evokes .OH generation via xanthine oxidase reaction.

Allopurinol↗

PubMed1998

Genomic cloning and characterization of the mouse POZ/zinc-finger protein ZF5.

We isolated genomic DNA containing the entire sequence of ZF5, which was originally identified by its ability to repress the mouse c-myc promoter and which was characterized as one of the POZ (Poxvirus and zinc finger) proteins. The POZ motif is a protein-protein interaction interface found at the N-terminal region of zinc finger proteins. Sequence analysis demonstrated that the ATG translation initiation codon was separately located from the remainder of the coding sequence. Using both RNase protection and primer extension assay, a single major transcription start site was determined. Promoter analysis by transient transfection assay suggested positive autoregulation by ZF5 itself. The ZF5 N-terminal region, including the POZ domain, was required for this regulation. Sp1 also activated the ZF5 promoter and this activity was repressed by addition of ZF5. ZF5 expression was stronger in mouse ovary, lung and brain than in other organs.

Animals↗

PubMed1998

Lung as reservoir for antidepressants in pharmacokinetic drug interactions.

BACKGROUND: Although high-affinity imipramine binding sites have been reported in both rat and human lung, the role of the lungs in the pharmacokinetics of antidepressants has not received much attention. Substantial accumulation of selective serotonin-reuptake inhibitors (SSRIs) in the lungs has been reported. We have investigated the role of the lungs in pharmacokinetic drug interactions between tricyclic antidepressants and SSRIs. METHODS: We used a carbon-11-labelled form of the imipramine derivative cyanoimipramine to measure uptake in the lungs and brain of healthy volunteers by positron emission tomography. Clomipramine (50 mg) was administered to measure the effect of antidepressants with high affinity for the serotonin transporter on lung and brain uptake. FINDINGS: A large proportion of the injected 11C-cyanoimipramine (68-86% in the four volunteers) was extracted by the lungs. Clomipramine decreased the lung uptake from 68% to 35% and from 81% to 54% in the two volunteers studied. By contrast, whole-brain uptake was low in control studies (1.7-2.0% in three volunteers) and increased after clomipramine administration (to 4.5-4.9%). Plasma radioactivity was also higher after clomipramine than in control studies. INTERPRETATION: The lungs may function as a reservoir for antidepressants with high affinity to the serotonin transporter. The accumulated antidepressants may be displaced by other antidepressants, and this displacement would substantially increase plasma concentrations and thus cause toxic effects.

Adult↗

PubMed1998

Development and biochemical characterization of a 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC)-resistant variant of the human fibrosarcoma cell line HT-1080.

2'-C-Cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is an antitumor nucleoside with a novel chemical structure that exerts potent antitumor activity against various human tumor cells in vitro and in vivo. In order to be active it needs to be phosphorylated by deoxycytidine (dCyd) kinase. We induced resistance to CNDAC in the human fibrosarcoma cell line HT-1080 by exposure to increasing concentrations of CNDAC. The resistant cells showed over 560 times higher resistance as compared to that of the parental HT-1080 cells and were cross-resistant to the other 2'-deoxycytidine derivatives. The dCyd kinase mRNA expression of the resistant cells decreased and there was the expression of aberrant mRNA of dCyd kinase which contained a 116-nucleotide deletion within the coding region, corresponding to the fifth exon of the gene. The dCyd kinase enzymatic activity of the resistant cells was deficient. The initial uptake of CNDAC into the resistant cells was similar to that of the parental cells. However, the incorporation of CNDAC into the DNA fraction of the resistant cells was significantly less than that of the parent cells. These results led us to conclude that the acquired resistance to CNDAC can be attributed to a deficiency of dCyd kinase activity, which should be based on a remarkable decrease in mRNA expression and genetic mutation of the dCyd kinase gene, but not on cellular CNDAC accumulation.

Antimetabolites, Antineoplastic↗

PubMed1998

NO and cGMP facilitate adenosine production in rat hearts via activation of ecto-5'-nucleotidase.

We examined whether nitric oxide (NO), a possible cardioprotective substance, can increase the production of interstitial adenosine in the ventricular myocardium. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at a rate of 1.0 microl min-1. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography. Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5'-nucleotidase, and the level of adenosine reflected the activity of ecto-5'-nucleotidase in the tissue. S-Nitroso-N-acetylpenicillamine (SNAP, 0.3-3 mM), an NO donor, increased the dialysate adenosine measured in the presence of AMP (100 microM) in a concentration-dependent manner. However, in the presence of an NO-oxidizing agent, 2-(4-carboxyphenyl-4,4,5, 5-tetramethylimidazoline)-1-oxyl 3-oxide (carboxy-PTIO, 1 mM), the effect of SNAP was abolished. Another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409, 1 mM) also increased adenosine production. 8-Bromo-cGMP (0.1-3 mM), a membrane-permeable cGMP analogue and a potent activator of cGMP-dependent protein kinase, increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. These results suggest that NO facilitates the production of interstitial adenosine in rat hearts in situ, via cGMP-mediated activation of ecto-5'-nucleotidase.

5'-Nucleotidase↗

PubMed1998

Effect of .OH scavenging action by non-SH-containing angiotensin converting enzyme inhibitor imidaprilat using microdialysis.

We examined the effect of non-SH-containing angiotensin converting enzyme (ACE) inhibitor imidaprilat on hydroxyl radical (.OH) generation using microdialysis. Salicylic acid in Ringer's solution containing sodium salicylate (0.5 n mol microL-1 min-1) was infused directly through a microdialysis probe to detect the generation of .OH as reflected by the formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. We compared the ability of two non-SH-containing ACE inhibitors (imidaprilat and enalaprilat) with an -SH-containing ACE inhibitor (captopril) to scavenge the .OH. When iron (II) was administered to animals pretreated with these three ACE inhibitors, a decrease in 2,3-DHBA of all three compounds was observed, as compared with the iron (II) only-treated group. All three ACE inhibitors were able to scavenge .OH generated by the action of iron (II). However, imidaprilat is a free radical scavenger more potent than enalaprilat. These results suggested that ACE inhibitors are probably not only related to the presence of the SH radical.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Adenosine deaminase activity in rat intestine: assay with a microdialysis technique.

Using microdialysis, we measured adenosine deaminase activity in rat intestine by detecting inosine, a breakdown product of adenosine. The dialysis probe consisted of a 3 x 0.22 mm dialysis fiber with a 50,000 mol wt cut off. When the probe was perfused at 1 microl/min in vitro, the average relative recovery rate of inosine was 22.1+/-0.9%). The dialysis probe was implanted in the intestinal mucosa and perfused with Tyrode solution containing adenosine at 1 microl/min. The dialysate samples were analyzed for inosine by high-performance liquid chromatography with ultraviolet (HPLC-UV) detection at 260 nm. When adenosine (100-1000 microM) was perfused, the level of inosine increased dose-dependently and was saturatable at about 1 mM adenosine. The ED50 of adenosine was 192.6 microM, with a maximum attainable inosine concentration of 59.7 microM. In the presence of aminoguanidine, a adenosine deaminase inhibitor (10 mM or 10 n mol/microl/min), the elevation of inosine was not observed. The dialysis technique makes it possible to measure adenosine deaminase activity in intestinal mucosa.

Adenosine↗

PubMed1998

Effects of lysophosphatidylcholine on the production of interstitial adenosine via protein kinase C-mediated activation of ecto-5'-nucleotidase.

1. Adenosine plays a crucial role in the evolution of ischemic preconditioning. With the use of microdialysis techniques in in situ rat hearts, we assessed the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production), and examined the effects of lysophosphatidylcholine (LPC) on the production of interstitial adenosine. 2. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP, 100 microM). With this system, the dialysate adenosine originates from the dephosphorylation of AMP, catalyzed by endogenous ecto-5'-nucleotidase. The level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in vivo. 3. LPC at concentrations of 25 and 50 microM significantly increased the level of dialysate adenosine to 122.7+/-4.3% (n=4, P<0.05) and 158.6+/-7.2% (n=5, P<0.05) of the control, respectively. Chelerythrine (200 microM), a protein kinase C (PKC) inhibitor, completely abolished the increase of dialysate adenosine afforded by LPC (50 microM) (n=5). 4. These data provide the first evidence that LPC does increase the concentration of interstitial adenosine in rat hearts in situ, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase.

Adenosine↗