Lipid content and G6PDH activity in relation to ooplasm morphology and oocyte maturational competence in the domestic cat model.

The aim of the study was to investigate the relationship between ooplasm morphology, lipid content, glucose-6-phosphate dehydrogenase activity (G6PDH) and maturation potential of domestic cat oocytes. Cumulus-oocyte complexes were classified according to ooplasm morphology: evenly dark (dCOC), heterogeneous/mosaic (hCOC), or light/transparent (lCOC), however only dCOCs are thought to be the best-quality, the remaining ones are usually rejected, therefore little is known about their intracellular properties. Lipid droplets (LDs) were visualized and quantified using Oil Red O. G6PDH activity was assessed before in vitro maturation (IVM), using the brilliant cresyl blue (BCB) test. IVM-control oocytes underwent IVM without BCB staining. The dCOCs and hCOCs had different patterns of LD spatial distribution, but similar amounts of lipid, although this tended towards being lower in hCOCs. Low G6PDH activity (BCB+) was observed in 74 %, 60 % and 24 % (P < 0.01) of dCOCs, hCOCs, and lCOCs, respectively. Significantly more BCB+ /oocytes than BCB-/oocytes reached the metaphase II stage in all groups. The maturation rate of BCB+ /hCOCs was higher than that of IVM/hCOC-controls (40 % v.s. 20 %, P < 0.001), and was comparable to that of BCB+ /dCOCs (54 %; P > 0.05). lCOCs were the smallest (P < 0.01), contained fewer (P < 0.01) lipids than dCOCs or hCOCs, and displayed reduced maturational potential. Overall, LD content and distribution, as well as G6PDH activity, in cat oocytes were strongly associated with ooplasm morphology and oocyte maturational competence. Deeper understanding of the intrinsic properties of oocytes with different ooplasm morphology using the domestic cat model, may be particularly important in the context of the conservation of endangered felids.

Comprehensive review on glucose 6 phosphate dehydrogenase: A critical immunometabolic and redox switch in insects.

Mounting an active immune response is energy intensive and demands the reallocation of nutrients to maintain the body's resistance and tolerance against infections. Central to this metabolic adaptation is Glucose-6-phosphate dehydrogenase (G6PDH), a housekeeping enzyme involve in pentose phosphate pathway (PPP). PPP play an essential role in generating ribose, which is critical for nicotinamide adenine dinucleotide phosphate (NADPH). It is vital for physiological and cellular processes such as generating nucleotides, fatty acids and reducing oxidative stress. The G6PDH is extremely conserved enzyme across species in PP shunt. The deficiency of enzymes leads to serious consequences on organism, particularly on adaptation and development. Acute deficiency can lead to impaired cell development, halted embryonic growth, reduce sensitivity to insulin, hypertension and increase inflammation. Historically, research focusing on G6PDH and PPP have primarily targeted diseases on mammalian. However, our review has investigated the unique functions of the G6PDH enzyme in insects and greatly improved mechanistic understanding of its operations. This review explore how G6PDH in insects plays a crucial role in managing the redox balance and immune related metabolism. This study aims to investigate the enzyme's role in different metabolic adaptations.

Implementation of π-π interaction in AuNPs@GDY to boost the bioelectrocatalysis in enzymatic biofuel cells.

The main challenges (sluggish electron transfer, low energy density) hinder the future application of enzymatic biofuel cells (EBFCs), which urgent to take effective measures to solve these issues. In this work, a composite of Au nanoparticles decorated graphdiyne (AuNPs@GDY) is fabricated and employed as the carrier of enzyme (G6PDH), and a mechanism based on π-π interaction of electron transfer is proposed to understand bioelectrocatalysis processes. The results show that the AuNPs@GDY composite exhibits the highest current density among the three materials (GDY, AuNPs, and AuNPs@GDY), which is 3.4 times higher than that of GDY and 2.5 times higher than that of AuNPs. Furthermore, the results reveal that the AuNPs could increase the loading of enzymes and provide more active site for reaction, while GDY provides highly π-conjugated structure and unique sp/sp2-hybridized linkages interface. This work provides new insights to explore a theoretical basis for the development of more efficient bioelectrocatalytic systems.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Butyrate induces oxidative burst mediated apoptosis via Glucose-6-Phosphate Dehydrogenase (G6PDH) in macrophages during mycobacterial infection.

Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.

Metabolic priming in G6PDH isoenzyme-replaced tobacco lines improves stress tolerance and seed yields via altering assimilate partitioning.

We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH-isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress-related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non-stressed plants contained twofold elevated fructose-2,6-bisphosphate (F2,6P2 ) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose-to-sucrose ratios, but also more lipids. Above-ground biomass lay in between susceptible Xanthi and resistant SNN, with photo-assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid-to-carbohydrate ratios, resulting in increased harvest yields - also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non-stressed plants. In leaves of salt-watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non-stressed plants showed enhanced PSII-induction kinetics (upon dark-light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids - especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi-cP2 lines. Apparently, metabolic priming by F2,6P2 -combined with sustained NADP(H) turnover-bypasses the genetically fixed growth-defence trade-off, rendering tobacco plants more stress-resilient and productive.

Analysis and modification of central carbon metabolism in Hypsizygus marmoreus for improving mycelial growth performance and fruiting body yield.

Hypsizygus marmoreus is one of the main industrially cultivated varieties of edible fungi, with a delicious taste and high nutritional value. However, the long harvest period of 130-150 days greatly limits its large-scale expansion. This study aimed to investigate the effects of central carbon metabolism (CCM) on the mycelial growth performance and fruiting body formation of H. marmoreus. Nine edible fungi with different harvest periods were collected and used to evaluate their intracellular carbon metabolic differences in the CCM, which revealed that the imbalanced distribution of intracellular carbon metabolic levels in the CCM of H. marmoreus might be one of the key factors resulting in a slow mycelial growth rate and a long harvest period. Further analysis by three strategies, including metabolomics, adaptation of different carbon sources, and chemical interference, confirmed that low carbon flux into the pentose phosphate pathway (PPP) limited the supply of raw materials, reduced power, and thus influenced the mycelial growth of H. marmoreus. Furthermore, four transformants with increased expression levels of glucose-6-phosphate dehydrogenase (G6PDH), a key rate-limiting enzyme in the PPP of H. marmoreus, were developed and showed more extracellular soluble protein secretion and higher sugar assimilation rates, as well as improved mycelial growth rates in bottle substrate mixtures. Finally, cultivation experiments indicated that the maturation periods of the fruiting body with ~4-5 days in advance and the maximum fruiting body yield of 574.8 g per bag with an increase of 7.4% were achieved by improving the G6PDH expression level of the PPP in H. marmoreus. This study showed that CCM played an important role in the mycelial growth and development of H. marmoreus, which provided new insights for future advancements in cultivating and breeding edible fungi.

A Mitochondria-Targeted Fluorescent Probe for Monitoring NADPH Overproduction during Influenza Virus Infection.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor in the progress of antioxidant synthesis and biosynthesis, and an abnormal NADPH level has been observed in many viral infection processes. However, efficient tools to monitor NADPH in living cells after viral infection have not been reported. In this work, we present a fluorescent probe, NAFP4, that could detect NADPH ex vivo with a low detection limit of 3.66 nM and image mitochondrial NADPH level changes in living cells. The probe exhibits excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. Using NAFP4, we reveal that the NADPH is overproduced in the host cells infected by influenza virus, which was caused by an elevated level of G6PDH during the virus infection. Moreover, there was positive association between the G6PDH level and virus replication. With the proposed probe NAFP4, our study highlights that the virus infection would influence the host metabolism in NADPH production and also suggests that G6PDH is expected to be a promising target for antiviral therapy.

Mitochondrial ferritin alleviates apoptosis by enhancing mitochondrial bioenergetics and stimulating glucose metabolism in cerebral ischemia reperfusion.

Oxidative stress and deficient bioenergetics are key players in the pathological process of cerebral ischemia reperfusion injury (I/R). As a mitochondrial iron storage protein, mitochondrial ferritin (FtMt) plays a pivotal role in protecting neuronal cells from oxidative damage under stress conditions. However, the effects of FtMt in mitochondrial function and activation of apoptosis under cerebral I/R are barely understood. In the present study, we found that FtMt deficiency exacerbates neuronal apoptosis via classical mitochondria-depedent pathway and the endoplasmic reticulum (ER) stress pathway in brains exposed to I/R. Conversely, FtMt overexpression significantly inhibited oxygen and glucose deprivation and reperfusion (OGD/R)-induced apoptosis and the activation of ER stress response. Meanwhile, FtMt overexpression rescued OGD/R-induced mitochondrial iron overload, mitochondrial dysfunction, the generation of reactive oxygen species (ROS) and increased neuronal GSH content. Using the Seahorse and O2K cellular respiration analyser, we demonstrated that FtMt remarkably improved the ATP content and the spare respiratory capacity under I/R conditions. Importantly, we found that glucose consumption was augmented in FtMt overexpressing cells after OGD/R insult; overexpression of FtMt facilitated the activation of glucose 6-phosphate dehydrogenase and the production of NADPH in cells after OGD/R, indicating that the pentose-phosphate pathway is enhanced in FtMt overexpressing cells, thus strengthening the antioxidant capacity of neuronal cells. In summary, our results reveal that FtMt protects against I/R-induced apoptosis through enhancing mitochondrial bioenergetics and regulating glucose metabolism via the pentose-phosphate pathway, thus preventing ROS overproduction, and preserving energy metabolism.

Short-term exposure to radiofrequency radiation and metabolic enzymes' activities during pregnancy and prenatal development.

Radiofrequency radiation (RFR) as an environmental and physical pollutant may induce vulnerability to toxicity and disturb fetal development. Therefore, the potential health effects of short-term mobile phone like RFR exposure (GSM 1800 MHz; 14 V/m, 2 mW/kg specific absorption rate (SAR) during 15 min/day for a week) during pregnancy and also the development of fetuses were investigated. Hepatic glucose regulation and glutathione-dependent enzymes' capacities were biochemically analyzed in adult (female) and pregnant New Zealand White rabbits. Pregnant rabbits' two-day-old offspring were included to understand their developmental stages under short-term maternal RFR exposure. We analyzed two regulatory enzymes in the oxidative phase of phosphogluconate pathways to interpret the cytosolic NADPH's biosynthesis for maintaining mitochondrial energy metabolism. Moreover, the efficiencies of maternal glutathione-dependent enzymes on both the removal of metabolic disturbances during pregnancy and fetus development were examined. Whole-body RFR exposures were applied to pregnant animals from the 15th to the 22nd day of their gestations, i.e., the maturation periods of tissues and organs for rabbit fetuses. There were significant differences in hepatic glucose regulation and GSH-dependent enzymes' capacities with pregnancy and short-term RFR exposure. Consequently, we observed that intrauterine exposure to RFR might lead to cellular ROS- dependent disturbances in metabolic activity and any deficiency in the intracellular antioxidant (ROS-scavenging) system. This study might be a novel insight into further studies on the possible effects of short-term RF exposure and prenatal development.

Genome-Wide Investigation of G6PDH Gene in Strawberry: Evolution and Expression Analysis during Development and Stress.

As one of the key enzymes in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides NADPH and plays an important role in plant development and stress responses. However, little information was available about the G6PDH genes in strawberry (Fragaria × ananassa). The recent release of the whole-genome sequence of strawberry allowed us to perform a genome-wide investigation into the organization and expression profiling of strawberry G6PDH genes. In the present study, 19 strawberry G6PDH genes (FaG6PDHs) were identified from the strawberry genome database. They were designated as FaG6PDH1 to FaG6PDH19, respectively, according to the conserved domain of each subfamily and multiple sequence alignment with Arabidopsis. According to their structural and phylogenetic features, the 19 FaG6PDHs were further classified into five types: Cy, P1, P1.1, P2 and PO. The number and location of exons and introns are similar, suggesting that genes of the same type are very similar and are alleles. A cis-element analysis inferred that FaG6PDHs possessed at least one stress-responsive cis-acting element. Expression profiles derived from transcriptome data analysis exhibited distinct expression patterns of FaG6PDHs genes in different developmental stages. Real-time quantitative PCR was used to detect the expression level of five types FaG6PDHs genes and demonstrated that the genes were expressed and responded to multiple abiotic stress and hormonal treatments.

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Neurons undergo pathogenic metabolic reprogramming in models of familial ALS.

OBJECTIVES: Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis [ALS]), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We investigated intermediary metabolism of neurons expressing familial ALS (fALS) genes and interrogated the functional consequences of glycolysis genes in fitness assays and neuronal survival. METHODS: We created a pure neuronal model system for isotopologue investigations of fuel utilization. In a yeast platform we studied the functional contributions of glycolysis genes in a growth fitness assay iafter expressing of a fALS gene. RESULTS: We find in our rodent models of fALS, a reduction in neuronal lactate production with maintained or enhanced activity of the neuronal citric acid cycle. This rewiring of metabolism is associated with normal ATP levels, bioenergetics, and redox status, thus supporting the notion that gross mitochondrial function is not compromised in neurons soon after expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. CONCLUSIONS: We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated alteration in mitochondrial function in a baleful manner and targeting this process can be healthful.

The ginger extract could improve diabetic retinopathy by inhibiting the expression of e/iNOS and G6PDH, apoptosis, inflammation, and angiogenesis.

Diabetic retinopathy is a complication of diabetes, caused by high blood sugar levels damaging the retina. It is the result of damage to the small blood vessels and neurons of the retina. Ginger and its phytochemical compounds can improve oxidative damage and inflammation. However, the effects of this plant on ocular expression G6PDH and e/iNOS, eye cell apoptosis, and angiogenesis are not well known in this tissue. Therefore, the aim of this study was to evaluate the therapeutic potential of ginger extract on rats with type 2 diabetic retinopathy. Thirty-two Wistar rats were randomly divided into four controlled and treated groups. The serum level of metabolic factors such as lipid profiles, insulin and glucose, and the level of oxidative biomarkers along with the TNF-α level in eye tissue were measured. The expression of NF-κB, VEGF, BAX, Bcl-2, caspase-3, e/iNOS, and G6PDH in eye tissue was measured. Serum levels of lipid profiles, glucose, and insulin, oxidative and inflammatory markers were significantly increased in the diabetic group compared to control. While, treatment with ginger extract could significantly improve these factors in diabetic rats. Moreover, the ocular expression of e/iNOS, G6PDH, VEGF, NF-κB, and genes involved in apoptosis was changed in diabetic rats. However, treatment with ginger extract could ameliorate these changes in the diabetic-treated group. It can be concluded that ginger extract could improve diabetic retinopathy by inhibiting oxidative damage, inflammation, iNOS, VEGF, apoptosis, and improving eNOS and G6PDH. PRACTICAL APPLICATIONS: Microvascular complications of diabetes such as retinopathy can be one of the main causes of disability in people with diabetes. Chronic hyperglycemia, oxidative stress, inflammation, and apoptosis cause diabetic retinopathy through retinal damage. Ginger, on the other hand, is an available, inexpensive, and uncomplicated medicinal plant that contains more than 20 different phytochemicals, such as gingerol and shogaol, which have anti-inflammatory, antioxidant, antihypertensive, hypoglycemic, and hypolipidemic properties. The results of our study showed well that the ginger extract could improve diabetic retinopathy by inhibiting the expression of e/iNOS and G6PDH and oxidative damage, apoptosis, inflammation, and angiogenesis. Therefore, ginger and its compounds can be a good option to improve the complications of diabetes.

Naringin prevents cyclophosphamide-induced erythrocytotoxicity in rats by abrogating oxidative stress.

Earlier reports have shown that Cyclophosphamide (CYCP), an anti-malignant drug, elicited cytotoxicity; and that naringin has several beneficial potentials against oxidative stress and dyslipidaemias. We investigated the influence of naringin on free radical scavenging, cellular integrity, cellular ATP, antioxidants, oxidative stress, and lipid profiles in the CYCP-induced erythrocytotoxicity rat model. Rats were pretreated orally by gavage for fourteen consecutive days with three doses (50, 100, and 200 mg/kg) naringin before single CYCP (200 mg/kg, i.p.) administration. Afterwards, the rats were sacrificed. Naringin concentrations required for 50 % scavenging hydrogen peroxide and nitric oxide radical were 0.27 mg/mL and 0.28 mg/mL, respectively. Naringin pretreatment significantly (p < 0.05) protected erythrocytes plasma membrane architecture and integrity by abolishing CYCP-induced decrease in the activity of erythrocyte LDH (a marker of ATP). Pretreatment with naringin remarkably (p < 0.05) reversed CYCP-induced decreases in the erythrocytes glutathione levels, activities of glutathione-S-transferase, catalase, glutathione peroxidase, and glutathione reductase; attenuated CYCP-mediated increases in erythrocytes levels of malondialdehyde, nitric oxide, and major lipids (cholesterol, triacylglycerol, phospholipids, and non-esterified fatty acids). Taken together, different acute pretreatment doses of naringin might avert CYCP-mediated erythrocytes dysfunctions via its antioxidant, free-radical scavenging, and anti-dyslipidaemia properties.

Effect of CAX1a TILLING mutations on photosynthesis performance in salt-stressed Brassica rapa plants.

Salinity is an important environmental factor that reduces plant productivity in many world regions. It affects negatively photosynthesis causing a growth reduction. Likewise, calcium (Ca2+) is crucial in plant stress response. Therefore, the modification of Ca2+ cation exchangers (CAX) transporters could be a potential strategy to increase plant tolerance to salinity. Using Targeting Induced Local Lesions in Genomes (TILLING), researchers generated three mutants of Brassica rapa CAX1a transporter: BraA.cax1a-7, BraA.cax1a-4, and BraA.cax1a-12. The aim of this study was to test the effect of those mutations on salt tolerance focusing on the response to the photosynthesis process. Thus, the three BraA.cax1a mutants and the parental line (R-o-18) were grown under salinity conditions, and parameters related to biomass, photosynthesis performance, glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), and soluble carbohydrates were measured. BraA.cax1a-4 provided higher biomass and a better photosynthetic performance manifested by higher water use efficiency (WUE), Fv/Fm, electron fluxes, and Rubisco (EC 4.1.1.39) values. In addition, BraA.cax1a-4 presented increased osmotic protection through myo-inositol accumulation. On the other hand, BraA.cax1a-7 produced some negative effects on photosynthesis performance and lower G6PDH and Rubisco accumulations. Therefore, this study points out BraA.cax1a-4 as a useful mutation to improve photosynthetic performance in plants grown under saline conditions.

Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase genes of winter wheat enhance the cold tolerance of transgenic Arabidopsis.

In this study, winter wheat G6PDH (TaG6PDH) and 6PGDH (Ta6PGDH) were investigated. Both their expression and their activity were upregulated under cold stress, suggesting that TaG6PDH and Ta6PGDH positively respond to cold stress in winter wheat. Exogenous abscisic acid (ABA) treatment markedly increased the expression and activity levels of TaG6PDH and Ta6PGDH in winter wheat under cold stress. Subsequently, TaG6PDH-and Ta6PGDH were overexpressed in Arabidopsis, and showed stronger reactive oxygen species (ROS)-scavenging ability and higher survival rate compared with wild-type (WT) plants under cold stress. In addition, we found that TaG6PDH and Ta6PGDH overexpression can promote the oxidative pentose phosphate pathway (OPPP) in the cytoplasm and peroxisomes of Arabidopsis. In summary, Arabidopsis overexpressing TaG6PDH and Ta6PGDH showed improved cold tolerance.

Insights into the ameliorative effect of oleic acid in rejuvenating phenylhydrazine induced oxidative stress mediated morpho-functionally dismantled erythrocytes.

Phenylhydrazine (PHZ), an intermediate in the synthesis of fine chemicals is toxic for human health and environment. Despite of having severe detrimental effects on different physiological systems, exposure of erythrocytes to PHZ cause destruction of haemoglobin and membrane proteins leading to iron release and complete haemolysis of red blood cells (RBC). Involvement of oxidative stress behind such action triggers the urge for searching a potent antioxidant. The benefits of consuming olive oil is attributed to its 75% oleic acid (OA) content in average. Olive oil is the basic component of Mediterranean diet. Hence, OA has been chosen in our present in vitro study to explore its efficacy against PHZ (1 mM) induced alterations in erythrocytes. Four different concentrations of OA (0.01 nM, 0.02 nM, 0.04 nM and 0.06 nM) were primarily experimented with, among which 0.06 nM OA has shown to give maximal protection. This study demonstrates the capability of OA in preserving the morphology, intracellular antioxidant status and the activities of metabolic enzymes of RBCs that have been diminished by PHZ, through its antioxidant mechanisms. The results of the present study firmly establish OA as a promising antioxidant for conserving the health of erythrocyte from PHZ toxicity which indicate toward future possible use of OA either singly or in combination with other dietary components for protection of erythrocytes against PHZ induced toxic cellular changes.

Caffeine Targets G6PDH to Disrupt Redox Homeostasis and Inhibit Renal Cell Carcinoma Proliferation.

Glucose-6-phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) and plays a crucial role in the maintenance of redox homeostasis by producing nicotinamide adenine dinucleotide phosphate (NADPH), the major intracellular reductant. G6PDH has been shown to be a biomarker and potential therapeutic target for renal cell carcinoma (RCC). Here, we report a previously unknown biochemical mechanism through which caffeine, a well-known natural small molecule, regulates G6PDH activity to disrupt cellular redox homeostasis and suppress RCC development and progression. We found that caffeine can inhibit G6PDH enzymatic activity. Mechanistically, caffeine directly binds to G6PDH with high affinity (K D = 0.1923 µM) and competes with the coenzyme NADP+ for G6PDH binding, as demonstrated by the decreased binding affinities of G6PDH for its coenzyme and substrate. Molecular docking studies revealed that caffeine binds to G6PDH at the structural NADP+ binding site, and chemical cross-linking analysis demonstrated that caffeine inhibits the formation of dimeric G6PDH. G6PDH inhibition abrogated the inhibitory effects of caffeine on RCC cell growth. Moreover, inhibition of G6PDH activity by caffeine led to a reduction in the intracellular levels of NADPH and reactive oxygen species (ROS), and altered the expression of redox-related proteins in RCC cells. Accordingly, caffeine could inhibit tumor growth through inhibition of G6PDH activity in vivo. Taken together, these results demonstrated that caffeine can target G6PDH to disrupt redox homeostasis and inhibit RCC tumor growth, and has potential as a therapeutic agent for the treatment of RCC.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.