Effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in isolated rat hepatocytes

α-Ketobutyrate, an intermediate in the catabolism of threonine and methionine, is metabolized to CO₂ and propionyl-CoA. Recent studies have suggested that propionyl-CoA may interfere with normal hepatic oxidative metabolism. Based on these observations, the present study examined the effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in hepatocytes isolated from fed rats. α-Ketobutyrate (10 mM) inhibited the oxidation of palmitic acid by 34%. In the presence of 10 mM carnitine, the inhibition of palmitic acid oxidation by α-ketobutyrate was reduced to 21%. These observations are similar to those previously reported using propionate as an inhibitor of fatty acid oxidation, suggesting that propionyl-CoA may be responsible for the inhibition. α-Ketobutyrate (10 mM) inhibited ¹⁴CO₂ generation from [¹⁴C]pyruvate by more than 75%. This inhibition was quantitatively larger than seen with equal concentrations of propionate. Carnitine (10 mM) had no effect on the inhibition of pyruvate oxidation by α-ketobutyrate despite the generation of large amounts of propionylcarnitine during the incubation. α-Ketobutyate inhibited [¹⁴C]glucose formation from [¹⁴C]pyruvate by more than 60%. This contrasted to a 30% inhibition caused by propionate. These results suggest that α-ketobutyrate inhibits hepatic pyruvate metabolism by a mechanism independent of propionyl-CoA formation. The present study demonstrates that tissue accumulation of α-ketobutyrate may lead to disruption of normal cellular metabolism. Additionally, the production of propionyl-CoA from α-ketobutyrate is associated with increased generation of propionylcarnitine. These observations provide further evidence that organic acid accumulation associated with a number of disease states may result in interference with normal hepatic metabolism and increased carnitine requirements.     

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.     

Peptide nucleic acid probe for protein affinity purification based on biotin–streptavidin interaction and peptide nucleic acid strand hybridization

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin–streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the “warhead”) and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl–PNA:PNA–biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.     

Effect of alpha-resorcylic acid–l-phenylalanine amide on collagen synthesis and matrix metalloproteinase expression in fibroblasts

Regulation of collagen synthesis and matrix metalloproteinases (MMPs) expression levels has been an important issue in medicinal, pharmaceutical and cosmetic industries. Herein, α-Resorcylic acid–phenylalanine amide (α-RA-F) was prepared and its biological activities were observed. We found that α-RA-F boosted collagen synthesis and reduced MMPs expression levels in human fibroblasts without cytotoxicity.     

Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, α-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes

Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [³H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A₂ and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A₂, cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

G protein and PLDδ are involved in JA to regulate osmotic stress responses in Arabidopsis thaliana

Jasmonic acid (JA) is regarded as an endogenous regulator which plays an important role in regulating plant growth, development and stress response. Using the seedlings of A. thaliana ecotype Col-0 (wild-type, WT), phospholipase Dδ (PLDδ) deficient mutant (pldδ), the G protein α subunit (GPA1) deficient mutant (gpa1-4), 9-Lipoxygenase (9-LOX) deficient mutants (lox1 and lox5) as materials, the effects of JA responding to osmotic stress and the functions of G protein and PLDδ in this response were investigated. The results showed that GPA1 involved in the regulation of JA to PLDδ under osmotic stress. Both GPA1 and PLDδ participated in the regulation of JA on the seed germination and osmotic tolerance. Exogenous MeJA reduced the EL and MDA in WT, but increased the EL and MDA in gpa1-4 and pldδ, indicating that GPA1 and PLDδ were involved in the protection of JA on the membrane. The genes expression levels, and the activities of PLDδ and LOX1 were significantly induced by osmotic stress. The LOX activity and JA content in pldδ seedings were lower obviously than those in WT, but were markedly increased and were higher than WT after applying phosphatidic acid (PA). These results demonstrated that JA responded to osmotic stress by regulating G protein and PLDδ in A. thaliana. PLDδ was located upstream of 9-LOX and involved in the JA biosynthesis.     

Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress?

Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain a relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine (N-trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.     

Effect of β-adrenergic agonist L-644,969 on the impact of the thermal environment on in vitro fatty acid synthesis and lipogenic enzymes in sheep

The effect of temperature and β-adrenergic agonist (BAA) on in vitro rates of fatty acid synthesis and catalytic activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) was examined in wether lambs after 5 weeks at either 0 or 20°C. Feeding BAA increased (P<0.05) rate of fatty acid synthesis by 38% in subcutaneous adipose (SC) tissue from cold-acclimated animals but the rate decreased (P<0.05) by 27% in SC tissue from warm-acclimated animals. In mesenteric fat (MS), BAA increased (P<0.05) fatty acid synthesis in the cold environment. In perirenal (PR) fat, rate of fatty acid synthesis was reduced (P<0.05) by 20% by BAA in the warm but had no effect in the cold. Activity of ACC in longissimus muscle was depressed (P<0.05) when BAA was fed in the warm environment. In adipose tissues BAA reduced (P<0.05) ACC activity in the warm, but reduced activity in the cold was limited to SC tissue. In PR tissue FAS activity was reduced (P<0.05) in the cold environment, while BAA increased FAS activity in the warm environment. Western blot analysis showed two isoforms of ACC with MW of 280 000 and 265 000 Da in longissimus muscle whereas only one isoform was recognized in each of Biceps femoris (280 000 Da) and adipose tissues (265 000 Da). Feeding BAA in the cold environment reduced (P<0.05) ACC and FAS immunoprotein expression in both MS and PR adipose tissues. The studies indicate that the effect of BAA on fatty acid synthesis and lipogenic enzymes is influenced by acclimation temperature.     

Effect of UV-C irradiation on mesophyll protoplasts of Cucumis sativus

In the present work we studied the effect of UV-C irradiation on short-term protoplast physiology, with the aim of identifying and assessing parameters which can provide valuable information for asymmetric fusion experiments. Protoplast viability, cell wall regeneration, density of cell suspension and intensity of DAPI signal were followed by using microscopy and by the detection of specific fluorescent or spectroscopic signals in a microplate reader. The control and irradiated mesophyll protoplasts of Cucumis sativus were used for this experiment. In contrast to control cells, viability of irradiated cells significantly decreased. Intensive cell wall regeneration was observed only in control cells, which also showed significantly higher DAPI fluorescence signal. Microscopy for determination of viability by FDA and cell wall regeneration by Calcofluor White were modified for microplate reader instrumentation. These methods are simple, fast and suitable for detection of the effectiveness of UV-C irradiation of cells intended to be used in asymmetric fusion experiments.     

Effect of “restricted ovulator” gene on uptake of yolk-precursor protein

Summary The bulk of the yolk proteins and lipoproteins constituting the yolks of mature oocytes in birds are synthesized by the liver and transported via the plasma to the oocytes where they are incorporated by micropinocytosis. Evidence is presented indicating that oocytes of hens possessing a mutation identified by Jones, Briles, and Schjeide as a restricted ovulator gene fail to incorporate normal amounts and proportions of low density lipoproteins, lipovitellin and possibly other proteins making up the bulk of the yolk material. Plasma albumin is taken into the yolks but the other proteins synthesized by the liver for deposition within the oocytes accumulate in the plasma, attaining very high levels. The possible nature of the lock preventing normal deposition of the excluded yolk proteins is discussed.     

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells

Summary Protoplasts were enzymatically isolated from Paul's scarlet rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was greater when the protoplasts were cultured in medium plus 12% sucrose than with 24% sucrose. Budding was observed. In medium plus 12% sucrose about 45% of the protoplasts divided but in medium plus 24% sucrose far fewer divided. Cytokinesis was abnormal: the phragmoplast disappeared soon after cytokinesis began and the cell plate became a groove and then a fibril-lined or filled tongue which progressed across the vacuole, unconnected by strands to other parts of the protoplast. The wall regenerated after several days culture in medium plus 12% sucrose fluoresced with calcofluor. The wall regenerated in medium with 24% sucrose fluoresced usually only after several weeks culture. Cytokinesis hastened formation of a wall fluorescing with calcofluor. In the electron microscope the wall was seen to contain fibrils and non-fibrillar material. The latter was the minor component in medium plus 12% sucrose but was usually the major component in medium plus 24% sucrose. The growth in plasmolysing and nonplasmolysing medium of the cells from which protoplasts are isolated was also studied.It appears that loss of the wall alters the potential of protoplasts to expand and possibly also to regenerate a wall and to divide. Wall regeneration is initially linked with expansion and cytokinesis. Osmotic pressure of the external medium is also an important factor.This work formed part of a thesis by one of us (R.S.P.) approved for the degree of Ph. D. in the University of Nottingham. The work was supported by the Agricultural Research Council.     

Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments

Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.     

Effect of cyclopentene ��,�¡�-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.     

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.     

Effect of theβ-adrenoceptor agonist BRL 26830 on fatty acid synthesis and on the activities of pyruvate dehydrogenase and acetyl-CoA carboxylase in adipose tissues of the rat

BRL 26830 is a thermogenic-adrenoceptor agonist which stimulates lipolysis and fatty acid oxidationin vivo. It also stimulates insulin secretion, and hence promotes glucose utilisationin vivo. The effect of this agent on white and brown adipose tissue of the rat was investigated. BRL 26830 increased the rate of fatty acid synthesisin vivo in white adipose tissue by 135% but reduced the rate of fatty acid synthesisin vivo in brown adipose tissue by 78%. The increase was abolished in white adipose tissue of streptozotocin-diabetic rats, indicating that the effect involved a rise in circulating insulin levels. The reduction in fatty acid synthesis in brown adipose tissues was associated with a reduction in the activity of acetyl-CoA carboxylase in the tissue consistent with a direct-adrenoceptor-mediated effect. BRL 26830 also increased the proportion of pyruvate dehydrogenase in its active formin vivo in brown adipose tissue and this increase was abolished in streptozotocin-diabetic rats. These findings illustrate different sensitivities of white and brown adipose tissues to combined-adrenergic and insulin stimulation.     

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[¹⁴C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[¹⁴C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.     

Comment on ‘Thermodynamic database supports deciphering protein–nucleic acid interactions’

Mei and colleagues reported a thermodynamic database, PNATDB for protein-nucleic acid interactions, which contains 12 635 experimentally determined thermodynamic parameters. They claimed that extracting data from existing databases is difficult. ProNAB, which has more than 20 000 experimental data points for binding affinities of protein–nucleic acid complexes and other information, was not discussed.     

Effect of tranexamic acid and δ-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or δ-aminovaleric acid (δ-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or δ-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1.5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1.4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.