Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser ²²⁵ and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A ₂ and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.     

Complexation of retroviruses with charged polymers enhances gene transfer by increasing the rate that viruses are delivered to cells

BACKGROUND: We have previously found that retrovirus transduction is enhanced when an anionic polymer (chondroitin sulfate C) is added to virus stocks that contain an equal weight concentration of a cationic polymer (Polybrene). This observation was unexpected given that previous work has shown that cationic polymers enhance transduction while anionic polymers have the opposite effect. METHODS: Using model recombinant retroviruses and lentiviruses that encode for the Escherichia coli lacZ gene and quantitative assays of virus adsorption and transduction, we examined the mechanism of enhancement. RESULTS: We found that addition of oppositely charged polymers (Polybrene and chondroitin sulfate C) to virus stocks enhanced gene transfer by increasing the flux of active viruses to the cells. Virus-polymer complexes formed that did not reduce the stability of the viruses, yet were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion. The size of the complexes, the rate of sedimentation, and the levels of gene transfer increased with increasing concentrations of polymers. The degree to which transduction was enhanced ranged from 2- to nearly 40-fold, and varied depending on the type of cells and viruses used. Interestingly, we found that association of the viruses with the polymer complexes did not significantly hinder their ability to complete post-binding steps of transduction. CONCLUSIONS: Complexation of retroviruses with charged polymers significantly improves the efficiency of ex vivo gene transfer by increasing the number of active viruses that reach the cells.     

Induction of epidermal proliferation and expressions of PKC and NF-kappaB by betel quid extracts in mouse: the role of lime-piper additives in betel quid

Components of betel quid (BQ) have been investigated for genotoxicity, mutagenicity, and animal toxicity. However, little information exists regarding their carcinogenic characteristics. Considerable attention has already been focused on tumor promoters that occur environmentally for human uptake. In this study, the promoting effects of BQ and lime-piper additives (LPA) in BQ on epidermal hyperplasia in CD-1 mouse skin are investigated. In the present study, we found that BQ and LPA at concentrations of 25,50,75 mg/ml caused significant induction of hyperplasia, but only LPA caused an increase of epidermal ornithine decarboxylase (ODC). Treatment of mouse skin with LPA caused remarkable increases in the production of H(2)O(2) by 2.41-, 3.90-, and 3.76-fold (for the above-indicated concentrations respectively); as well as marked increases of myeloperoxidase (MPO) by 1.43-, 2.70-, and 2.29-fold. Application of LPA or BQ (50,100,150 mg/ml) also caused induction of protein kinase C-alpha (PKC-alpha) and NF-kappaB. LPA exhibited more significant effect than BQ. Thus, LPA might make a major contribution to the BQ-induced expression of PKC and NF-kappaB. These results indicated that BQ has the potential of being promoting agents, and that LPA should play a major role in increasing the effects of BQ-caused skin hyperplasia and inflammation. The promoting effects of BQ and LPA on mouse skin were associated with the induction of the expressions of PKC and NF-kappaB.     

Effects of supplementation of rumen-protected choline on growth performance,meat quality and gene expression in longissimus dorsi muscle of lambs

This study determined the effects of rumen-protected choline (RPC) on growth performance, blood lipids, meat quality and expression of genes involved in fatty-acid metabolism in young lambs. A total of 24 Dorper × Hu lambs (about 20 kg body weight) were kept in individual pens and fed diets with 0%, 0.25%, 0.50% and 0.75% RPC for 60 d. Supplementation of 0.25% RPC increased average daily gain of lambs, whereas treatments had no significant effect on feed intake. The pH values of meat were increased at 0.25% RPC and both, dripping loss and shear force of meat, were significantly decreased in RPC-supplemented lambs. No significant changes were observed for dressing percentage and intramuscular fat. RPC supplementations had no significant effect on the concentrations of triglycerides and cholesterols in serum, but the concentration of high-density lipoprotein was decreased at 0.50% RPC and that of low-density lipoprotein was increased at 0.75% RPC. In m. longissimus dorsi, the expressions of cluster of differentiation 36 (CD36), acetyl-CoA carboxylase (ACC) and fatty-acid synthase (FASN) genes were increased at 0.25% RPC. Supplementation of 0.75% RPC increased the expressions of lipoprotein lipase (LPL) and FASN genes, decreased the expression of ACC gene and had no effect on CD36 gene. The results of this study showed that supplementation of 0.25% RPC could promote growth performance of lambs and improve meat quality. This may be mediated by effects on blood lipid profiles and the metabolism of fatty acids in skeleton muscles. However, the beneficial effects of 0.25% RPC supplementation need to be validated with a larger number of animals. Higher doses, particularly 0.75% RPC, showed adverse effects on live weight gain and ACC expression.     

The effect of vitamin E and plant extract mixture composed of carvacrol, cinnamaldehyde and capsaicin on oxidative stress induced by high PUFA load in young pigs

The objective of our study was to determine the antioxidative potential of a plant extract (PE) mixture composed of carvacrol, capsicum oleoresin and cinnamaldehyde against high n-3 polyunsaturated fatty acid (PUFA)-induced oxidative stress in young pigs. Thirty-two weaned castrated male crossbred pigs (BW 10.9 kg; n = 32) were randomly assigned to four dietary treatments (n = 8). The negative control diet (Cont) contained 17.2% energy from fat. Oxidative stress was induced in three of the four experimental groups with the inclusion of n-3 PUFA rich linseed oil. Linseed oil substituted wheat starch in the diet to elevate the amount of energy from fat to 34.1%. One of these diets served as a positive control (Oil), one was additionally supplemented with 271.2 mg/kg of PE mixture and one with 90.4 mg/kg α-tocopheryl acetate (Vit E). After 14 days of treatment, blood and urine were collected for the determination of lipid peroxidation and DNA damage. Lipid peroxidation was studied by plasma malondialdehyde (MDA) concentrations, 24 h urinary MDA and F2-isoprostane (iPF2α-VI) excretion, total antioxidant status of plasma and glutathione peroxidase assays. Lymphocyte DNA fragmentation and 24 h urinary 8-hydroxy-2'-deoxyguanosine excretion were measured to determine DNA damage. Consumption of n-3 PUFA rich linseed oil increased the amount of MDA in plasma and urine, and induced DNA damage in lymphocytes, but did not elevate the amount of iPF2α-VI excreted in the urine. The supplementation with PE and with Vit E did not reduce MDA levels in plasma and urine, but it decreased the percentage of DNA damage in lymphocytes (P < 0.001). The PE reduced the urinary iPF2α-VI excretion in comparison to the Cont diet. The results show that PE and Vit E supplemented to pigs in concentrations of 271.2 mg/kg and 90.4 mg/kg, respectively, can effectively protect pig's blood lymphocytes against oxidative DNA damage, thus suggesting their potentially beneficial effects on the immune system under dietary-induced oxidative stress.     

Effect of Basic and Acidic Additives on the Separation of Some Basic Drug Enantiomers on Polysaccharide‐Based Chiral Columns With Acetonitrile as Mobile Phase

The separation of enantiomers of 16 basic drugs was studied using polysaccharide‐based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate‐based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several β‐blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase. Chirality 27:228–234, 2015. © 2015 Wiley Periodicals, Inc.     

Effects of PFOS and PFOA on L-type Ca2+ currents in guinea-pig ventricular myocytes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are amphiphiles found ubiquitously in the environment, including wildlife and humans, and are known to have toxic effects on physiological functions of various tissues. We investigated the effects of PFOS and PFOA on action potentials and L-type Ca(2+) currents, I(CaL), in isolated guinea-pig ventricular myocytes using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the rate of spike, action potential duration, and peak potential at doses over 10 microM. In voltage-clamp experiments, PFOS increased the voltage-activated peak amplitude of I(CaL), and shifted the half-activation and inactivation voltages of I(CaL) to hyperpolarization. PFOA had similar effects PFOS, but showed significantly lower potency. These findings are consistent with previous observations for anionic n-alkyl surfactants, suggesting that PFOS and PFOA may change membrane surface potential, thereby eliciting general effects on calcium channels. These findings provide further insights into the mechanisms of PFOA and PFOS toxicities.     

脂溶性维生素的安全性

脂溶性维生素是很重要的营养素,在食品或饲料业中作为添加剂,使用范围广泛。这类维生素可在体内贮存,缺乏或过量都会影响机体生长和代谢功能。本文综述脂溶性维生素的生理功能与来源、缺乏与过量时的危害,以及添加时的注意事项,以保证其使用的安全性。