Kouichi Katayama

Involvement of tumor necrosis factor-α in the pathogenesis of activated macrophage-mediated hepatitis in mice

The possible involvement of tumor necrosis factor-alpha in the pathogenesis of an experimentally induced hepatitis was investigated. Balb/c mice were primed with Propionibacterium acnes to induce the infiltration of mononuclear cells into the liver. Immunohistochemical study showed that most of the accumulated mononuclear cells at 7 days were Mac-2 positive, suggesting that they were activated macrophages. An injection of lipopolysaccharide resulted in massive hepatic necrosis and high mortality in the mice within 24 hours. Plasma tumor necrosis factor-alpha activity initially rose sharply and then declined over 3 hours. The increase in plasma aminotransferase activity correlated well with the elevation of plasma tumor necrosis factor-alpha activity. Pretreatment with dexamethasone or 16,16-dimethyl-prostaglandin E2 attenuated not only the elevation of plasma tumor necrosis factor-alpha activity but also the increase in plasma aminotransferase activity and improved the survival rate. Passive immunization against tumor necrosis factor-alpha showed protective effects. These findings suggest that tumor necrosis factor-alpha released from activated macrophages may play a crucial role in the pathogenesis of this murine hepatitis.

Simultaneous determination of reduced and oxidized ubiquinones

Publisher Summary Quantitative analyses of individual ubiquinones (Q) homologs in biological samples have been performed by high-performance liquid chromatography (HPLC) combined with an ultraviolet spectrometric detector (UVD) or by mass spectrometry (MS). An electrochemical detector (ECD) for HPLC was confirmed to be simple and sensitive for the determination of Q. However, only Q was determined by these methods. For the determination of QH 2 (ubiquinol) and Q in mitochondria, submitochondrial particle and cell-free bacterial homogenates, the dual-wavelength spectrometric method has been generally used. The method, however, cannot simultaneously measure the amounts of QH 2 and Q in whole tissues owing to the presence of vitamin A and other interfering compounds, which have an absorbance in the same spectral region as Q and undergo an absorption change by chemical reduction. The dual-wavelength spectrometric method cannot separately determine individual Q homologs. The analytical procedure described was developed to provide a rapid, sensitive, and direct assay method for QH 2 and Q in biological samples. This method is based on extraction from tissues, mitochondria, microsomal fractions, or plasma with organic solvents, followed by quantitation by means of reversed-phase chromatography with UVD and ECD.

Increased lung uptake of liposomes coated with polysaccharides.

Liposomes labeled with [14C]coenzyme Q10 in the lipid bilayer were coated with various polysaccharide derivatives, i.e., palmitoyl conjugates of pullulan, pullulan phosphate, amylopectin, amylopectin phosphate and amylopectin sulfate. The kinetics of disposition and the tissue distribution of [14C]coenzyme Q10 after intravenous injection of the liposomes into guinea pigs were investigated. Lung uptake of radioactivity after injection of the O-palmitoyl amylopectin- and O-palmitoyl amylopectin phosphate-coated liposomes was 5- and 3-times higher, respectively, at 30 min after injection than that of the conventional liposomes. For doubly labeled liposomes with [3H]inulin and [14C]coenzyme Q10, the 3H/14C ratios in the lung, spleen and heart were similar to one another. Urinary excretion of [3H]inulin encapsulated in O-palmitoyl amylopectin-coated liposomes was much lower than that of unencapsulated [3H]inulin. These observations suggest that the O-palmitoyl amylopectin-coated liposomes are rather stable in vivo and are taken up into tissues in the intact form.

Effect of Azelastine on the Release and Action of Leukotriene C4 and D4

The effect of azelastine on the release of leukotriene C4 and D4 (LTC4 and LTD4), and the antagonistic action of the drug against the leukotrienes were determined by using in vitro tests and compared with those of ketotifen and chlorpheniramine. Azelastine inhibited LTC4 and LTD4 release from guinea pig lung fragments passively sensitized with homologous anti-ovalbumin IgGl-b antibody. The 50% inhibitory concentration (IC50) of azelastine was 6.4 X 10(-5) M for a 15-min preincubation or 4.7 X 10(-5) M for a 30-min preincubation. Ketotifen and chlorpheniramine were inhibitory only at the highest concentration tested (3 X 10(-4) M), giving inhibitions of 35.6 and 21.3%, respectively. Azelastine also inhibited calcium ionophore A23187-induced release of leukotrienes from human polymorphonuclear leukocytes; the IC50 values were 3.6 X 10(-5) M for 15 min and 2.3 X 10(-6) M for 30 min of preincubation. Ketotifen and chlorpheniramine were inhibitory only after a 30-min preincubation, with IC50 values of 2.1 X 10(-5) and 5.9 X 10(-5) M, respectively. The potent inhibition by azelastine might be partly a result of the inhibition of 5-lipoxygenase, since 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemia cell homogenate was inhibited by azelastine. Pretreatment of guinea pig ileum with azelastine antagonized LTC4- and LTD4-induced contraction of the ileum with IC50 values of 7.0 X 10(-6) and 1.1 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptotic cell death of cultured cerebral cortical neurons induced by withdrawal of astroglial trophic support

Peripheral neurons which depend on NGF for their survival undergo apoptosis after NGF deprivation. However, a convenient in vitro method for assessing the programmed cell death of the central neurons has not been established, because the dependence of particular central neurons on neurotrophic factors has been clarified only for small populations of neurons. Based on the fact that cortical neurons survive in culture for many weeks in the presence of astroglial cells, we have established an in vitro cell death model in which the neurons die through apoptosis. Cortical neurons were maintained on a cover slip for 1 week on top of astroglial cells, and then cell death was induced by separation of the neurons from the astroglial cells. The cortical neurons died within 2-4 days. Nuclei of the dying neurons showed the morphological features of apoptosis, and DNA fragmentation was observed by the TUNEL method and by in situ nick translation (ISNT) staining. The cell death was significantly suppressed by neurotrophic factors, NT-3, NT-4, BDNF, and GDNF, but not by NGF. The neuronal survival was prolonged, as in the case of peripheral neurons, by bFGF, elevated potassium, cAMP, forskolin, and metabotropic glutamate receptor agonist. The cell death was inhibited by inhibitors of interleukin-1 beta-converting enzyme and CPP32. CPP32-like proteolytic activity was increased prior to the appearance of apoptotic cells. These results suggest that cortical neurons die after separation from glial cells through apoptosis caused by deprivation of neurotrophic factors produced by the astroglial cells.

Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria, microsomes and plasma by high-performance liquid chromatography.

Reduced and oxidized coenzyme Q10 (Q10H2 and Q10) in guinea-pig liver mitochondria were rapidly extracted and determined by high-performance liquid chromatography (HPLC). The percentages of Q10H2 as compared to the total (sum of Q10 and Q10H2) were increased by the addition of respiratory substrates such as succinate, malate and beta-hydroxybutyrate (State 4). The levels of Q10H2 in State 4 were increased more extensively with electron-transport inhibitors such as KCN, NaN3 and antimycin A. These results indicate that the method for determining Q10H2 and Q10 by HPLC is quite useful for investigation of the physiological function of coenzyme Q in mitochondria and other organelles. The reduced and oxidized coenzyme Q levels of rat liver mitochondria, which contain both coenzyme Q9 and coenzyme Q10, were measured simultaneously. The results suggest that coenzymes Q9 and Q10 play a similar role as an electron carriers. The liver microsomes of guinea-pig contained approx. 133 nmol total coenzyme Q10 per g protein. The Q10H2 levels of microsomes were increased from 46.5 to 67.5 and 64.8% with NADH and NADPH, respectively. The plasma levels of total coenzyme Q were 0.92 microgram/ml for man, 0.35 microgram/ml for guinea-pig and 0.27 microgram/ml for rat. The reduced coenzyme Q were also present in those plasma samples. The levels of reduced coenzyme Q were 51.1, 48.9 and 65.3%, respectively.

Involvement of P-type calcium channels in high potassium-elicited release of neurotransmitters from rat brain slices

Several types of voltage-dependent calcium channels appear to occur in neurons, although coupling of the particular subtype of calcium channels to the release of neurotransmitter has not been clearly understood. We have examined the effects of subtype-specific inhibitors of the calcium channels on depolarization-induced release of endogenous neurotransmitters from brain slices. High potassium-induced release of glutamate and aspartate from hippocampal and striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from the hippocampal slices with almost the same potency as in the case of glutamate, whereas the potency in blocking the release of serotonin and dopamine from striatal slices was lower than that from the hippocampal slices. Another calcium channel blocker, omega-agatoxin TK, that was recently found to block P-type channels with very similar selectivity and potency to omega-agatoxin IVA, also inhibited the release of amino acid transmitters and monoamines, though its potency was lower than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA, partially inhibited the neurotransmitter release, but an L-type channel blocker, nifedipine was ineffective. We propose that the activation of P-type calcium channels makes a major contribution to depolarization-elicited neurotransmitter release in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and omega-agatoxin TK modulate the neurotransmitter release.

An improved method for the determination of γ-carboxyglutamic acid in proteins, bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem. 117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.

Simultaneous determination of ubiquinone-10 and ubiquinol-10 in tissues and mitochondria by high performance liquid chromatography

Abstract A method of high performance liquid chromatography with both of a UV detector and an electrochemical detector for the simultaneous determination of ubiquinone and ubiquinol was established. This method could sensitively and specifically measure the redox state of ubiquinone in mitochondria and tissues.

High-performance liquid chromatographic method for the determination of dolichols in tissues and plasma

High-performance liquid chromatographic methods for the determination of dolichols in tissues and plasma have been developed. The tissue concentration of dolichols was measured by high-performance liquid chromatography with uv detection and plasma levels of dolichols were determined fluorometrically after derivatization with anthracene-9-carboxylic acid. In both methods, 2,2-didecaprenylethanol was used as an internal standard. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichols in human plasma.