Metabolism of platelet-activating factor in primary cultured adult rat hepatocytes by a new pathway involving phospholipase C and alkyl monooxygenase

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) upon interaction with primary cultured adult rat hepatocytes was investigated. [3H]PAF-acether was transformed time-dependently into [3H]lyso-PAF-acether, 1-O-[3H]alkylglycerol and finally converted to 3H-labeled fatty aldehyde. 1-O-[3H]Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) was formed after a long incubation time and with a smaller amount compared with that formed in platelets and neutrophils. When lipids from cells, cell surfaces and incubation medium were analyzed separately, most of the transformed products of [3H]PAF-acether remained in the cells. When 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine was incubated with hepatocytes, it was mainly converted into 1-O-[3H]alkylglycerol. 3H-labeled fatty aldehyde and [3H]alkylacyl-GPC were also found. Hepatocytes metabolized slowly from 1-O-[1-14C]hexadecylglycerol to 3H-labeled fatty aldehyde and 3H-labeled phospholipid. These findings suggest that cultured hepatocytes mainly catabolize exogeneous PAF-acether by removing the acetyl residue and the polar head group and, finally, by cleaving an ether bond. The deacetylation-reacylation step, which is important in platelets and neutrophils, was not shown to be a main metabolic pathway of PAF-acether in cultured hepatocytes.     

The effect of high sodium concentration on the action potential of the skate heart

It already has been well documented that the maximum rate of depolarization and amplitude of action potentials are directly dependent on [Na+]o in the vertebrate myocardium. Almost all studies have been carried out at low sodium concentration ranges by substituting NaCl for other substances. Action potentials should be demonstrable in higher sodium concentrations, but cells are inevitably damaged by osmotic changes. The blood of elasmobranchs is nearly isosmotic with sea water, but NaCl accounts for 54.5% of the osmotic pressure and 38.7% of it is maintained by urea molecules. Utilizing this special situation in elasmobranchs, the effect of high sodium concentration was studied up to 170% of normal sodium concentration, while still retaining isosmotic condition. The rate of depolarization, amplitude, and duration of the myocardial action potential all increased in direct proportion to [Na+]o, and no depressant effect on transmembrane action potentials was observed in solutions of high sodium concentration. With regard to depolarization rate, the regression curve fitted by the least squares method passed through zero within two standard errors. At high sodium levels, the overshoot changed as expected theoretically, but at lower ranges it deviated from the theoretical values. [Na+]i and [K+]i in this tissue have been determined, and these data are explained on the basis of the Na theory.     

Effect of cholestanol feeding on sterol concentrations in the serum, liver, and cerebellum of mice

In order to elucidate the mechanism of xanthoma formation in cerebrotendinous xanthomatosis, mice were fed for 32 weeks with a diet rich in 5 alpha-cholestan-3 beta-ol (cholestanol) (1%, w/w). The concentrations of sterols in the serum, liver, and cerebellum were determined using high performance liquid chromatography. In the cholestanol-fed mice, the cholestanol concentrations in the serum and liver reached maxima in the first 2 to 4 weeks; the levels were about 30- to 100-fold higher than in the control diet mice. The cholestanol concentrations declined thereafter, finally to 50-60% of the maxima. Cholesterol concentrations were slightly lower in the cholestanol-fed mice throughout the experiments than in the control diet mice. On the other hand, the levels of cholestanol in the cerebellum increased almost linearly in parallel to the feeding time, and no decline was observed. These results suggest that the capacity of the liver to remove or degrade cholestanol was increased by long-term intake of this compound, whereas the cerebellum had no such feed-back regulation. Histological examinations using an electron microscope revealed the enlargement of lysosomal granules in the liver of the cholestanol-fed mice.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Insulin-induced tyrosine-phosphorylation in intact rat adipocytes

Insulin-induced tyrosine-phosphorylation in intact isolated rat adipocytes was studied using immunoblotting method with antiphosphotyrosine antibodies. Insulin-stimulated adipocytes were solubilized with Triton X-100. The lysate was incubated with wheat germ agglutinin, then with hydroxylapatite. Insulin stimulated tyrosine-phosphorylation of a 95 KDa protein which adsorbs to wheat germ agglutinin and appears to be the beta-subunit of the insulin receptor. Among the proteins adsorbed to hydroxylapatite, tyrosine-phosphorylation of 170 KDa and 60 KDa proteins was stimulated. 170 KDa was also stimulated by polyclonal anti-insulin receptor antibodies B-10 Ig G, IGF-I and H2O2. The detection of these proteins in rat adipocytes may lead to the elucidation of a common signal transduction pathway in insulin-responsive cells.     

Mg‐dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY‐GREEN (SGR) gene encodes Mg‐dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion‐mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F_v/F_m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light‐harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.     

Biodegradable nanogel formation of polylactide-grafted dextran copolymer in dilute aqueous solution and enhancement of its stability by stereocomplexation

Monodisperse stereocomplex nanogels were obtained through the self-assembly of an equimolar mixture of dextran-graft-poly(L-lactide) (Dex-g-PLLA) and dextran-graft-poly(D-lactide) (Dex-g-PDLA) amphiphilic copolymers with well-defined composition in a dilute aqueous solution. The stereocomplex nanogel possessed partially crystallized cores of hydrophobic polylactide (PLA) and the hydrophilic dextran skeleton by intra- and/or intermolecular self-assembly between PLLA and PDLA chains. The stereocomplex nanogels exhibited significantly lower critical aggregation concentration (CAC) value as well as stronger thermodynamic stability compared with those of the corresponding L- or D-isomer nanogels. The mean diameter of the stereocomplex nanogels was 70 nm with narrow size distribution, implying they were well-defined and presumably nanogels. Furthermore, stereocomplex nanogel exhibited strong kinetic stability. The tunable degradation properties of Dex-g-PLA nanogels were achieved by varying the number of grafted PLA chains as well as applying stereocomplexation. This study demonstrates the advantage of stereocomplexation in the design of biodegradable nanogels with enhanced stability.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.