Increased plasma glutamic acid in a genetic model of epilepsy

A significant increase in the plasma levels of glutamic acid and a significant decrease in aspartic acid and taurine in epileptic patients and their first degree relatives was reported more than a decade ago and an underlying genetic basis for these amino acid changes was suggested. The main objective of the present study was to determine the plasma levels of glutamic acid, aspartic acid and taurine in El mice which are an inbred epileptic mutant mouse strain. The results show a significant increase in plasma glutamic acid but no changes in aspartic acid or taurine in the epileptic mice as compared to controls. The data provide the first evidence of a significant increase in plasma glutamic acid in an animal model of hereditary epilepsy and substantiate the hypothesis that a genetic defect underlies the elevated plasma glutamic acid levels in association with epilepsy. The findings are also compatible with neurochemical and neurophysiological evidence implicating glutamic acid in the mechanism of seizures.     

Acid increases NHE8 surface expression and activity in NRK cells

We previously demonstrated that there is a paucity of brush-border membrane NHE3 in neonates, the predominant Na(+)/H(+) exchanger in the adult proximal tubule, while NHE8 is relatively highly expressed in neonates compared with adults. We recently showed that metabolic acidosis in neonatal rodents can increase brush-border membrane NHE8 protein expression and Na(+)/H(+) exchange activity. To further examine the regulation of NHE8 by acid, we incubated NRK cells, which express NHE8 but not NHE3, with either acid or control media (6.6 vs. 7.4). There was an increase in Na(+)/H(+) exchanger activity within 6 h of incubation with acid media assessed as the rate of sodium-dependent recovery of pH from an acid load (dpH(i)/dt). The acid stimulation persisted for at least 24 h. The increase in Na(+)/H(+) exchange activity was paralleled by an increase in surface expression of NHE8, assessed by surface biotinylation and streptavidin precipitation. The increase in both apical membrane NHE8 protein expression and Na(+)/H(+) exchange activity with pH 6.6 media compared with 7.4 media was not affected by actinomycin D or cycloheximide consistent with an increase in surface expression independent of mRNA or protein synthesis. Furthermore, there was no increase in total cellular NHE8 protein abundance or mRNA abundance with acid media. Finally, we demonstrate that the increase in surface expression of NHE8 with acid media was blocked by colchicine and cytochalasin D and mediated by acid increasing the rate of exocytosis. In conclusion, NHE8 surface expression and activity are regulated by acid media by increasing the rate of trafficking to the apical membrane.     

Exogenous isoleucine and fatty acid shortening ensure the high content of anteiso-C15:0 fatty acid required for low-temperature growth of Listeria monocytogenes

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C15:0 solely at the expense of anteiso-C17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10 degrees C. Under this condition, the increase of anteiso-C15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain alpha-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of beta-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C15:0 at the expense of anteiso-C17:0.     

Regulation of fatty acid oxidation and triglyceride and phospholipid metabolism by hypolipidemic sulfur-substituted fatty acid analogues

The mechanisms behind the hypotriglyceridemic effect of 1,10-bis(carboxymethylthio)decane (3-thiadicarboxylic acid) and tetradecylthioacetic acid and the development of fatty liver caused by 3-tetradecylthiopropionic acid (Aarsland et al. 1989. J. Lipid Res. 30: 1711-1718.) were studied in the rat. Repeated administration of S-substituted non-beta-oxidizable fatty acid analogues to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids, and free fatty acids. This was accompanied by an acute reduction in the liver content of triglycerides and an increase in the hepatic concentration of phospholipids. Mitochondrial fatty acid oxidation was stimulated, whereas lipogenesis was inhibited. The activity of phosphatidate phosphohydrolase decreased while the activity of CTP:phosphocholine cytidylyltransferase increased. These results suggest that the observed triglyceride-lowering effect was due to increased mitochondrial fatty acid oxidation accompanied by a reduction in the availability of the substrate i.e., free fatty acid, along with an enzymatic inhibition (phosphatidate phosphohydrolase). Administration of 3-tetradecylthiopropionic acid led to a drastic increase in the hepatic triglyceride content. Levels of plasma triglyceride phospholipid and free fatty acid also increased. Phosphatidate phosphohydrolase activity was stimulated whereas CTP:phosphocholine cytidylyltransferase was inhibited. Mitochondrial fatty acid oxidation was decreased. These data indicate that the development of fatty liver as an effect of 3-tetradecylpropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by an increase in the availability of fatty acid along with stimulation of phosphatidate phosphohydrolase. The results of the present study speak strongly in favor of the hypothesis that phosphatidate phosphohydrolase is a major rate-limiting enzyme in triglyceride biosynthesis. Furthermore, they point out that the biosynthesis of triglycerides and phospholipids might be coordinately regulated. Such regulation is possibly mediated via phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase. Whether the increase in hepatic phospholipids via increased CDP-pathway accounts for an increase of lipid components for proliferation of peroxisomes (3-thiadicarboxylic acid and tetradecylacetic acid) should be considered.     

Regulation by dietary choline of hepatic fatty acid synthetase in the rat

Fatty acid synthetase activity was measured in the high-speed supernatant fraction of liver homogenates from rats fed a semisynthetic diet low in lipotropic factors. If choline was omitted from the diet, a significant increase of fatty acid synthetase activity was observed after two feedings of the deficient diet. Compared with controls, the increase of fatty acid synthetase activity was of a magnitude that could account for the amount of triglyceride accumulating in the hepatic floating lipid fraction. Gas-liquid chromatographic analysis of the floating lipid triglycerides showed an increased content of palmitic acid due to choline deficiency; this increase could be predicted from the increased fatty acid synthetase activity and its known characteristic yield of palmitic acid.     

The mechanism of estrogen-induced increase in hyaluronic acid biosynthesis,with special reference to estrogen receptor in the mouse skin

The increase in hyaluronic acid and water contents induced by estradiol treatment in the mouse skin was dependent on dose and the number of treatments of estradiol. The anti-estrogen administered together with estradiol blocked the increase in hyaluronic acid content produced by treatment with estradiol alone. It was suggested that the anti-estrogen may act as an antagonist by competing for the cytoplasmic estrogen receptor on increase in hyaluronic acid synthesis. It was observed that the sensitivity in increase in hyaluronic acid biosynthesis by estradiol was related to the age of the mouse and the content of the cytoplasmic estrogen receptor in the mouse skin.It was suggested that there was a possible relationship between the increase in hyaluronic acid and cytoplasmic estrogen receptor in the mouse skin.     

Interdependence of growth, water relations and abscisic acid level in Phaseolus vulgaris during waterlogging

The interdependence between changes in growth and water relations after waterlogging was investigated by recording simultaneously growth, transpiration, water potential, turgor, leaf diffusion resistance and abscisic acid content in Phaseolus vulgaris L. cv. bruine Noord-Hollandse. Growth was inhibited immediately after flooding, whereas transpiration decreased gradually to a low level in about three days. The first two days after flooding a small increase in abscisic acid content in the leaves was observed which was accompanied by an increase in diffusion resistance. The increase in abscisic acid content could result from an inhibited export from the leaves. After the first two days a decrease in water potential and turgor was accompanied by a drastic increase in both abscisic acid content and diffusion resistance. This large increase in abscisic acid content occurred before the turgor had reached its minimum value. The change in diffusion resistance kept showing a lag of about one day with the change in abscisic acid content. The possibility is discussed that besides abscisic acid also its metabolite phaseic acid is involved in stomatal closure. After the formation of adventitious roots on the hypocotyl, abscisic acid level, diffusion resistance, water potential and turgor returned to the control values. Transpiration showed a slow recovery from the sixth day after flooding, whereas growth was inhibited for at least nine days. A remarkable similarity exists between our observations on the responses of bean plants to flooding and the well known responses to drought.     

l-Valine biosynthesis during batch and fed-batch cultivations of Corynebacterium glutamicum: Relationship between changes in bacterial growth rate and intracellular metabolism

A transition in the bacterial growth rate to below maximum was found to be an optimum parameter of cellular physiology to increase the activity of acetohydroxy acid synthase, a regulatory enzyme in l-valine synthesis, and amino acid overproduction by Corynebacterium glutamicum ATCC 13032 recombinants under batch and fed-batch cultivation conditions. An increase in l-valine synthesis under transient situations when cellular growth rate was downregulated was correlated to a decrease in the activity of aconitase, a key enzyme in the tricarboxylic acid cycle (TCA) of C. glutamicum, and, in contrast, to an increase in the activity of glucose-6-phosphate dehydrogenase, a key enzyme in the pentose phosphate pathway (PPP). The increase in amino acid synthesis was also directly related to a drastic increase in intracellular pyruvate concentration. Thus, an increase in intracellular pyruvate availability and NADPH₂ generation by PPP could be the metabolic origins of the increased l-valine overproduction by growth restrained C. glutamicum cell culture.     

The effect of immunization with protein-hapten conjugate on the intestinal uptake of p-aminobenzoic acid as a hapten

Intestinal uptake of p-aminobenzoic acid was examined by means of an in vitro everted sac technique in rats immunized with ovalbumin-p-aminobenzoic acid conjugate. A dose-dependent and antigen-specific decrease in the serosal transfer of p-aminobenzoic acid was observed in rats immunized 6 times with protein-hapten conjugate compared with the control. There was a significant increase in the recovery of p-acetamidobenzoic acid, a metabolite of p-aminobenzoic acid, in mucosal fluid, tissue, and serosal fluid in the jejunum. In the case of ileum, increase of p-acetamidobenzoic acid was observed in mucosal fluid. However, there was no significant effect in the ileal p-acetamidobenzoic acid in tissue and serosal fluid between immunized and non-immunized rats. To examine the increased metabolism of immunized rats, N-acetyltransferase activity of the small intestinal mucosa was examined. There was a significant increase in mucosal N-acetyltransferase activity in immunized rats compared with the control animals. These observations suggested that the mucosal immune system may play an important role in regulating the intestinal uptake of the low molecular weight compounds.     

Delta6 desaturase inhibition : a novel mode of action of norflurazone

The herbicide Norflurazone was shown to be an inhibitor of fatty acid of the Δ6 desaturation system. Treating cultures of the microalgae Spirulina platensis or Monodus subterraneus with this herbicide brought about a reduction in the level of γ-linolenic acid and in an increase in the level of linoleic acid. In Monodus, the increase in linoleic acid made it more available for ω3 desaturation, resulting in an increase in the proportion of eicosapentaenoic acid. The proportion of arachidonic acid did not decrease albeit the drastic decrease in its precursor γ-linolenic acid.     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).     

Effect of fatty acids on the synthesis and secretion of apolipoprotein B by rat hepatocytes

The modulation of apolipoprotein B synthesis and secretion by fatty acids in rat hepatocytes was studied. Maximum apolipoprotein B production was obtained in the case of oleic acid followed by linoleic, stearic and palmitic/linolenic acid when compared to control which was not supplemented with any fatty acids. Oleic acid was found to exert a concentration dependent increase in the secretion of [³H] apolipoprotein B into the medium while that associated with the cell layer was not affected. Pulse chase experiments in the presence of oleic acid showed that it caused an increase in the secretion of apolipoprotein B into the medium.¹⁴C-acetate incorporation into cholesterol and cholesteryl ester associated with the cell layer and secreted very low density lipoproteins also showed an increase in the presence of oleic acid indicating an increase in cholesterogenesis. The effect of oleic acid on [³H] apolipoprotein B and very low density lipoproteins secretion appeared to be mediated through cholesterol as (i) ketoconazole, an inhibitor of cholesterol synthesis caused significant reduction in the stimulatory effect of oleic acid on apolipoprotein secretion and (ii) mevinolin, another inhibitor of cholesterol synthesis also reversed the stimulatory effect of oleic acid on apolipoprotein B secretion. These results indicated that oleic acid may influence apolipoprotein B synthesis and secretion in hepatocytes probably by affecting cholesterol/cholesteryl ester formation which may be a critical component in the secretion of apolipoprotein B as lipoproteins     

Amino acid transport in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

A study was made of the transport of a variety of amino acids by uninfected and Rous sarcoma virus-infected chicken embryo fibroblasts. Following a period of amino acid starvation, transformed, but not normal cells, showed increased levels of transport for alpha-aminoisobutyric acid, proline and alanine, three amino acids which are transported primarily by the A transport system. There was no starvation-induced increase in the transport of leucine, phenylalanine, lysine, or cycloleucine. In the absence of starvation, normal and transformed cells exhibited comparable rates of amino acid transport. Cycloheximide was able to block the increase in uptake. The enhanced uptake was characterized by an increase in Vmax for transport and little change in Km. The data demonstrate that an alteration in the regulation of the A amino acid transport system is an early event in malignant transformation by Rous sarcoma virus. However, since this alteration in made manifest only following a period of starvation, our findings suggest that increased amino acid uptake does not play a role in generating the other manifestations of the transformed state seen in cell culture.     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.     

Arachidonic acid stimulates 45calcium efflux and hPL release in isolated trophoblast cells

Previous investigations from this laboratory have indicated that arachidonic acid stimulates a rapid, dose-dependent and reversible increase in hPL release which is not dependent on cyclooxygenase or lipoxygenase metabolism. To investigate further the mechanism by which arachidonic acid stimulates the release of hPL, the effect of arachidonic acid on the release of 45Ca from perifused cells prelabelled with 45CaCl was examined in an enriched cell culture population of term human syncytiotrophoblast. Arachidonic acid (10-100 microM) stimulated a dose-dependent, rapid, and reversible increase in the release of both 45Ca and hPL from the perifused placental cells. On the other hand, palmitic acid had little effect on either hPL release or 45Ca release even at concentrations as high as 100 microM. Ionophore A23187 (1-10 microM) also stimulated a dose-dependent and reversible increase in hPL release. Since arachidonic acid increases the mobilization of cellular calcium, as reflected by the increased 45calcium efflux, and since an increase in the intracellular calcium concentration appears to stimulate an increase in hPL release, these results suggest that the stimulation of hPL release by arachidonic acid may be due, at lease in part, to the effects of the fatty acid on cellular calcium mobilization.     

Evaluating laccase-facilitated coupling of phenolic acids to high-yield kraft pulps

In an effort to alter the physical properties of high-yield kraft, fibers were treated at high consistency (20%) with laccase and syringic, vanillic, or 4-hydroxybenzoic acid. Treatment with laccase and 4-hydroxybenzoic acid resulted in a 20-point increase in kappa number and a 100% increase in bulk acid groups. ESCA analysis of the treated and untreated pulp revealed that the laccase-grafted fibers had a two-fold enrichment in acid groups, strongly suggesting a laccase-facilitated coupling of 4-hydroxybenzoic acid to the fiber surface. A model system consisting of lignin-coated cellulosic fibers was developed to determine changes to the lignin structure during laccase grafting. ³¹P NMR analysis of lignin from the model system revealed an increase in acid groups with a concomitant decrease in phenolic hydroxyl groups.     

Regulation of palmitoyl-CoA chain elongation and linoleoyl-CoA chain elongation in rat liver microsomes and the differential effects of peroxisome proliferators, insulin and thyroid hormone

Treatment of rats with p-chlorophenoxyisobutyric acid (clofibric acid), 2,2'-(decamethylenedithio)diethanol, di(2-ethylhexyl)phthalate or acetylsalicylic acid caused an increase in activity of palmitoyl-CoA chain elongation in hepatic microsomes. The activity of palmitoyl-CoA chain elongation decreased in both hypothyroid-state and diabetic-state rats, increased in hyperthyroid-state rats and did not change in adrenalectomized rats. The administration of clofibric acid to these rats in an altered hormonal state caused an increase in the activity of palmitoyl-CoA chain elongation, but no additional increase in the activity was observed with treatment of hyperthyroid rats with clofibric acid. The activity of linoleoyl-CoA chain elongation did not respond to the changes in either the nutritional conditions or the hormonal state of insulin so sensitively as the activity of palmitoyl-CoA chain elongation. The treatment of rats with triiodothyronine caused a marked increase in the activity of linoleoyl-CoA chain elongation; nevertheless, the activity of linoleoyl-CoA chain elongation was not changed by the treatment of rats with clofibric acid. The results suggest that rat liver microsomes contain at least two fatty acid chain elongation systems and that these chain elongation systems are regulated differently by hormones and drugs.     

The Role of Abscisic Acid in Flower Abscission of Lupinus luteus

The role of abscisic acid in the control of flower abscission in Lupinus luteus L. was examined. Using a modified extraction and purification technique, endogenous abscisic acid levels in the upper flowers of an inflorescence were found to increase markedly some days before abscission could be detected. When abscisic acid was injected into flower-bearing nodes or fed via the roots, no increase in the abscission rate was obtained at any position in the flowerhead. Application of abscisic acid to only the leaves resulted in a marked increase in flower abscission. The role of abscisic acid per se as a primary controlling factor of flower abscission in yellow lupin is questioned.