Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi.

In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family.

Immunocytochemical localization of calretinin-containing neurons in the rat periaqueductal gray and colocalization with enzymes producing nitric oxide: a double, double-labeling study.

The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 µm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 µm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity.

NADPH-diaphorase expression in the rat jejunum after intestinal ischemia/reperfusion.

The purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase (NADPH-d) activity in the rat jejunum after a mesenteric ischemia/reperfusion injury. Nitric oxide, synthetised from L-arginine by the enzyme nitric oxide synthase, is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle. It plays an important role in the process of plasticity after the ischemia/reperfusion injury. Experimental animals were divided in two groups: the control group and the ischemic/reperfusion group, with different period of the reperfusion. The NADPH-d histochemical method has been used as a marker for the nitric oxide synthase. NADPH-d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion. Differences were predominantly detected in the myenteric plexus; they were seen in change of the neuronal shape, in the arrangement of neurons and in intensity of their staining. The NADPH-d positivity was absent in the intestinal crypts. After 1 h ischemia and 24 h reperfusion, the NADPH-d activity was gradually increased, but it was lower in comparison with the control group. On the 30th day following the ischemia/reperfusion there were no changes in NADPH-d positivity compared with the control animals. These results indicated that the jejunal ischemia/reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of NADPH-d positivity 1 h of the reperfusion insult. The gradual increasing of NADPH-d activity in 24 h following the reperfusion could be considered as a result of the plasticity process. On the 30(th) day after the ischemia/reperfusion all histochemical changes were returned to the control levels.

Effects of moderate and chronic exercise on the nitrergic system and behavioral parameters in rats.

Several reports suggest that nitric oxide (NO) could play a critical role on synaptic plasticity related to physical activity improving learning and memory; thus, physical exercise would have important effects on cerebral health. In order to analyze the long-term effects of chronic moderate physical training on the morphology and activity of nitrergic neurons belonging to the cerebral cortex, hippocampus and striatum, and their relationship with behavioral parameters. Wistar rats were aerobically trained (AT) up to the age of 18months and compared to sedentary controls (SC). At the end of the training protocol behavioral parameters were analyzed in an eight-arms radial maze. Rats were sacrificed by perfusion fixation with 4% paraformaldehyde. Brains were dissected out and coronal sections containing the three mentioned areas were obtained. The neurons expressing nitric oxide synthase (NOS) were stained using the technique of NADPH-diaphorase (NADPH-d) and their morphological and densitometric parameters were quantified by image analysis. Afterwards, the isoforms of NOS were determined by immunofluorescence. Results revealed AT rats learned faster, performed less mistakes and were more successful than SC rats in the maze. The nitrergic neurons of the cerebral cortex were larger and they had an increased number of dendrites. The NADPH-d reactivity in the cortex and striatum was upregulated. Colocalization was significant for the neuronal nitric oxide synthase (nNOS), in both groups. In conclusion, moderate and chronic exercise had a positive effect on cognitive performance and anxiety related behavior. An upregulation of the nitrergic system was detected in AT rats and this fact could be involved in this beneficial action on the aged subjects.

Nitroxidergic nerve fibers of intracerebral vessels.

Light and electron histochemical methods were used to study the structure and distribution of neurons containing NADPH diaphorase and their processes in the parietal area of the cortex in rats. Most neurons were found to be characterized by tight associations with intracerebral vessels. The smallest distances between the axon plasmalemma and the smooth myocytes of intracerebral arteries in the cerebral cortex were at least 0.3-0.5 microm. Neuron bodies were located at functionally important locations of vessels (sites at which subsidiary vessels branched off, the origins of arterioles), and their processes accompanied vessels, densely entwining the vessels with their branches. Neurons whose dendrites contacted the bodies or process of above- or below-lying neurons often sent nerve conductors to arteries, veins, or capillaries. Thus, nitroxidergic neurons or groups of these neurons may monitor the state of the circulation at different points in the vascular bed, functioning as local nerve centers.

Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis.

The consequences of the infection caused by Toxoplasma gondii in myenteric neurons of the jejunum of swines reactive to NADH-diaphorase and NADPH-diaphorase were evaluated in this study. Ten 88-day-old mixed-breed swines (Pietrain and Wessex) were assigned into two groups: Control (n=5) and Experimental (n=5), which orally received 5000 sporulated oocysts from a genotype III T. gondii strain. After 30days, the animals were anesthetized, having part of their jejunum removed and stained with NADPH-diaphorase and NADH-diaphorase. NADPHd-p neurons (nitrergic) presented increase of the number of cells per ganglion and hypertrophy. The number of NADHd-p neurons (metabolic more active) and their nuclear area decreased.

Verifying of participation of nitric oxide in morphine place conditioning in the rat medial septum using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d).

BACKGROUND: Role of nitric oxide (NO) in morphine-induced conditioned place preference (CPP) has already been proposed in the rat medial septum (MS), but no molecular evidence has been provided to clear this fact. METHODS: Effects of intraseptal injections of L-arginine and/or NG-nitro-L-arginine methyl ester (L-NAME) on morphine place conditioning in Wistar rats were examined. Morphine (2.5-7.5 mg/kg) was injected s.c. using a three-day schedule of an unbiased place preference. All of the brain samples were examined histochemically by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), the main marker for NO activation. RESULTS: Morphine induced a significant CPP in the rats. Single injections of L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not induce CPP. In addition, co-administration of morphine (5.0 mg/kg) with L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not affect morphine response. However, administration of L-arginine (0.3, 1.0 and 3.0 µg/rat) prior to morphine conditioning testing enhanced the expression of morphine response. Moreover, pre-injection of L-NAME (0.3, 1.0 and 3.0 µg/rat) to L-arginine (0.3 µg/rat) did not reverse the response to the agent. The expression of NADPH-d was observed in the rat brain samples treated by L-arginine. A decreased expression of NADPH-d was also observed in rats pre-injected by L-NAME. CONCLUSION: This finding strongly suggests that NO system in the rat MS has an impact on the expression of morphine rewarding, and that the NO participates in place conditioning induced of morphine.

[Nitroxidergic nerve fibers of intracerabral blood vessels].

Methods of light and electron microscopic histochemistry were applied to study the structure and distribution of NADPH-diaphorase-positive neurons and processes in the parietal area of rat cerebral cortex. It was found that the most of the neurons displayed close connections with the intracerebral vessels. In the cerebral cortex, the smallest distance between the axonal plasma membrane and smooth muscle cells of the intracerebral arteries was found to be no less than 0.3-0.5 microm. Neuronal cell bodies were located in the functionally important areas of the vessels (in the areas of lateral trunk branching and in arteriolar sources), while their processes accompanied the vessels, tightly embracing them with their branches. Quite often, the neurons, the dendrites of which make contacts with the bodies or processes of over- or underlying neurons, sent their nerve fibers to the arteries, veins and capillaries. Thus, nitroxidergic neurons or their groups may control the blood flow in the different areas of vascular bed, performing the functions of the local nerve center.

Distribution and structure of efferent synapses in the chicken retina.

The visual system of birds includes an efferent projection from a visual area, the isthmo-optic nucleus in the midbrain, back to the retina. Using a combination of anterograde labeling of efferent fibers, reconstruction of dye-filled neurons, NADPH-diaphorase staining, and transmission electron microscopy, we have examined the distribution of efferent fibers and their synaptic structures in the chicken retina. We show that efferent fibers terminate strictly within the ventral retina. In two completely mapped retinas, only 2 fibers from a total of 15,359 terminated in the dorsal retina. The major synapse made by each efferent fiber is with a single efferent target amacrine cell (TC). This synapse consists of 5-25 boutons of 2 microm diameter, each with multiple active zones, pressed into the TC soma or synapsing with a basketwork of rudimentary TC dendrites in the inner nuclear layer (INL). This basketwork, which is sheathed by Muller cell processes, defines a private neuropil in the INL within which TCs were also seen to receive input from retinal neurons. In addition to the major synapse, efferent fibers typically produce several very thin processes that terminate nearby in single small boutons and for which the soma of a local amacrine cell is one of the likely postsynaptic partners. A minority of efferent fibers also give rise to a thicker process, terminating in a strongly diaphorase-positive ball about 5 microm in diameter.

Increase in NADPH-diaphorase-positive and neuronal NO synthase immunoreactive neurons in the rat spinal trigeminal nucleus following infusion of a NO donor--evidence for a feed-forward process in NO production involved in trigeminal nociception.

Nitric oxide (NO) donors, which cause delayed headaches in migraineurs, have been shown to activate central trigeminal neurons with meningeal afferent input in animal experiments. Previous reports indicate that this response may be due to up-regulation of NO-producing cells in the trigeminal brainstem. To investigate this phenomenon further, we determined nitric oxide synthase (NOS)-containing neurons in the rat spinal trigeminal nucleus (STN), the projection site of nociceptive trigeminal afferents, following infusion of the NO donor sodium nitroprusside (SNP). Barbiturate anaesthetized rats were infused intravenously with SNP (50 microg/kg) or vehicle for 20 min or 2 h, and after periods of 3-8 h fixed by perfusion. Cryostat sections of the medulla oblongata containing the caudal STN were histochemically processed for detection of nicotineamide adenine dinucleotide phosphate (NADPH)-diaphorase or immunohistochemically stained for NOS isoforms and examined by light and fluorescence microscopy. The number of neurons positive for these markers was determined. Various forms of neurons positive for NADPH-diaphorase or immunoreactive to neuronal NOS (nNOS) were found in superficial and deep laminae of the STN caudalis and around the central canal. Neurons were not immunopositive for endothelial (eNOS) or inducible (iNOS) NOS isoforms. The number of NADPH-diaphorase-positive neurons increased time dependently after SNP infusion by a factor of more than two. Likewise, the number of nNOS-immunopositive neurons was increased after SNP compared with vehicle infusion. Around the central canal the number of NADPH-diaphorase-positive neurons was slightly increased and the number of nNOS+ neurons not changed after SNP treatment. NO donors increase the number of neurons that produce NO in the STN, possibly by induction of nNOS expression. Increased NO production may facilitate neurotransmitter release and promote nociceptive transmission in the STN. This mechanism may explain the delayed increase in neuronal activity and headache after infusion of NO donors.

Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice.

Naturally occurring coumarins possess anti-carcinogenic activities in part by inducing carcinogen-detoxifying enzymes glutathione S-transferase (GST) and/or NAD(P)H quinone oxidoreductase (NQO1). Our goal was to determine whether citrus coumarins induce hepatic GST and/or NQO1 via activation of Nrf2 and the antioxidant response element (ARE). First, HepG2 cells stably transfected with the ARE and a green fluorescent protein (GFP) reporter were treated with increasing concentrations of coumarins and compared to positive controls. tert-Butylhydroquinone (TBHQ) and oltipraz increased GFP fluorescence, as did coumarin, limettin, auraptene, imperatorin, and 7,8-benzoflavone, suggesting that they activate the ARE, whereas isopimpinellin did not increase GFP fluorescence. Next, the effects of orally administered coumarins and oltipraz on hepatic GST and NQO1 activities were compared in Nrf2 knockout mice or Nrf2 heterozygous mice exhibiting the wild-type phenotype. Oltipraz, auraptene, imperatorin, isopimpinellin, and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. This effect was abrogated in Nrf2(-/-) mice dosed with oltipraz, attenuated in mice Nrf2(-/-) mice treated with auraptene and imperatorin, and still significant in Nrf2(-/-) mice treated with isopimpinellin. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2(-/-) mice. These results strongly suggest that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism. Although structurally similar, isopimpinellin did not appear to activate HepG2-ARE-GFP and the Nrf2 knockout mouse study suggests that isopimpinellin may induce GST and NQO1 via additional mechanisms.

Nrf2 regulates antioxidant gene expression evoked by oxidized phospholipids in endothelial cells and murine arteries in vivo.

Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.

Activation of Nrf2 in defense against cadmium-induced oxidative stress.

Exposure to cadmium (Cd) elicits a range of adverse responses including oxidative damage and cancer. The molecular targets of Cd remain largely unidentified. Here, we analyzed the function and signal transduction of transcription factor Nrf2 in protection against Cd-induced oxidative stress. Wild-type (Nrf2 (+/+)) mouse embryonic fibroblasts (MEF) produced reactive oxygen species (ROS) at a low level, whereas treatment with Cd significantly increased the ROS production. On the other hand, Nrf2 knockout (Nrf2 (-/-)) MEF cells exhibited an elevated level of ROS under a basal condition, and Cd dramatically increased the ROS production at concentrations as low as 2 microM, resulting in increased sensitivity to Cd-induced cell death. Cd induced the basal and inducible expression of cytoprotective enzymes NQO1 and HO1 in WT MEF cells, but induction was lost in Nrf2 (-/-) MEF cells. Induction of the genes required antioxidant response elements (ARE) as Cd drove ARE-dependent reporter expression and Cd-activated Nrf2 bound to endogenous AREs in mouse hepa1c1c7 cells. Activation of Nrf2 by Cd involved stabilization of the Nrf2 protein, increased formation of Nrf2/Keap1 complex in the cytoplasm, translocation of the complex into the nucleus, and subsequently disruption of the complex. Lastly, Nrf2 was found ubiquitinated in the cytoplasm but deubiquitinated in the nucleus. The study provided a mechanistic transcriptional model in which Cd activates Nrf2 through a metal-activated signaling pathway involving a dynamic interplay between ubiquitination/deubiquitination and complex formation/dissociation of Nrf2 and Keap1.

Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a disease that compromises hepatic function and the capacity to metabolize numerous drugs. Aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor-E2 related factor 2 (Nrf2) are xenobiotic activated transcription factors that regulate induction of a number of drug metabolizing enzymes (DMEs). The purpose of the current study was to determine whether experimental NASH alters the xenobiotic activation of these transcription factors and induction of downstream DME targets Cyp1A1, Cyp2B10, Cyp3A11, Cyp4A14 and NAD(P)H:quinone oxidoreductase 1 (Nqo1), respectively. Mice fed normal rodent chow or methionine-choline-deficient (MCD) diet for 8 weeks were then treated with microsomal enzyme inducers beta-naphoflavone (BNF), 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16alpha-carbonitrile (PCN), clofibrate (CFB) or oltipraz (OPZ), known activators of AhR, CAR, PXR, PPARalpha and Nrf2, respectively. Results of this study show that (1) Hepatic PXR mRNA levels were significantly increased (1.4-fold) in mice fed MCD diet, while AhR, CAR, PPARalpha and Nrf2 were not affected. (2) The MCD diet did not alter hepatic inducibility of Cyp1A1, Cyp2B10, Cyp3A11 mRNA levels by their respective microsomal inducers. (3) Constitutive levels of Cyp4A14 mRNA were significantly increased in mice fed the MCD diet, yet further induction by clofibrate was not observed. (4) Hepatic Nqo1 mRNA levels were significantly increased by the MCD diet; however, additional induction of Nqo1 was still achievable following treatment with the Nrf2 activator OPZ.

The Nrf2 activator oltipraz also activates the constitutive androstane receptor.

Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Water-deprivation-induced expression of neuronal nitric oxide synthase in the hypothalamic paraventricular nucleus of rat.

This study was conducted to define the molecular mechanism by which dehydration induces expression of neuronal nitric oxide synthase (nNOS) in the hypothalamic paraventricular nucleus (PVN). Rats were deprived from water for 48 hr and then sacrificed immediately or 1 hr after ad libitum access to water. Another group of rats had free access to food and water and was included as euhydrate control group. The PVN sections fixed with 4% paraformaldehyde were processed for nNOS immunohistochemistry and NADPH-diaphorase (NADPH-d)/pCREB or NADPH-d/c-Fos double staining. nNOS-ir neurons significantly increased with water deprivation and decreased with rehydration, both in the posterior magnocellular (pM)- and the medial parvocellular (mP)-PVN. Most NADPH-d histostained neurons in the PVN appeared to exhibit pCREB-ir as well. Water deprivation markedly increased, and rehydration decreased, NADPH-d/pCREB neurons both in the pM- and in the mP-PVN. Gel shift assay demonstrated that dehydration may promote CREB binding to nNOS promoter in the PVN neurons. Significant amounts of NADPH-d-stained neurons in the PVN of water-deprived rats (67-68% in both the mP and the pM) exhibited c-Fos-ir. NADPH-d/c-Fos neurons in the pM-PVN were increased by water deprivation but not changed by rehydration. NADPH-d/c-Fos double-stained neurons in the mP-PVN did not significantly change depending on different water conditions. These results suggest that pCREB may play a role in dehydration-induced nNOS gene expression in the PVN neurons, and c-Fos might not be implicated in the regulatory pathway.

Filopodia are required for cortical neurite initiation.

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Quinone formation as a chemoprevention strategy for hybrid drugs: balancing cytotoxicity and cytoprotection.

Cellular defense mechanisms that respond to damage from oxidative and electrophilic stress, such as from quinones, represent a target for chemopreventive agents. Drugs bioactivated to quinones have the potential to activate antioxidant/electrophile responsive element (ARE) transcription of genes for cytoprotective phase 2 enzymes such as NAD(P)H-dependent quinone oxidoreductase (NQO1) but can also cause cellular damage. Two isomeric families of compounds were prepared, including the NO-NSAIDs (NO-donating nonsteroidal anti-inflammatory drugs) NCX 4040 and NCX 4016; one family was postulated to release a quinone methide on esterase bioactivation. The study of reactivity and GSH conjugation in model and cell systems confirmed the postulate. The quinone-forming family, including NCX 4040 and conisogenic bromides and mesylate, was rapidly bioactivated to a quinone, which gave activation of ARE and consequent induction of NQO1 in liver cells. Although the control family, including NCX 4016 and conisogenic bromides and mesylates, cannot form a quinone, ARE activation and NQO1 induction were observed, compatible with slower SN2 reactions with thiol sensor proteins, and consequent ARE-luciferase and NQO1 induction. Using a Chemoprevention Index estimate, the quinone-forming compounds suffered because of high cytoxicity and were more compatible with cancer therapy than chemoprevention. In the Comet assay, NCX 4040 was highly genotoxic relative to NCX 4016. There was no evidence that NO contributes to the observed biological activity and no evidence that NCX 4040 is an NO donor, instead, rapidly releasing NO3- and quinone. These results indicate a strategy for studying the quinone biological activity and reinforce the therapeutic attributes of NO-ASA through structural elements other than NO and ASA.

Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes.

Electrophile and free radical detoxifying enzymes including NAD(P)H:quinine oxidoreductase 1 (Nqo1) play an important role in the defense system by enhancing cellular antioxidant capacity. Chemopreventive efficacy of 3H-1,2-dithiole-3-thione (D3T) is mediated through activation of the transcription factor Nrf2 and subsequent elevation of detoxifying enzymes. In the present study, we have investigated the potential role of extracellular signal-regulated kinase (ERK) in regulation of D3T-induced and Nrf2-dependent gene expression in murine keratinocytes. Expression levels of Nqo1 were highly inducible by D3T treatment and increased nuclear levels of Nrf2 were observed in these cells. Treatment with pharmacological inhibitor of ERK1/2 largely blocked nuclear accumulation of Nrf2, ARE-driven reporter gene expression, and induction of Nqo1, as well as other phase 2 genes. Activation of ERK1/2 has been demonstrated following treatment with D3T. While, inhibitors of p38, PKC and PI3K did not affect ARE-driven gene expression. Involvement of the ERK1/2 cascade in inducible ARE-transcription activities was also observed in cells treated with other types of inducers oltipraz, sulforaphane and hydrogen peroxide. Collectively, current study suggests that phosphorylation cascade via ERK1/2 is associated with the activation process of Nrf2 and subsequent transactivation of its target gene Nqo1 following treatment with dithiolethione in murine keratinocyte.