A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis.

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L-1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice.

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

Localized Heat Therapy Improves Mitochondrial Respiratory Capacity but Not Fatty Acid Oxidation.

AIM: Mild heat stress can improve mitochondrial respiratory capacity in skeletal muscle. However, long-term heat interventions are scarce, and the effects of heat therapy need to be understood in the context of the adaptations which follow the more complex combination of stimuli from exercise training. The purpose of this work was to compare the effects of 6 weeks of localized heat therapy on human skeletal muscle mitochondria to single-leg interval training. METHODS: Thirty-five subjects were assigned to receive sham therapy, short-wave diathermy heat therapy, or single-leg interval exercise training, localized to the quadriceps muscles of the right leg. All interventions took place 3 times per week. Muscle biopsies were performed at baseline, and after 3 and 6 weeks of intervention. Mitochondrial respiratory capacity was assessed on permeabilized muscle fibers via high-resolution respirometry. RESULTS: The primary finding of this work was that heat therapy and exercise training significantly improved mitochondrial respiratory capacity by 24.8 ± 6.2% and 27.9 ± 8.7%, respectively (p < 0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p < 0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. CONCLUSION: Six weeks of muscle-localized heat therapy significantly improves mitochondrial respiratory capacity, comparable to exercise training. However, unlike exercise, heat does not improve fatty acid oxidation capacity.

Mechanistic assessment of tolerance to iron deficiency mediated by Trichoderma harzianum in soybean roots.

AIMS: Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. METHODS AND RESULTS: Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2 •- ) and hydrogen peroxide (H2 O2 ) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. CONCLUSIONS: TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.

N-acetylcysteine supplementation did not reverse mitochondrial oxidative stress, apoptosis, and inflammation in the salivary glands of hyperglycemic rats.

BACKGROUND/OBJECTIVES: Previous studies have shown that N-acetylcysteine (NAC) supplementation with the simultaneous inclusion of HFD prevents salivary glands from oxidative stress and mitochondrial dysfunction. In this experiment, we examined if NAC supplementation could reverse the harmful effect of HFD on mitochondrial function, reduce the severity of apoptosis, and the activity of pro-oxidative enzymes in the salivary glands of rats with confirmed hyperglycemia. SUBJECTS/METHODS: Wistar rats were fed the standard or high-fat (HFD) diet for 10 weeks. After 6 weeks of the experiment, HFD rats were diagnosed with hyperglycemia and for the next 4 weeks, the animals were given NAC intragastrically. In the mitochondrial fraction of the parotid (PG) and submandibular salivary glands (SMG), we assessed redox status, inflammation, and apoptosis. RESULTS: The inclusion of NAC increased the activity of mitochondrial complexes I and II + III as well as decreased the concentration of interleukin-1ß, tumor necrosis factor α, and caspase-3, but only in the parotid glands of rats with hyperglycemia compared to the HFD group. However, N-acetylcysteine supplementation did not reduce the activity of caspase-9 or the Bax/Bcl-2 ratio in PG and SMG mitochondria. In both salivary glands we observed reduced activity of cytochrome c oxidase, NADPH oxidase, and xanthine oxidase, as well as hindered production of ROS and lower ADP/ATP radio, but the levels of these parameters were not comparable to the control group. CONCLUSIONS: We demonstrated that NAC supplementation restores the glutathione ratio only in the mitochondria of the submandibular salivary glands. The supply of NAC did not significantly affect the other measured parameters. Our results indicate that NAC supplementation provides little protection against free radicals, apoptosis, and inflammation in the salivary gland mitochondria of HFD rats. Stimulated salivary secretion in hyperglycaemic rats supplemented with NAC seems to be closely related to mitochondrial respiratory capacity and appropriate ATP level.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

Anti-Inflammatory Effects Induced by Near-Infrared Light Irradiation through M2 Macrophage Polarization.

Near-infrared (NIR) can penetrate the dermis. NIR is able to regulate cutaneous component cells and immune cells and shows significant anti-inflammatory therapeutic effects. However, the mechanisms of these effects are largely unknown. The purpose of this study is to elucidate NIR-induced molecular mechanisms on macrophages because macrophages play initial roles in directing immune responses by their M1 or M2 polarizations. Proteomic analysis revealed that NIR radiation enhanced the expression of mitochondrial respiratory gene citrate synthase. This increased citrate synthase expression was triggered by NIR-induced H3K4 hypermethylation on the citrate synthase gene promoter but not by heat, which led to macrophage M2 polarization and finally resulted in TGFß1 release from CD4+ cells. These cellular effects were validated in human primary macrophages and abdominal NIR-irradiated mouse experiments. In a phorbol 12-myristate 13-acetate‒induced inflammatory model on mouse ear, we confirmed that NIR irradiation induced significant anti-inflammatory effects through decreased M1 counts, reduced TNF-α, and increased CCL22 and/or TGFß1 levels.

Preventive effects of medium-chain triglycerides supplementation on the oxidative capacity in skeletal muscle under cachectic condition.

Cachexia is a multifactorial condition characterized by muscle mass loss and induces metabolic dysfunction of the skeletal muscles. The preventive effects of medium-chain triglycerides (MCT) supplementation on the oxidative capacity in skeletal muscle under cachectic condition were investigated in the present study. ICR mice were randomly divided into four groups; control, lipopolysaccharide (LPS), LPS plus long-chain triglycerides (LCT) and LPS plus MCT supplementation. LCT and MCT oil were administered to the LPS + LCT and LPS + MCT groups orally (5.0 g/kg body weight/day) by a catheter for one week. Cachexia was induced in the LPS, LPS + LCT, and LPS + MCT groups via LPS injection (7.5 mg/kg body weight, i.p.) after the supplementation. LPS induced a reduction of ketone bodies concentration in blood plasma. LPS also induced a decrease in succinate dehydrogenase activity and PGC-1α expression level in tibialis anterior muscles. Meanwhile, MCT supplementation suppressed a decrease in ketone bodies concentration and succinate dehydrogenase activity. In addition, MCT supplementation increased the level of citrate synthase activity in the muscles. These results suggested the preventive effect of MCT supplementation on oxidative capacity in skeletal muscle and the involvements of ketone bodies regulation under cachectic condition.

Leucine improves α-amylase secretion through the general secretory signaling pathway in pancreatic acinar cells of dairy calves.

The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased (P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.

Tricarboxylic acid cycle dehydrogenases inhibition by naringenin: experimental and molecular modelling evidence.

The study of polyphenols' effects on health has been gaining attention lately. In addition to reacting with important enzymes, altering the cell metabolism, these substances can present either positive or negative metabolic alterations depending on their consumption levels. Naringenin, a citrus flavonoid, already presents diverse metabolic effects. The objective of this work was to evaluate the effect of maternal naringenin supplementation during pregnancy on the tricarboxylic acid cycle activity in offspring's cerebellum. Adult female Wistar rats were divided into two groups: (1) vehicle (1 ml/kg by oral administration (p.o.)) or (2) naringenin (50 mg/kg p.o.). The offspring were euthanised at 7th day of life, and the cerebellum was dissected to analyse citrate synthase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities. Molecular docking used SwissDock web server and FORECASTER Suite, and the proposed binding pose image was created on UCSF Chimera. Data were analysed by Student's t test. Naringenin supplementation during pregnancy significantly inhibited IDH, α-KGDH and MDH activities in offspring's cerebellum. A similar reduction was observed in vitro, using purified α-KGDH and MDH, subjected to pre-incubation with naringenin. Docking simulations demonstrated that naringenin possibly interacts with dehydrogenases in the substrate and cofactor binding sites, inhibiting their function. Naringenin administration during pregnancy may affect cerebellar development and must be evaluated with caution by pregnant women and their physicians.

Ellagic acid promotes browning of white adipose tissues in high-fat diet-induced obesity in rats through suppressing white adipocyte maintaining genes.

Promoting brown adipose tissue (BAT) formation and function reduces obesity. Ellagic Acid (EA), located abundantly in plant extracts and fruits, has been shown to modulate formation and differentiation of adipocytes, although its role in the process of browning of white adipose tissue (WAT) has not been elucidated. In this study, fifty-six five-week old SD rats were randomly assigned to receive normal diet (ND, 10% lipids) or high-fat diet (HFD, 60% lipid) with or without various dosages of EA for 24 weeks. Our results showed that high fat diet intake triggered overweight, glucose intolerance and white adipocyte hypertrophy, the effects of which were mitigated by EA treatment. Meanwhile, EA supplementation reduced serum resistin levels, improved hepatic steatosis and serum lipid profile in DIO (high fat diet induced obesity) rats. Moreover, EA supplementation significantly decreased mRNA expression of Zfp423 and Aldh1a1, the key determinants of WAT plasticity. EA also increased mRNA expression of brown adipocyte markers including UCP1, PRDM16, Cidea, PGC1α, Ppar-α; beige markers including CD137and TMEM26; mitochondrial biogenesis markers including TFAM in inguinal WAT (iWAT) when compared to their counterparts. EA treatment significantly improved mitochondrial function, as measured by citrate synthase activity. More importantly, EA markedly elevated the expression of UCP1 in iWAT, which is a specific protein of brown adipocyte. In conclusion, our results provided evidence that EA improved obesity-induced dyslipidemia and hepatic steatosis in DIO rats via browning of iWAT through suppressing white adipocyte maintaining genes and promoting expression of key thermogenic genes. These findings suggest that EA could be a promising therapeutic avenue to treat metabolic diseases.

Hydrangea serrata Tea Enhances Running Endurance and Skeletal Muscle Mass.

SCOPE: Skeletal muscle mass and quality can be negatively affected by aging, inactivity, and disease, while a loss of muscle mass is associated with chronic disease status, falls, and mortality. We investigate the effects of Hydrangea serrata on skeletal muscle mass and function, along with the underlying mechanisms. METHODS AND RESULTS: H. serrata, identified through MyoD transcription activity screening, increases myogenic differentiation via Akt and p38. C57BL/6 mice are fed a 0.25% or 0.5% H. serrata diet for 8 weeks. H. serrata increased treadmill running distance and maximum speed, as well as skeletal muscle mass. H. serrata promotes the expression of myosin heavy chain 1 (MHC1) and MHC2A but not MHC2B. H. serrata also upregulates the protein expression of peroxisome proliferator-activated receptor δ (PPARδ) and mitochondrial complexes, and enhances citrate synthase and mitochondrial complex І activity. Transforming growth factor-ß (TGF-ß), myostatin, and growth differentiation factor 11 (GDF11) are attenuated by H. serrata, together with associated downstream signaling factors including phospho-Smad3 and NADPH oxidase 4 (NOX4). CONCLUSION: H. serrata enhances exercise endurance by upregulating PPARδ and downregulating TGF-ß, myostatin, and GDF11. H. serrata is a potential candidate for the development of functional food to maintain skeletal muscle mass and function.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia.

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα-/-) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O2) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα-/- mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Homeologue Specific Gene Expression Analysis of Two Vital Carbon Metabolizing Enzymes-Citrate Synthase and NADP-Isocitrate Dehydrogenase-from Wheat (Triticum aestivum L.) Under Nitrogen Stress : Homeologue Specific gene expression of CS and NADP-ICDH.

Citrate synthase (CS) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) have been considered as candidate enzymes to provide carbon skeletons for nitrogen assimilation, i.e., production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The CS and NADP-ICDH cDNAs were encoded for polypeptides of 402 and 480 amino acids with an estimated molecular weight of 53.01 and 45 kDa and an isoelectric point of 9.08 and 5.98, respectively. Phylogenetic analysis of these proteins in wheat across kingdoms confirmed the close relationship with Aegilops tauschii and Hordeum vulgare. Further, their amino acid sequences were demonstrated to have some conserved motifs such as Mg2+ or Mn2 binding site, catalytic sites, NADP binding sites, and active sites. In-silico-identified genomic sequences for the three homeologues A, B, and Dof CS and NADP-ICDH were found to be located on long arm of chromosomes 5 and 3, and sequence analysis also revealed that the three homeologues consisted of 13 and 15 exons, respectively. The total expression analysis indicated that both genes are ubiquitously expressed in shoot and root tissues under chronic as well as transient nitrogen stress. However, they are differentially and contrastingly expressed but almost in a coordinated manner in both the tissues. Under chronic as well as transient stress, both the genes in shoot tissue showed downregulation, lowest at 6 h of transient stress. However, in the root tissue, trend was found opposite except with exceptions. Moreover, all the three homeologues of both the genes were transcribed differentially, and the ratio of the individual homeologues transcripts to total homeologues transcripts also varied with the tissue, i.e., shoots or roots, as well as with nitrogen stress treatments. Thus, cDNA as well as genomic sequence information, apparent expression at different time point of nitrogen stress, and coordination between these enzymes would be ultimately linked to nitrate assimilation and nitrogen use efficiency in wheat.

p-Hydroxybenzyl alcohol inhibits four obesity-related enzymes in vitro.

Recently, antiobesity studies using the method of inhibiting enzymatic activity of obesity-related enzymes as targets have received considerable attention. The aims of the current study were to investigate whether p-hydroxybenzyl alcohol (HBA), identified from Cudrania tricuspidata fruits with antiobesity effects, inhibits the activity of digestive and obesity-related enzymes and acts as an inhibitor against four target enzymes in kinetic studies. In vitro enzyme assays showed HBA at the highest concentration significantly reduced the enzymatic activity of four targets: pancreatic lipase (IC50 = 2.34-3.70 µM), α-glycosidase (IC50 = 9.08 µM), phosphodiesterase IV (IC50 = 4.99 µM), and citrate synthase (IC50 = 2.07 µM) enzymes. Based on the results of kinetic assays, the types of inhibition were investigated. Our findings indicate that HBA could have antiobesity efficacy, and it deserves further study.

Differential response of hexaploid and tetraploid wheat to interactive effects of elevated [CO2] and low phosphorus.

KEY MESSAGE: Hexaploid wheat is more responsive than tetraploid to the interactive effects of elevated [CO2] and low P in terms of carboxylate efflux, enzyme activity and gene expression (TaPT1 and TaPAP). Availability of mineral nutrients to plants under changing climate has become a serious challenge to food security and economic development. An understanding of how elevated [CO2] influences phosphorus (P) acquisition processes at the whole-plant level would be critical in selecting cultivars as well as to maintain optimum yield in limited-P conditions. Wheat (Triticum aestivum and T. durum) grown hydroponically with sufficient and low P concentration were exposed to elevated and ambient [CO2]. Improved dry matter partitioning towards root resulted in increased root-to-shoot ratio, root length, volume, surface area, root hair length and density at elevated [CO2] with low P. Interaction of low P and [CO2] induced activity of enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase and citrate synthase) in root tissue resulting in twofold increase in carboxylates and acid phosphatase exudation. Physiological absorption capacity of roots showed that plants alter their uptake kinetics by increasing affinity (low Km) in response to elevated [CO2] under low P supply. Increased relative expression of genes, purple acid phosphatase (TaPAP) and high-affinity Pi transporter (TaPT1) in roots induced by elevated [CO2] and low P supported our physiological observations. Hexaploid wheat (PBW-396) being more responsive to elevated [CO2] at low P supply as compared to tetraploid (PDW-233) necessitates the ploidy effect to be explored further which might be advantageous under changing climate.

Critical Role of Flavin and Glutathione in Complex I-Mediated Bioenergetic Failure in Brain Ischemia/Reperfusion Injury.

BACKGROUND AND PURPOSE: Ischemic brain injury is characterized by 2 temporally distinct but interrelated phases: ischemia (primary energy failure) and reperfusion (secondary energy failure). Loss of cerebral blood flow leads to decreased oxygen levels and energy crisis in the ischemic area, initiating a sequence of pathophysiological events that after reoxygenation lead to ischemia/reperfusion (I/R) brain damage. Mitochondrial impairment and oxidative stress are known to be early events in I/R injury. However, the biochemical mechanisms of mitochondria damage in I/R are not completely understood. METHODS: We used a mouse model of transient focal cerebral ischemia to investigate acute I/R-induced changes of mitochondrial function, focusing on mechanisms of primary and secondary energy failure. RESULTS: Ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a reversible oxidative modification of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration of glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester. CONCLUSIONS: Our results unveil a key role of mitochondrial complex I in the development of I/R brain injury and provide the mechanistic basis for the well-established mitochondrial dysfunction caused by I/R. Targeting the functional integrity of complex I in the early phase of reperfusion may provide a novel therapeutic strategy to prevent tissue injury after stroke.

Mitochondrial content is preserved throughout disease progression in the mdx mouse model of Duchenne muscular dystrophy, regardless of taurine supplementation.

Mitochondrial dysfunction is a pathological feature of Duchenne muscular dystrophy (DMD), a debilitating and fatal neuromuscular disorder characterized by progressive muscle wasting and weakness. Mitochondria are a source of cellular ATP involved in Ca2+ regulation and apoptotic signaling. Ameliorating aberrant mitochondrial function has therapeutic potential for reducing DMD disease severity. The dystrophic mdx mouse exhibits peak muscle damage at 21-28 days, which stabilizes after 8 wk. The amino acid taurine is implicated in mitochondrial health and function, with endogenous concentrations low when measured during the cycle of peak muscle damage in mdx mice. Using whole soleus and extensor digitorum longus (EDL) muscle homogenates from 28- and 70-day mdx mice, we found that there was no change in native state mitochondrial complexes using blue native-PAGE. NADH:ubiquinone oxidotreductase subunit-A9 (NDUFA9) protein abundance was lower in soleus muscle of 28- and 70-day mdx mice and EDL muscle of 70-day mdx mice compared with same muscles in WT (C57/BL10ScSn) animals. There were age-dependent increases in both NDUFA9 protein abundance and citrate synthase activity in soleus muscles of mdx and wild-type mice. There was no change in abundances of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49). Taurine administration essentially did not affect any measurements of mitochondria. Collectively, these findings suggest mitochondrial content and dynamics are not reduced in the mdx mouse regardless of disease severity. We also elucidate that taurine affords no significant benefit to mitochondrial content or dynamics in the mdx mouse at either 28 or 70 days.

Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells.

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72 h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.