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K Oikawa

Publications and source records attributed to K Oikawa.

At least 37 records · Page 2Linked to original sources
PubMed2000

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1 +/- 11.6% (mean +/- S.E., n = 4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37 degrees C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy.

Animals↗
PubMed2000

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging.

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Animals↗
PubMed2000

The effects of the elimination of Kupffer cells in the isolated perfused liver from non-heart-beating rat.

We examined the effect of elimination of Kupffer cells on the sinusoidal microcirculation in graft harvested from non-heart-beating donors (NHBD), focusing on the arachidonic acid cascade and cytokines. Cardiac arrest was induced by thoracotomy. Livers were harvested 30 min after thoracotomy and perfused by Krebs-Henseleit bicarbonate buffer for 60 min after 6 h cold preservation. For the elimination of Kupffer cells, rats were pretreated liposome-encapsulated dichloromethylene diphosphonate (KE group). Eicosanoids (TXB2, 6-keto-PGF1alpha, LTB4) and cytokines (TNFalpha, IL-1beta) in the perfusate were measured. Histological examination was also carried out. In the KE group, the value of TXB2 was suppressed completely and cytokines were reduced, and sinusoidal structures and hepatocytes were well protected. These results indicated that the elimination of Kupffer cells improved sinusoidal microcirculation in NHBD and liver transplantation using grafts from NHBD could be made to succeed by modulation of Kupffer cells.

Animals↗
PubMed2000

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system.

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.

Animals↗
PubMed2000

Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III.

Apolipophorin III (apoLp-III) from the silkmoth, Bombyx mori, has been over-expressed in Escherichia coli, purified and characterized. Far-UV CD spectroscopic analysis revealed 65% alpha-helix secondary structure. Near-UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp-III alpha-helices reorient upon interaction with lipid, indicative of a protein conformational change. In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 M was observed, corresponding to a DeltaGDH2O = 2.46 kcal. mol-1. Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp-III revealed an emission lambdamax = 327 nm. Compared to free tryptophan, Stern-Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20-fold and sevenfold, respectively. In studies of apoLp-III-DMPC disc complexes, far-UV CD spectroscopy revealed an increase in alpha-helix content to approximately 85% and a ninefold increase in the GdnHCl-induced denaturation transition midpoint to 3 M. In studies of lipid interaction, apoLp-III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes. Characterization of apoLp-III-DMPC discs, using 5-doxyl or 12-doxyl stearic acid as lipid-based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups. KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp-III-DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers. Given the large amount of alpha-helix in apoLp-III, the data presented support a model in which amphipathic alpha-helical segments are stabilized by helix-helix interactions and lipid association induces a protein conformational change which results in substitution of helix-helix interactions for helix-lipid contacts.

Animals↗
PubMed2000

Comparison of receptor-mediated endocytosis kinetics between wild-type t-PA and recombinant pamiteplase in isolated rat hepatocytes and liver cell plasma membranes.

1. Differences in receptor-mediated endocytosis kinetics between pamiteplase, an engineered t-PA, and an unmodified rt-PA were examined using liver cell plasma membranes and isolated rat hepatocytes. 2. Whereas the binding site of pamiteplase on hepatocytes was the same as that of rt-PA, the Kd of pamiteplase was 5.1-7.7 times larger than that of rt-PA, indicating a lower affinity of pamiteplase for the t-PA receptor. 3. ke for pamiteplase measured using parenchymal cells or non-parenchymal cells was slightly smaller than that for rt-PA, whereas kon for pamiteplase were much lower than that of rt-PA, suggesting that the interaction between pamiteplase and the receptor is slower than that of rt-PA because of its structural modification. 4. Therefore, the difference in drug disposition between pamiteplase and rt-PA is mainly due to the difference in the hepatic clearance caused by a change in the interaction rate between the ligand and its cell-surface receptor.

Animals↗
PubMed2000

Determination, pharmacokinetics and protein binding of a novel tissue-type plasminogen activator, pamiteplase in human plasma.

1. An enzyme-linked immunosorbent assay (ELISA) and functional bioassay to determine immunoreactive and bioactive concentrations of pamiteplase, a novel thrombolytic agent, in human plasma were developed. The ELISA and functional bioassay showed satisfactory accuracy and precision within a concentration range of 0.5-25 ng.ml(-1) and of 0.127-16.2 ng.ml(-1) respectively. 2. The pharmacokinetics of pamiteplase in healthy human subjects were evaluated using the ELISA and functional bioassay. Irrespective of the method used, plasma concentrations declined bi-exponentially. Half-lives in the beta phase were 1.25 and 0.78 h, and AUCs were 507.9 and 286.4 ng.h.ml(-1) respectively. Total clearances of pamiteplase decreased to 19 and 31% of those of the wild-type tissue-type plasminogen activator. 3. The protein binding of pamiteplase in human plasma was investigated by gel filtration chromatography. Pamiteplase formed three high molecular weight complexes with alpha2-macroglobulin, C1-esterase inhibitor and alpha2-plasmin inhibitor in human plasma. This complex formation was relatively slow, and was thought to be irreversible and covalently bounded. Furthermore, this protein binding in humans resulted in the termination of biological action.

Adult↗
PubMed2000

Comparison of anthocyanin distribution in different blueberry sources by capillary zone electrophoresis.

Capillary zone electrophoretic separation of blueberry anthocyanins was studied using a Na-borate buffer containing trans-1,2-diaminocyclohexane-N,N,N',N'-tetra acetic acid monohydrate (CyDTA) as the carrier buffer. The separation conditions were precisely examined using an aqueous extract of bilberry (wild type blueberry) as the separation sample which is rich in this type and amount of anthocyanins. Each separated peak was identified by comparing the mobility with that of anthocyanin standards after normalization against the mobility of malvidin 3-o-glucoside (Mv 3-Glc) added as an internal standard. As salt concentrations of the running buffer increased, the peak resolution was markedly improved over the whole range of separation, especially, among the fast moving components (petunidin 3-glucoside, cyanidin 3-glucoside and malvidin 3-galactoside). Inversely, the peak separation both between petunidin 3-glucoside and peonidin 3-glucoside, and between delphinidin 3-glucoside and petunidin 3-galactoside, respectively, were decreased. The anthocyanins were, however, successfully separated by decreasing the buffer pH. Good separation of anthocyanins was finally achieved by 30 mM Na-borate (pH 8.78) containing 7.5 mM CyDTA within 10 min. Under this separation condition, anthocyanins from different blueberry sources were analyzed. The results revealed that different blueberry sources had their own patterns of anthocyanin distribution and amounts in the extracts, thus the present method is suitable for the quality control of anthocyanin-containing food materials.

Anthocyanins↗
PubMed2000

Comparison of drug disposition between wild-type and novel tissue-type plasminogen activator pamiteplase in rats.

The pharmacokinetics of pamiteplase in rats was compared with the pharmacokinetics of recombinant wild-type tissue-type plasminogen activator (rwt-PA). The half-life in the beta-phase and total clearance after administration of (125)I-labeled pamiteplase ((125)I-pamiteplase) to rats were 480 and 22% of those of (125)I-labeled rwt-PA ((125)I-rwt-PA), respectively. The amount of radioactivity distributed in the liver after administration of (125)I-pamiteplase was lower than that of (125)I-rwt-PA; consequently, a possible difference in metabolism between the drugs was assessed by an integration plot and a tissue-sampling single-injection technique. Use of these two methods revealed that the hepatic clearances of both compounds accounted for almost all of the total clearance and also revealed that the hepatic clearance of (125)I-pamiteplase was markedly lower than that of (125)I-rwt-PA. Therefore, the lower distribution of pamiteplase in the liver compared with rwt-PA is thought to contribute greatly to the higher plasma concentration of pamiteplase. Additionally, the uptake of (125)I-pamiteplase in the liver was inhibited by rwt-PA, suggesting that there is a common uptake mechanism for both compounds.

Animals↗
PubMed2000

In vivo imaging of increased oxidative stress in the liver by electron spin resonance-computed tomography.

The aim of this study was to investigate whether increased hepatic oxidative stress could be visualised in living animals before the onset of obvious liver injury. Acute hepatic injury was induced in mice by priming with heat-killed Corynebacterium parvum followed by injection of a low dose of lipopolysaccharide (LPS). Low frequency band electron spin resonance-computed tomography (ESR-CT) with 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was used to visualize hepatic oxidative stress. Biochemical and histological investigations performed 3 h after injection of LPS revealed no obvious injury to the liver. Conversely, significant hepatic oxidative stress could be detected at this time. Nitroxides such as carbamoyl-PROXYL are rapidly reduced to the corresponding hydroxylamine in vivo. resulting in the disappearance of their ESR signals. The kinetic clearance of carbamoyl-PROXYL after intravenous administration was delayed significantly in mice that had received LPS, due to impairment of the reduction system by hepatic oxidative stress. ESR-CT of the murine abdomen revealed a high intensity area of carbamoyl-PROXYL which consisted mainly of the liver and enlarged spleen. Time-course observations with ESR-CT using carbamoyl-PROXYL showed that the high intensity area in the liver disappeared rapidly due to reduction of carbamoyl-PROXYL. Three hours after LPS injection into the same mouse, ESR-CT images were obtained again by intravenous injection of carbamoyl-PROXYL. The ESR-CT images of the mouse with hepatic oxidative stress clearly showed that the high intensity area of carbamoyl-PROXYL in the liver persisted for a long period of time. This study is the first report to describe the use of in vivo ESR-CT for visualizing the state of increased oxidative stress in the liver before the onset of obvious hepatic injury.

Animals↗
PubMed1999

The FinO repressor of bacterial conjugation contains two RNA binding regions.

Conjugative transfer of F-like plasmids in Escherichia coli is repressed by a plasmid-encoded protein, FinO. FinO blocks the translation of TraJ, a positive activator of transcription of genes required for conjugation. FinO binds a traJ antisense RNA, FinP, thereby protecting it from degradation, and catalyzes FinP-traJ mRNA hybridization. Interactions between these two RNAs are predicted to block the traJ ribosomal binding site. In this paper, we use limited proteolysis, circular dichroism spectroscopy, and an electrophoretic mobility shift assay to map the regions within FinO that are required for interactions with RNA. Our results show that FinO is largely helical, binds to its highest affinity binding site within FinP as a monomer, and contains two distinct RNA binding regions, one of which is localized between residues 26 and 61, and a second which is localized between residues 62 and 186.

Amino Acid Sequence↗
PubMed1999

Fragility of the electron transport chain and superoxide generation in mitochondria of the liver graft after cold ischemia.

BACKGROUND: After cold ischemia, electrons transferred in the electron transport chain may leak out of the mitochondria in proportion to the deterioration of mitochondrial oxidative phosphorylation. This seems to be one major cause of the lipid peroxidation that occurs mainly in the hepatocytes at reperfusion in liver transplantation. To examine this hypothesis, we investigated superoxide generation and the amount of oxidative phosphorylation in the mitochondria isolated from rat livers after cold preservation. METHODS: Rat liver was preserved in University of Wisconsin solution at 4 degrees C for 24 hr. The mitochondrial fraction was prepared, and the amount of ATP synthesis and superoxide generation was investigated. Superoxide generation in the electron transport chain of submitochondrial particles was also measured by a chemiluminescence recorder. RESULTS: The amount of ATP synthesis was significantly decreased after 12 hr of cold preservation. In the whole mitochondria, superoxide production in the presence of succinate was approximately 1/2000 to 1/3000 less than that observed in the submitochondrial particles at any determination point, and superoxide production was not affected by cold preservation. In the presence of antimycin A, superoxide production in the mitochondria after 18 hr of preservation increased significantly. CONCLUSION: These results indicate that the electron transfer in the complex III of the mitochondrial membrane becomes leaky after long periods of cold ischemia, but that leakage of superoxide anion did not increase, although the mitochondrial respiratory phosphorylation was deteriorated. We conclude that superoxide through the mitochondrial membrane cannot cause lipid peroxidation in hepatocytes at reperfusion even after a long period of cold ischemia.

Adenosine Triphosphate↗
PubMed1999

Ca2+ regulation of interactions between endoplasmic reticulum chaperones.

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.

Animals↗
PubMed1999

Negative U-wave as a predictor of antihypertensive treatment effect on regression of echocardiographic hypertrophy in hypertensive patients.

To clarify the mechanism and the predictors of the reduction in left ventricular mass (LVM) induced by antihypertensive drugs, forty hypertensive patients were classified according to the presence of left ventricular hypertrophy (LVH) as a defined by echocardiographic LVM findings (LVH group: 27 patients, non-LVH group: 13 patients) and according to the presence of negative U-waves (NU) (NU group: 10 patients, non-NU group: 30 patients). Negative U-waves appeared in the LVH group only (10 of 27 patients). The hemodynamic determinants were investigated as a mechanism of LVM reduction in 38 of these patients who were treated for 2 years with antihypertensive drugs. In the LVH group, thickness of interventricular septum (IVST), posterior wall thickness (PWT) and LVM increased significantly compared to the non-LVH group. In the NU group, the left ventricular end-diastolic dimension (LVDD) was significantly larger and the relative wall thickness was significantly smaller compared to the non-NU group in the LVH group, with no difference in LVM between the two groups. Negative U-waves disappeared in all cases after treatment. Significant decreases in LVDD and LV wall thickness were observed in the NU group and significant decrease in LV wall thickness in the non-NU group. LVM index was reduced by 24.0% in the NU group and 9.5% in the non-NU group. The disappearance of negative U-waves was an independent predictor of the reduction of LVH.

Adult↗
PubMed1998

Spatiotemporal measurement of free radical elimination in the abdomen using an in vivo ESR-CT imaging system.

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Abdomen↗
PubMed1998

Two types of differentially photo-regulated nuclear genes that encode sigma factors for chloroplast RNA polymerase in the red alga Cyanidium caldarium strain RK-1.

A nuclear gene, sigA, that encodes a sigma factor for chloroplast RNA polymerase has previously been identified and characterized in the primitive red alga Cyanidium caldarium strain RK-1. Southern hybridization analysis indicated the presence of two additional sigma factor genes, which have now been cloned and shown to encode virtually identical proteins that are homologous to eubacterial sigma factors. These genes, which are also present in the nuclear genome, have therefore been named sigB and sigC. The substantial sequence similarity of sigB and sigC to sigA of the same strain as well as to cyanobacterial principal sigma factors and other chloroplast sigma factors strongly suggests that the nuclear genome of C. caldarium contains three genes that encode two types of chloroplast sigma factors. Each of the three recombinant Sig proteins showed sigma factor activity in vitro when combined with the Escherichia coli RNA polymerase core enzyme. Northern blot analysis revealed that, whereas the overall abundance of sigA transcripts was not affected by light, the amount of sigB and sigC mRNAs was greater in the light than in the dark. Thus, multiple sigma factors appear to contribute to light-regulated gene expression in the chloroplast.

Amino Acid Sequence↗