NAD(P)-Driven Redox Status Contributes to Desiccation Tolerance in Acer seeds.

Desiccation tolerance is a developmental program enabling seed survival in a dry state and is common in seeds categorized as orthodox. We focused on NAD and its phosphorylated form (NADP) because their continual switching between reduced (NAD(P)H) and oxidized (NAD(P)+) forms is involved in the modulation of redox signaling and the determination of the reducing power and further antioxidant responses. Norway maple and sycamore seeds representing the orthodox and recalcitrant categories, respectively, were used as models in a comparison of responses to water loss. The process of desiccation up to 10% water content (WC) was monitored in Norway maple seeds, while dehydration up to 30% WC was monitored in desiccation-sensitive sycamore seeds. Norway maple and sycamore seeds, particularly their embryonic axes, exhibited a distinct redox status during dehydration and desiccation. High NADPH levels, NAD+ accumulation, low and stable NAD(P)H/NAD(P)+ ratios expressed as reducing power and high NADPH-dependent enzyme activity were reported in Norway maple seeds and were considered attributes of orthodox-type seeds. The contrasting results of sycamore seeds contributed to their low antioxidant capacity and high sensitivity to desiccation. NADPH deficiency, low NADPH-dependent enzyme activity and lack of NAD+ accumulation were primary features of sycamore seeds, with implications for their NAD(P)H/NAD(P)+ ratios and reducing power and with effects on many seed traits. Thus, we propose that the distinct levels of pyridine nucleotides and their redox status contribute to orthodox and recalcitrant phenotype differentiation in seeds by affecting cellular redox signaling, metabolism and the antioxidant system.

Glucose 6-phosphate dehydrogenase and the kidney.

PURPOSE OF REVIEW: Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. G6PD is the main source of the essential cellular reductant, NADPH. The purpose of this review is to describe the biochemistry of G6PD and NADPH, cellular factors that regulate G6PD, normal physiologic roles of G6PD, and the pathogenic role altered G6PD/NADPH plays in kidney disease. RECENT FINDINGS: NADPH is required for many essential cellular processes such as the antioxidant system, nitric oxide synthase, cytochrome p450 enzymes, and NADPH oxidase. Decreased G6PD activity and, as a result, decreased NADPH level have been associated with diabetic kidney disease, altered nitric oxide production, aldosterone-mediated endothelial dysfunction, and dialysis-associated anemia. Increased G6PD activity is associated with all cancers including kidney cancer. Inherited G6PD deficiency is the most common mutation in the world that is thought to be a relatively mild disorder primarily associated with anemia. Yet, intriguing studies have shown an increased prevalence of diabetes mellitus in G6PD-deficient people. It is not known if G6PD-deficient people are at more risk for other diseases. SUMMARY: Much more research needs to be done to determine the role of altered G6PD activity (inherited or acquired) in the pathogenesis of kidney disease.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels.

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca(2+) from acidic intracellular endolysosomal Ca(2+) stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca(2+) release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca(2+) . Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca(2+) over K(+) than TPC1 and hence capable of releasing greater quantities of Ca(2+) from acidic stores. TPC1 is also permeable to H(+) and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca(2+) -release channels of the endolysosomal system.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

Calcium mobilizing second messengers derived from NAD.

Nicotinamide adenine dinucleotide (NAD) has been known since a long period of time as co-factor of oxidoreductases. However, in the past couple of decades further roles have been assigned to NAD. Here, metabolism of NAD to the Ca²âº mobilizing second messengers cyclic adenosine diphosphoribose, nicotinic acid adenine dinucleotide phosphate and adenosine diphosphoribose is reviewed. Moreover, the mechanisms of Ca²âº mobilization by these adenine nucleotides and their putative target Ca²âº channels, ryanodine receptors and transient receptor potential channels are discussed. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Teaching the Z-Scheme of electron transport in photosynthesis: a perspective.

This paper deals with how Govindjee taught the Z-Scheme of electron transport in oxygenic photosynthesis at Ravenshaw University, Cuttack, Odisha, India, in 2014, in a unique and highly effective fashion-using students to act as molecules, representing the entire electron transport chain from water to nicotinamide adenine dinucleotide phosphate (NADP(+)). It culminated in a show by B.Sc. students in the garden of the Department of Botany, Ravenshaw University. The first author (PKM) personally acted as Ferredoxin NADP Reductase (FNR) catalyzing the reduction of NADP(+) to NADPH, taking electrons from reduced ferredoxin at the end of Photosystem I. On the other hand, the Q-cycle was played by M.Sc. students, who acted as molecules running this ingenious cycle that produces extra protons. An interesting event was when a student, acting as a herbicide, who was dressed like a devil (fierce looking, in black clothes with a sword; "Yamaraj: The God of Death", as he called himself), stopped all reactions by throwing out QB, the second plastoquinone molecule of Photosystem II, and that too aggressively, taking its position instead. The second author was the major organizer of the Z-scheme show. We provide here a basic background on the process, a bit on Govindjee's teaching, and some selected pictures from the drama played in March, 2014 at Ravenshaw University. Here, we also recognize the teacher Govindjee for his ingenious and fun-filled teaching methods that touched the hearts and the souls of the students as well as the teachers of Ravenshaw University. He was rated as one of the most-admired teachers of plant biology at our university.

Regulation of ion channels by pyridine nucleotides.

Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion-transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide-binding ß-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP(+) to Kvß removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K(+) transporters. These nucleotides also have been shown to modify the activity of the plasma membrane K(ATP) channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit-the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP(+) metabolite, NAADP(+), regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias.

Overview of pyridine nucleotides review series.

Pyridine nucleotides are abundant soluble coenzymes and they undergo reversible oxidation and reduction in several biological electron-transfer reactions. They are comprised of two mononucleotides, adenosine monophosphate and nicotinamide mononucleotide, and are present as oxidized and reduced nicotinamide adenine dinucleotides in their unphosphorylated (NAD(+) and NADH) and phosphorylated (NADP(+) and NADPH) forms. In the past, pyridine nucleotides were considered to be primarily electron-shuttling agents involved in supporting the activity of enzymes that catalyze oxidation-reduction reactions. However, it has recently been demonstrated that pyridine nucleotides and the balance between the oxidized and reduced forms play a wide variety of pivotal roles in cellular functions as important interfaces, beyond their coenzymatic activity. These include maintenance of redox status, cell survival and death, ion channel regulation, and cell signaling under normal and pathological conditions. Furthermore, targeting pyridine nucleotides could potentially provide therapeutically useful avenues for treating cardiovascular diseases. This review series will highlight the functional significance of pyridine nucleotides and underscore their physiological role in cardiovascular function and their clinical relevance to cardiovascular medicine.

Pyridine nucleotide regulation of cardiac intermediary metabolism.

The pyridine nucleotides NAD(+) and NADP(+) play a pivotal role in regulating intermediary metabolism in the heart. The intracellular NAD(+)/NADH ratio controls flux through various dehydrogenase enzymes involved in both anaerobic and aerobic metabolism and also regulates posttranslational protein modification. The intracellular NADP(+)/NADPH ratio controls flux through the pentose phosphate pathway (PPP) and the polyol pathway, while also regulating ion channel function and oxidative stress. Not only does the NAD(+)/NADH ratio regulate the rates of ATP production, it can also modify energy substrate preference. For instance, in many forms of heart disease a greater contribution from fatty acids for oxidative energy metabolism increases fatty acid ß-oxidation-derived NADH, which can activate pyruvate dehydrogenase (PDH) kinase isoforms that inhibit PDH and subsequent glucose oxidation. As such, novel therapies that overcome fatty acid ß-oxidation-induced inhibition of PDH improve cardiac efficiency and subsequent function during ischemia/reperfusion and in heart failure. Furthermore, recent studies have implicated a pivotal role for increased PPP-derived NADPH in mediating oxidative stress observed in heart failure. In this article, we review the multiple actions of NAD(+)/NADH and NADP(+)/NADPH in regulating intermediary metabolism in the heart. A better understanding of the roles of NAD(+)/NADH and NADP(+)/NADPH in cellular physiology and pathology could potentially be used to exploit pyridine nucleotide modification in the treatment of a number of different forms of heart disease.

Regulation of cell survival and death by pyridine nucleotides.

Pyridine nucleotides (PNs), such as NAD(H) and NADP(H), mediate electron transfer in many catabolic and anabolic processes. In general, NAD(+) and NADP(+) receive electrons to become NADH and NADPH by coupling with catabolic processes. These electrons are utilized for biologically essential reactions such as ATP production, anabolism and cellular oxidation-reduction (redox) regulation. Thus, in addition to ATP, NADH and NADPH could be defined as high-energy intermediates and "molecular units of currency" in energy transfer. We discuss the significance of PNs as energy/electron transporters and signal transducers, in regulating cell death and/or survival processes. In the first part of this review, we describe the role of NADH and NADPH as electron donors for NADPH oxidases (Noxs), glutathione (GSH), and thioredoxin (Trx) systems in cellular redox regulation. Noxs produce superoxide/hydrogen peroxide yielding oxidative environment, whereas GSH and Trx systems protect against oxidative stress. We then describe the role of NAD(+) and NADH as signal transducers through NAD(+)-dependent enzymes such as PARP-1 and Sirt1. PARP-1 is activated by damaged DNA in order to repair the DNA, which attenuates energy production through NAD(+) consumption; Sirt1 is activated by an increased NAD(+)/NADH ratio to facilitate signal transduction for metabolic adaption as well as stress responses. We conclude that PNs serve as an important interface for distinct cellular responses, including stress response, energy metabolism, and cell survival/death.

N-terminal tagging of two-pore channels interferes with NAADP action.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is the most potent Ca2+-releasing second messenger known to date. Since its discovery in 1995 identifying the NAADP receptor protein/Ca2+ channel has been a major persuit of the Ca2+ signalling community. In their paper 'The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP' published in this issue of the Biochemical Journal Patel and colleagues describe that the N-terminus of one of the NAADP receptor protein/Ca2+ channel candidates, TPC1 (two-pore channel 1), is crucial for protein targeting and for sensitivity to NAADP.

The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP.

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor.

BACKGROUND AND PURPOSE: Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH: The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS: Pretreatment with 1µM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3ß (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS: Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.

Effects of NAD(P)H and its derivatives on the DNA-binding activity of NPAS2, a mammalian circadian transcription factor.

NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.

Mitochondrial activation at the onset of contractions in isolated myofibres during successive contractile periods.

At the onset of skeletal muscle repetitive contractions, there is a significant delay in the time to achieve oxidative phosphorylation steady state. The purpose of the present study was to examine the factors that limit oxidative phosphorylation at the onset of contractions. NAD(P)H was measured in real time during two contractile periods (2 min each) separated by 5 min of rest in intact single muscle fibres (n = 7) isolated from Xenopus laevis. The fibres were then loaded with the dye tetramethylrhodamine methyl ester perchlorate (TMRM) to evaluate the kinetics of the mitochondrial membrane potential (Δψ (m)) during two further successive contractile periods. At the onset of contractions in the first period, NAD(P)H exhibited a time delay (14.1 ± 1.3 s) before decreasing toward a steady state. In contrast, Δψ(m) decreased immediately after the first contraction and started to be reestablished after 10.7 ± 0.9 s, with restoration to the pre-stimulation values after approximately 32 s. In the second contractile period (5 min after the first), NAD(P)H decreased immediately (i.e. no time delay) after the first contraction and had a significantly shorter time constant compared to the first contractile bout (3.3 ± 0.3 vs. 5.0 ± 0.2 s, P < 0.05). During the second bout, Δψ(m) remained unchanged from pre-stimulation values. These results suggest: (1) that at the onset of contractions, oxidative phosphorylation is primarily limited by the activity of the electron transport chain complexes rather than by a limited level of substrates; and (2) when the muscle is 'primed' by previous contractile activity, the faster enhancement of the cellular respiratory rate is due to intrinsic factors within the myofibre.

Glucose-6-phosphate dehydrogenase, NADPH, and cell survival.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. Many scientists think that the roles and regulation of G6PD in physiology and pathophysiology have been well established as the enzyme was first identified 80 years ago. And that G6PD has been extensively studied especially with respect to G6PD deficiency and its association with hemolysis, and with respect to the role G6PD plays in lipid metabolism. But there has been a growing understanding of the central importance of G6PD to cellular physiology as it is a major source of NADPH that is required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system, and others. Indeed G6PD is essential for cell survival. It has also become evident that G6PD is highly regulated by many signals that affect transcription, post-translation, intracellular location, and interactions with other protein. Pathophysiologic roles for G6PD have also been identified in such disease processes as diabetes, aldosterone-induced endothelial dysfunction, cancer, and others. It is now clear that G6PD is under complex regulatory control and of central importance to many cellular processes. In this review the biochemistry, regulatory signals, physiologic roles, and pathophysiologic roles for G6PD that have been elucidated over the past 20 years are discussed.

Triggering of Ca2+ signals by NAADP-gated two-pore channels: a role for membrane contact sites?

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger implicated in many Ca2+-dependent cellular processes. It is highly unusual in that it appears to trigger Ca2+ release from acidic organelles such as lysosomes. These signals are often amplified by archetypal Ca2+ channels located in the endoplasmic reticulum. Recent studies have converged on the TPCs (two-pore channels) which localize to the endolysosomal system as the likely primary targets through which NAADP mediates its effects. 'Chatter' between TPCs and endoplasmic reticulum Ca2+ channels is disrupted when TPCs are directed away from the endolysosomal system. This suggests that intracellular Ca2+ release channels may be closely apposed, possibly at specific membrane contact sites between acidic organelles and the endoplasmic reticulum.

NOX1/nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase promotes proliferation of stellate cells and aggravates liver fibrosis induced by bile duct ligation.

UNLABELLED: Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1α mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of α-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27(kip1) , a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27(kip1) gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO. CONCLUSION: These findings indicate that reactive oxygen species derived from NOX1/NADPH oxidase oxidize and inactivate PTEN to positively regulate the Akt/FOXO4/p27(kip1) signaling pathway. NOX1 may thus promote proliferation of HSCs and accelerate the development of fibrosis following BDL-induced liver injury.

Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells.

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.