Increased carbonylation of the lipid phosphatase PTEN contributes to Akt2 activation in a murine model of early alcohol-induced steatosis

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is a key event in the pathogenesis of alcoholic liver disease (ALD), which ranges from simple steatosis to fibrosis. The lipid phosphatase PTEN plays a central role in the regulation of lipid metabolism in the liver. In this study, the effects of chronic ethanol feeding and carbonylation on the PTEN signaling pathway were examined in a 9-week mouse feeding model for ALD. Chronic ethanol consumption resulted in altered redox homeostasis as evidenced by decreased GSH, decreased Trx1, and increased GST activity. Both PTEN expression and PTEN phosphorylation were significantly increased in the livers of ethanol-fed mice. Carbonylation of PTEN increased significantly in the ethanol-fed mice compared to pair-fed control animals, corresponding to decreased PTEN 3-phosphatase activity. Concomitantly, increased expression of Akt2 along with increased Akt phosphorylation at residues Thr³⁰⁸, Thr⁴⁵⁰, and Ser⁴⁷³ was observed resulting in increased Akt2 activity in the ethanol-fed animals. Akt2 activation corresponded to a decrease in cytosolic SREBP and ChREBP. Subsequent LC/MS/MS analysis of 4-HNE-modified recombinant human PTEN identified Michael addition adducts of 4-HNE on Cys⁷¹, Cys¹³⁶, Lys¹⁴⁷, Lys²²³, Cys²⁵⁰, Lys²⁵⁴, Lys³¹³, Lys³²⁷, and Lys³⁴⁴. Computational-based molecular modeling analysis of 4-HNE adducted to Cys⁷¹ near the active site and Lys³²⁷ in the C2 domain of PTEN suggested inhibition of enzyme catalysis via either stearic hindrance of the active-site pocket or prevention of C2 domain-dependent PTEN function. We hypothesize that 4-HNE-mediated PTEN inhibition contributes to the observed activation of Akt2, suggesting a possible novel mechanism of lipid accumulation in response to increased reactive aldehyde production during chronic ethanol administration in mice.     

Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

The use of the iron chelating agent desferrioxamine in rice cell cryopreservation: a novel approach for improving recovery

The iron chelating drug, desferrioxamine is used to suppress oxidative stress in mammalian transplant organs subjected to cold storage. The efficacy of desferrioxamine in improving post-thaw survival in cryopreserved cells from two rice culture lines was evaluated. Unfrozen rice cells maintained proliferation capacity over a fifteen day time course when exposed to concentrations of desferrioxamine > 10 mg·l⁻¹. Albeit, growth was reduced compared to controls. Short-term applications of the drug at concentrations of 0.5 and 10 mg·l⁻¹ before cryopreservation and during the early post-thaw period had a positive affect on recovery as assessed by cell proliferation and triphenyl tetrazolium chloride reduction capabilities. The pharmaceutical properties of desferrioxamine are attributed to iron sequestration and the prevention of harmful Fenton and free radical chemistry. However, desferrioxamine did not significantly reduce lipid peroxidation in cryopreserved rice cells.     

Control of membrane polar lipid composition in Acholeplasma laidlawii a by the extent of saturated fatty acid synthesis

The low level of endogenous fatty acid synthesis in Acholeplasma laidlawii A strain EF22 was found to be caused by a deficiency of pantetheine in the lipid-depleted growth medium. By supplementing the oleic acid-containing medium with increasing concentrations of pantetheine, saturated fatty acid synthesis was stimulated (having an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 5 μM for pantetheine) and the incorporation of endogenously synthesized fatty acids in membrane lipids increased markedly. Furthermore, carotenoid biosynthesis was stimulated. Exogenous palmitic acid was found to inhibit partially the endogenous fatty acid synthesis. A gradual stimulation of fatty acid synthesis was accompanied by a linear increase in the molar proportion between the two dominating membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The total amount of charged membrane lipids decreased upon increasing the degree of fatty acid saturation. These regulations are discussed in terms of membrane stability, and influence of membrane molecular ordering and surface charge density on lipid polar head group synthesis.     

The developmentally regulated shift from membrane to secreted mu mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency.

To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.     

结合反相高效液相色谱和酶联免疫吸附法测定昆虫血淋巴中的蜕皮甾醇激素

【目的】昆虫体内的蜕皮酮和20羟基蜕皮酮(20E)是两种主要的蜕皮甾醇激素,其中蜕皮酮是20E的前体,而20E是有活性的蜕皮甾醇激素。本研究旨在建立一种测定昆虫血淋巴中蜕皮激素高效、稳定、准确的方法。【方法】采集家蚕血淋巴,抽提总蜕皮甾醇激素,利用反相高效液相色谱法(反相HPLC)分离并收集蜕皮酮与20E,进而利用酶联免疫吸附法(ELISA)测定家蚕血淋巴的总蜕皮甾醇激素、蜕皮酮和20E各自的滴度。【结果】计算出总蜕皮甾醇激素中蜕皮酮与20E的比例,推算出蜕皮酮合成和分泌、以及蜕皮酮转化为20E的能力。【结论】该方法高效、稳定、准确,可广泛应用于昆虫蜕皮甾醇激素滴度的测定。     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

The enhancement of glucose uptake caused by the collapse of gap junction communication is due to an increase in astrocyte proliferation

We have previously shown that several gap junction uncouplers increase the uptake of glucose in astrocytes. The aim of the present work was to study whether the increase in glucose uptake was a consequence of the inhibition of gap junction communication and the purpose of this effect. Our results show that alpha-glycyrrhetinic acid and endothelin-1 increase the uptake of glucose in highly, but not in poorly, coupled astrocytes. This effect depended on connexin 43 levels and was abolished when the inhibition of gap junction communication was prevented by tolbutamide or ouabain. The inhibition of gap junctions increased the rate of glucose incorporation into DNA and RNA, which was inhibited by treatment with dehydroepiandrosterone, an inhibitor of glucose-6-phosphate dehydrogenase, the regulatory enzyme of the pentose phosphate pathway. The inhibition of gap junctions significantly increased astrocyte proliferation, which was counteracted by tolbutamide. These effects were not observed in poorly coupled astrocytes expressing low levels of connexin 43. The increase in astrocyte proliferation caused by gap junction inhibition was prevented when either glucose uptake or the pentose phosphate pathway were inhibited. We conclude that the inhibition of gap junction communication induces astrocyte proliferation, resulting in an enhancement of glucose uptake and its utilization through the pentose phosphate pathway to provide ribose-5-phosphate for the synthesis of nucleic acids.     

The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.     

The proper localization of RESPONSIVE TO DESICCATION 20 in lipid droplets depends on their biogenesis induced by STRESS-RELATED PROTEINS in vegetative tissues

Arabidopsis LD surface proteins, SRPs are found only in higher plants and are important for LD biogenesis and abiotic stress signaling. However, the cellular mechanism of SRPs is still unclear. To investigate molecular functions of SRPs, we used tobacco transient expression system. Transient expression of SRPs was sufficient and synergistic for LD biogenesis, and SRPs participated in the formation step of LD in tobacco leaves. RESPONSIVE TO DESICCATION 20 (RD20), a known LD-localizing peroxygenase, localized to LD in the presence of an SRP, and its peroxygenase activity correlated with proper localization of RD20 to LD. Our data suggest that Arabidopsis SRPs play roles as positive factors for LD biogenesis to provide a proper localization of LD-localizing proteins in vegetative tissues.     

Mixture design as a potential tool in modeling the effect of light wavelength on Dunaliella salina cultivation: an alternative solution to increase microalgae lipid productivity for biodiesel production

Abstract

For a feasible microalgae biodiesel, increasing lipid productivity is a key parameter. An important cultivation parameter is light wavelength (λ). It can affect microalgal growth, lipid yield, and fatty acid composition. In the current study, the mixture design was used as an alternative to model the influence of the λ on the Dunaliella salina lipid productivity. The illumination was considered to be the mixture of different λ (the light colors blue, red, and green). All experiments were performed with and without sodium acetate (4?g/L), as carbon source, allowing the identification of the impact of the cultivation regimen (autotrophic or mixotrophic). Without sodium acetate, the highest lipid productivity was obtained using blue and red light. The use of mixotrophic cultivations significantly enhanced the results. The optimum obtained result was mixotrophic cultivation under 65% blue and 35% green light, resulting in biomass productivity of 105.06 mgL^?1day^?1, a lipid productivity of 53.47 mgL^?1day^?1, and lipid content of 50.89%. The main fatty acids of the oil obtained in this cultivation were oleic acid (36.52%) and palmitic acid (18.31%).     

The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress

Aims

Exposure to hyperbaric oxygen (HBO₂) causes an antinociceptive response in mice. However, breathing oxygen (O₂) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO₂ antinociception and the toxicity profile of HBO₂.

Main methods

Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O₂ at 1.0 or 3.5 atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to 3.5 ATA oxygen for 11 min, 60 min, and 60 min daily for 2 days (120 min) or 60 min daily for 4 days (240 min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung.

Key findings

Only the combination of 100% O₂ and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O₂ was pressure-dependent up to 3.5 ATA. In the oxygen toxicity test, mice exposed to HBO₂ for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air.

Significance

Treatment with 100% O₂ evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded that HBO₂ at 3.5 ATA produces antinociception in the absence of oxidative stress in mice.     

The shift of an increase in phosphofructokinase activity from protein synthesis-dependent to -independent mode during concanavalin A induced lymphocyte proliferation

Phosphofructokinase activity increased dramatically in cultured mouse spleen lymphocytes 8 hours after concanavalin A stimulation and preceded the onset of DNA synthesis by 8 hours. The increase in enzyme activity and [³H]-thymidine incorporation were mitogen-concentration dependent. Enzyme activity increased 12-fold over control level at 48 hours when DNA synthesis peaked. The protein synthesis inhibitor, cycloheximide, blocked the rise in phosphofructokinase only when given prior to the increase in enzyme activity. Once the increase began, later addition of cycloheximide became progressively less inhibitory. These observations suggest that the period of increase in phosphofructokinase activity involves the activation of preexisting enzyme molecules.     

The formation of helical tubular vesicles by binary monolayers containing a nickel-chelating lipid and phosphoinositides in the presence of basic polypeptides

Binary lipid monolayers consisting of equimolar proportions of a phosphoinositide and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by myelin basic protein (MBP). Another basic polypeptide, poly-L-lysine, had a similar effect but not to as great a degree as MBP; the proteins thus appeared to act as polycations. Although, the nickel-chelating lipid is a synthetic product, other endogenous divalent cations such as Zn(2+), as well as phosphoinositides, are integral and dynamic components of the myelin sheath in vivo. There, comparable helical tubular structures might represent a means for sequestration of these lipids into domains of high local concentration, perhaps in regions where the membrane is greatly curved.     

Effect of a negative energy balance induced by feed restriction in lactating sows on hepatic lipid metabolism,milk production and development of litters

In rodents, forced activation of hepatic peroxisome proliferator-activated receptor α (PPARα) by administration of exogenous PPARα activators during lactation leads to a reduction of milk triacylglycerol (TAG) production. Herein, we investigated whether a negative energy balance (NEB) induced by feed restriction (about 18% lower feed and energy intake) during lactation by increasing the release of fatty acids, which act as PPARα agonists, causes a disruption of hepatic lipid metabolism and thereby impairs milk TAG production in sows. Nutrient and energy content of the milk on day 20 of lactation and gains of litters during the first 14 d and the whole 21 d suckling period did not differ between Control and feed-restricted sows. The mRNA concentrations of several sterol regulatory element-binding protein target genes involved in lipid synthesis in the liver and the plasma concentration of TAG were reduced in the feed-restricted sows, whereas the mRNA concentrations of PPARα target genes involved in fatty acid oxidation in liver and skeletal muscle were not different between groups. In conclusion, it was shown that an NEB during lactation does not adversely affect milk composition and gains of litters, despite inhibiting hepatic expression of genes involved in lipid synthesis and reducing plasma TAG concentration. The finding that PPARα target genes involved in fatty acid utilisation in liver and muscle of sows are not induced by the NEB during lactation may explain that fatty acid availability in the mammary gland is sufficient to maintain milk TAG production and to allow normal litter gain.