NADPH-diaphorase Research Articles

Distribution of NADPH-diaphorase reactivity in the central nervous system of the common toad (Bufo bufo)

We examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive elements in the central nervous system (CNS) of the common toad, Bufo bufo. The investigation involved adult male and female toads collected during the breeding season. Labeled neurons of different morphological appearances (weakly or darkly stained, unipolar, bipolar, and multipolar) and fibers were observed across all subdivisions of the amphibian brain. Overall, a similar distribution of NADPH-d-labeled neurons was observed in the brain of male and female toads. In the secondary prosencephalon NADPH-d-labeled neurons were observed in the olfactory bulbs, pallial regions, nucleus accumbens, diagonal band of Broca, septum, striatum, amygdala, suprachiasmatic and magnocellular preoptic nuclei, dorsal and ventral hypothalamus. In the diencephalon, NADPH-d-positive neurons were seen in the anterior thalamic nuclei, ventromedial and ventrolateral nuclei, central and lateral thalamic nuclei, posterior tubercle, posterodorsal division of the lateral thalamic nucleus, and in the pretectal and pretoral gray. In the mesencephalon, heavily stained neurons were present in the anterodorsal and anteroventral tegmental nuclei, magnocellular, principal and laminar nuclei of the torus semicircularis, and nucleus profundus mesencephali. In the isthmus, stained cells were observed medially and ventrally in the posterodorsal and posteroventral tegmental nuclei. In the rhombencephalon, numerous NADPH-d-stained neurons were distributed in the cerebellar nucleus, sensory and descending trigeminal nuclei, motor nuclei of the glossopharyngeal and vagus nerves, the nucleus of the solitary tract, nuclei of the hypoglossal and octaval nerves, dorsal column nucleus, central gray region, and in reticular formation. However, the complete absence of NADPH-d-stained neurons in the cerebellar cortex was an unusual feature observed in this study. The widespread distribution of NADPH-d staining in diverse cell types, belonging to a variety of neuronal systems suggests a widespread role for NADPH-d in modulating diverse functions, including sensory coding in the amphibian nervous system.

Cerebral Structures Responsible for the Formation of Autonomic Reflexes Related to Realization of Motivated Operant Movements by Rats

We studied changes in the levels of Fos immunoreactivity and NADPH diaphorase reactivity in the autonomic nuclei and catecholaminergic (CA) groups of brainstem cells in rats that realized food-procuring movements by the forelimb. Fos-immunoreactivity in the medullary nuclei of the autonomic nervous system, ANS (Sol, IRt, CVL/RVL), of these animals was appreciably higher than that in control rats. Under such conditions, a considerable part of motoneurons in the brainstem autonomic motor nuclei (10, Amb, and RAmb) were activated. In the motor nuclei of the vagus nerve, large Fos-immunoreactive (Fos-ir) neurons were observed. In rats, therefore, intense autonomic reactions closely related to realization of a motor program develop during sessions of operant food-procuring movements. The expression of protein c-Fos in brainstem CA neurons during the performance of operant movements was intensified, mostly on the contralateral side. In the brainstem CA groups А5 and А6 (LC/SC), we observed a maximum number of Fos-ir neurons, as compared with the control values. These cell groups are formed mostly of noradrenergic neurons that are the main sources of descending inhibitory inputs to the spinal thorasic and lumbar intermediolateral sympathetic nuclei. Therefore, the ANS can directly influence the functioning of muscle spindles and proprioceptors and can, as a result, influence the realization of motor program.

Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3-propanediol and glycerol production based on in silico simulation of metabolic flux distribution

BackgroundProduction directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation.MethodsFirst, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain.Results and ConclusionsDuring 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.

Characterization of Detergent Insoluble Proteome in Chronic Traumatic Encephalopathy

Quantitative proteomics of postmortem human brain can identify dysfunctional proteins that contribute to neurodegenerative disorders like Alzheimer disease (AD) and frontotemporal dementia. Similar studies in chronic traumatic encephalopathy (CTE) are limited, therefore we hypothesized that proteomic sequencing of CTE frontal cortex brain homogenates from varying CTE pathologic stages may provide important new insights into this disorder. Quantitative proteomics of control, CTE and AD brains was performed to characterize differentially expressed proteins, and we identified over 4000 proteins in CTE brains, including significant enrichment of the microtubule associated protein tau. We also found enrichment and pathologic aggregation of RNA processing factors as seen previously in AD, supporting the previously recognized overlap between AD and CTE. In addition to these similarities, we identified CTE-specific enrichment of proteins which increase with increasing severity of CTE pathology. NADPH dehydrogenase quinone 1 (NQO1) was one of the proteins which showed significant enrichment in CTE and also correlated with increasing CTE stage. NQO1 demonstrated neuropathologic correlation with hyperphosphorylated tau in glial cells, mainly astrocytes. These results demonstrate that quantitative proteomic analysis of CTE postmortem human brain can identify disease relevant findings and novel cellular pathways involved in CTE pathogenesis.

Nitric Oxide Signaling in Neurodegeneration and Cell Death.

In this tribute to Solomon H. Snyder (Sol) we discuss the mechanisms by which nitric oxide (NO) kills neurons. We provide a historical perspective regarding the discovery that glutamate excitotoxicity is mediated by NO. It also contains a discussion of the discovery that neuronal nitric oxide synthase (nNOS) catalytic activity accounts for NADPH diaphorase activity and its localization in the central nervous system. NADPH diaphorase/nNOS neurons are unique in that they are resistant to toxic effects of excess glutamate and that they are resistant to neurodegeneration in a variety of neurodegenerative diseases. NADPH diaphorase/nNOS neurons are resistant to neurotoxicity and neurodegeneration through the overexpression of manganese superoxide dismutase. The review also delves into the mechanisms by which NO kills neurons including NO's activation of the glyceraldehyde-3-phosphate dehydrogenase-dependent cell pathway. In addition, there is a review of parthanatos in which NO combines with the superoxide anion ( [Formula: see text] ) to form peroxynitrite (ONOO-) that damages DNA and activates poly (ADP-ribose) (PAR) polymerase (PARP). This ultimately leads to activation of the PARP-dependent apoptosis-inducing factor-associated nuclease, the final executioner in NO-dependent cell death. Finally, there is a discussion of potential targets that are under development that target the mechanisms by which NO kills neurons.

Afferent Pathway-Mediated Effect of α1 Adrenergic Antagonist, Tamsulosin, on the Neurogenic Bladder After Spinal Cord Injury.

PurposeThe functions of the lower urinary tract (LUT), such as voiding and storing urine, are dependent on complex central neural networks located in the brain, spinal cord, and peripheral ganglia. Thus, the functions of the LUT are susceptible to various neurologic disorders including spinal cord injury (SCI). SCI at the cervical or thoracic levels disrupts voluntary control of voiding and the normal reflex pathways coordinating bladder and sphincter functions. In this context, it is noteworthy that α1-adrenoceptor blockers have been reported to relieve voiding symptoms and storage symptoms in elderly men with benign prostatic hyperplasia (BPH). Tamsulosin, an α1-adrenoceptor blocker, is also considered the most effective regimen for patients with LUT symptoms such as BPH and overactive bladder (OAB). MethodsIn the present study, the effects of tamsulosin on the expression of c-Fos, nerve growth factor (NGF), and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the afferent micturition areas, including the pontine micturition center (PMC), the ventrolateral periaqueductal gray matter (vlPAG), and the spinal cord (L5), of rats with an SCI were investigated. ResultsSCI was found to remarkably upregulate the expression of c-Fos, NGF, and NADPH-d in the afferent pathway of micturition, the dorsal horn of L5, the vlPAG, and the PMC, resulting in the symptoms of OAB. In contrast, tamsulosin treatment significantly suppressed these neural activities and the production of nitric oxide in the afferent pathways of micturition, and consequently, attenuated the symptoms of OAB. ConclusionsBased on these results, tamsulosin, an α1-adrenoceptor antagonist, could be used to attenuate bladder dysfunction following SCI. However, further studies are needed to elucidate the exact mechanism and effects of tamsulosin on the afferent pathways of micturition.

Modular-extramodular organization in developing multisensory shell regions of the mouse inferior colliculus.

The complex neuroanatomical connections of the inferior colliculus (IC) and its major subdivisions offer a juxtaposition of segregated processing streams with distinct organizational features. While the tonotopically layered central nucleus is well-documented, less is known about functional compartments in the neighboring lateral cortex (LCIC). In addition to a laminar framework, LCIC afferent-efferent patterns suggest a multimodal mosaic, consisting of a patchy modular network with surrounding extramodular domains. This study utilizes several neurochemical markers that reveal an emerging LCIC modular-extramodular microarchitecture. In newborn and post-hearing C57BL/6J and CBA/CaJ mice, histochemical and immunocytochemical stains were performed for acetylcholinesterase (AChE), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), glutamic acid decarboxylase (GAD), cytochrome oxidase (CO), and calretinin (CR). Discontinuous layer 2 modules are positive for AChE, NADPH-d, GAD, and CO throughout the rostrocaudal LCIC. While not readily apparent at birth, discrete cell clusters emerge over the first postnatal week, yielding an identifiable modular network prior to hearing onset. Modular boundaries continue to become increasingly distinct with age, as surrounding extramodular fields remain largely negative for each marker. Alignment of modular markers in serial sections suggests each highlight the same periodic patchy network throughout the nascent LCIC. In contrast, CR patterns appear complementary, preferentially staining extramodular LCIC zones. Double-labeling experiments confirm that NADPH-d, the most consistent developmental modular marker, and CR label separate, nonoverlapping LCIC compartments. Determining how this emerging modularity may align with similar LCIC patch-matrix-like Eph/ephrin guidance patterns, and how each interface with, and potentially influence developing multimodal LCIC projection configurations is discussed.

Regional Specific Modulation of Stress-Induced Neuronal Activation Associated with the PSD95/NOS Interaction Inhibitor ZL006 in the Wistar Kyoto Rat.

BackgroundTo determine brain areas involved in the antidepressant-related behavioral effects of the selective neuronal nitric oxide synthase inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) and experimental test compound 4-((3,5-dichloro-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid (ZL006), an inhibitor of the PSD of 95 kDa/neuronal nitric oxide synthase interaction in the N-methyl-D-aspartic acid receptor signalling pathway, regional specific expression of the neuronal activation marker c-FOS was assessed following exposure to the forced swimming test in the Wistar Kyoto rat.MethodsWistar Kyoto rats were subjected to a 15-minute swim pretest (pre-forced swimming test) period on day 1. At 24, 5, and 1 hour prior to the 5-minute test, which took place 24 hours following the pre-forced swimming test, animals were treated with TRIM (50 mg/kg; i.p.), ZL006 (10 mg/kg; i.p.), or saline vehicle (1 mL/kg i.p). Behavior was recorded during both pretest and test periods.ResultsBoth TRIM and ZL006 decreased immobility time in Wistar Kyoto rats in the forced swimming test. Exposure to the forced swimming test increased c-FOS immunoreactivity in the lateral septum, paraventricular nucleus of the hypothalamus, periaqueductal grey, dentate gyrus, and ventral CA1 of the hippocampus compared with non-forced swimming test-exposed controls. Forced swimming test-induced c-FOS immunoreactivity was further increased in the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus following treatment with TRIM or ZL006. By contrast, forced swimming test-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus and ventral CA1 following treatment with TRIM or ZL006. Exposure to the forced swimming test resulted in an increase in NADPH diaphorase staining in the paraventricular nucleus of the hypothalamus. This forced swimming test-induced increase was attenuated following treatment with ZL006 and points to the paraventricular nucleus as a brain region where ZL006 acts to attenuate forced swimming test-induced neuronal nitric oxide synthase activity while concomitantly regulating region specific neuronal activation associated with an antidepressant-related response.ConclusionsThis study identified a pattern of enhanced and reduced forced swimming test-related c-FOS immunoreactivity indicative of a regulated network where inhibition of nitric oxide coupled to the N-methyl-D-aspartic acid receptor leads to activation of the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus with concomitant inhibition of the hippocampus.

The higher plant plastid NAD(P)H dehydrogenase-like complex (NDH) is a high efficiency proton pump that increases ATP production by cyclic electron flow

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex, a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by 2-fold compared with CEF pathways involving non-proton-pumping plastoquinone reductases. By virtue of this proton-pumping stoichiometry, we hypothesize that NADPH dehydrogenase not only efficiently contributes to ATP production but operates near thermodynamic reversibility, with potentially important consequences for remediating mismatches in the thylakoid energy budget.

Anti-inflammatory effects of novel polygonum multiflorum compound via inhibiting NF-κB/MAPK and upregulating the Nrf2 pathways in LPS-stimulated microglia

The incorporation of Polygonum multiflorum into the diet can result in anti-aging effects owing to its wide range of biological and pharmaceutical properties. We investigated the anti-neuroinflammatory properties of CRPE56IGIH isolated from P. multiflorum by focusing on its role in the induction of phase II antioxidant enzymes and the modulation of upstream signaling pathways. In microglia, CRPE56IGIH significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide and prostaglandin E2 production with nonspecific cytotoxicity. CRPE56IGIH also markedly inhibited LPS-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 protein and mRNA expression in the same manner as it inhibited nitric oxide and prostaglandin E2 production. In the control cells, NF-κB transactivation and nuclear translocation occurred at a baseline level, which was significantly increased in response to LPS. However, pretreatment with CRPE56IGIH concentration-dependently inhibited the LPS-induced NF-κB transactivation and nuclear translocation. The phosphorylation of Janus kinase-signal transducers and activators of transcription and mitogen-activated protein kinases was markedly upregulated by LPS, but considerably and dose-dependently inhibited by pretreatment with CRPE56IGIH. Furthermore, CRPE56IGIH induced the expression of phase II antioxidant enzymes, including heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone-1 (NQO-1). The activation of upstream signaling pathways, such as the Nrf2 pathway, was significantly increased following CRPE56IGIH treatment. Furthermore, the anti-neuroinflammatory effect of CRPE56IGIH was reversed by transfection of Nrf2, HO-1, and NQO-1 siRNA. Our results indicated that CRPE56IGIH isolated from P. multiflorum could be used as a natural anti-neuroinflammatory agent that induces phase II antioxidant enzymes via Nrf2 signaling.

Biogenic Nanoselenium Particles Effectively Attenuate Oxidative Stress-Induced Intestinal Epithelial Barrier Injury by Activating the Nrf2 Antioxidant Pathway.

In the present study, a new form of selenium nanoparticle (biogenic nanoselenium (BNS) particles) was synthesized using bacteria. The protection of BNS particles against oxidative stress-induced intestinal barrier dysfunction and the inherent mechanisms of this process were investigated, and selenomethionine (SeMet) and chemically synthesized nanoselenium (Nano-Se) particles were used for comparison. Characterization of BNS particles revealed that they were monodispersed and homogeneous spheres, with an average size of 139.43 ± 7.44 nm. In the mouse model of intestinal oxidative stress, BNS particles were found to protect the mouse intestinal barrier function and preserve intestinal redox homeostasis more efficiently than SeMet and Nano-Se. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells verified the stronger epithelial barrier-protecting effect of BNS particles against oxidative stress, with reduced cell apoptosis and an improved cell redox state. BNS activated the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and increased the expression of its downstream genes, including thioredoxin reductase (TXNRD)-1, NADPH dehydrogenase (NQO)-1, heme oxygenase (HO)-1, and thioredoxin (Trx), in dose- and time-dependent manners. In contrast, SeMet and Nano-Se merely enhanced the activity of the selenoenzymes TXNRD-1 and glutathione peroxidase (GPx)-1, indicating the role of selenium donors. Moreover, the knock down of Nrf2 significantly blocked the antioxidative effect of BNS, confirming that BNS protects the intestinal barrier from oxidative stress-induced damage by activating Nrf2 and its downstream genes. Our results suggest that BNS is a promising selenium species with potential application in treating oxidative stress-related intestinal diseases.

Pattern of Nitrergic Neuronal System Organization in the Brain of Two Holostean Fishes (Actinopterygii: Ginglymodi)

The study of the nitrergic system, formed by the networks of neurons containing the enzyme nitric oxide synthase (NOS), has been extremely useful in unraveling neuroanatomical features of the organization of the central nervous system of vertebrates. Thus, data are available for representatives of most vertebrate classes and, in particular, several studies have detailed the organization of this system in teleosts. In contrast, no information is available regarding this neurotransmission system in the brains of holosteans, an early diverged and poorly understood group of actinopterygian fishes, currently considered a sister group of teleosts that contains only 8 species. The present study provides the first detailed information on the distribution of nitrergic cell bodies and fibers in 2 holostean species of the genus Lepisosteus, the spotted gar L. oculatus and the Florida gar L. platyrhincus. NOS immunohistochemistry and the NADPH diaphorase (NADPH-d) histochemical reaction were used, and both techniques yielded identical results, with the exception of the primary olfactory and terminal nerve fibers, which only labeled for NADPH-d exclusively in L. oculatus. Double immunohistochemistry was conducted for the simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin to accurately establish the localization of the nitrergic neurons and fibers in the brain of holosteans, the neuroanatomy of which has been mostly neglected, and to assess possible interactions between these neuroactive substances. Distinct groups of nitrergic cells were located in subpallial areas, the basal hypothalamus, posterior tubercle, optic tectum and mesencephalic tegmentum, reticular formation, solitary tract nucleus, spinal cord, and amacrine cells in the retina. In addition, low numbers of nitrergic cells were observed in the pallium, suprachiasmatic nucleus, prethalamic and thalamic areas, torus lateralis and torus semicircularis, cerebellar and laterodorsal tegmental nuclei, and the ventral octavolateral area. Comparison of these results with those from other classes of vertebrates, and including a segmental analysis to correlate cell populations, reveals that the pattern of the nitrergic system in holosteans is very close to that in ancestral actinopterygian fishes and highlights conserved and derived traits.

Concerted regulation of renal plasma flow and glomerular filtration rate by renal dopamine and NOS I in rats on high salt intake.

Under high sodium intake renal dopamine (DA) increases while NOS I expression in macula densa cells (MD) decreases. To explore whether renal DA and NOS I, linked to natriuresis and to the stability of the tubuloglomerular feedback, respectively, act in concert to regulate renal plasma flow (RPF) and glomerular filtration rate (GFR). Male Wistar rats were studied under a normal sodium intake (NS, NaCl 0.24%) or a high sodium intake (HS, NaCl 1% in drinking water) during the 5 days of the study. For the last two days, the specific D1‐like receptor antagonist SCH 23390 (1 mg kg bwt−1 day−1, sc) or a vehicle was administered. HS intake increased natriuresis, diuresis, and urinary DA while it decreased cortical NOS I expression (P < 0.05 vs. NS), Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‐d) activity in MD (P < 0.001 vs. NS) and cortical nitrates+nitrites (NOx) production (NS 2.04 ± 0.22 vs. HS 1.28 ± 0.10 nmol mg protein−1, P < 0.01). Treatment with SCH 23390 to rats on HS sharply decreased hydroelectrolyte excretion (P < 0.001 vs. HS) while NOS I expression, NADPH‐d activity and NOx production increased (P < 0.05 vs. HS for NOS I and P < 0.001 vs. HS for NADPH‐d and NOx). SCH 23390 increased RPF and GFR in HS rats (P < 0.01 HS+SCH vs. HS). It did not cause variations in NS rats. Results indicate that when NS intake is shifted to a prolonged high sodium intake, renal DA through the D1R, and NOS I in MD cells act in concert to regulate RPF and GFR to stabilize the delivery of NaCl to the distal nephron.

Fine Structure and Synaptology of the Nitrergic Neurons in Medial Septum of the Rat Brain

The nitrergic neuron population and certain aspects of their connectivity (peptidergic inputs, co-localization with GABA, synaptic target distribution) were studied in the medial septum of the rat brain. The histochemical localization of NADPH diaphorase and immunohistochemical identification of nNOS at light and electron microscopic level was applied. Double-labeling experiments with galanin and leucine enkephalin, moreover the postembedding GABA immunogold staining was also carried out. NADPH diaphorase- and nNOS-immunopositive neurons could be identified inside the borders of medial septum. Out of their peptidergic inputs galanin- and leucine enkephaline-immunopositive varicose fibers were found in close apposition with nNOS-immunopositive neurons. Based on fine structural characteristics (large indented nucleus, thin cytoplasmic rim, lack of axosomatic synapses) the nitrergic neurons are suggested to be identical with the septal cholinergic nerve cells. Their boutons established asymmetrical synapses mainly on dendritic shafts and spines, some of which were also nNOS-immunopositive. A lower amount of nNOS-immunopositive boutons of presumably extrinsic origin were found to be GABAergic.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2–10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

Characterization of NADPH Diaphorase- and Doublecortin-Positive Neurons in the Lizard Hippocampal Formation

The lizard cortex has remarkable similarities with the mammalian hippocampus. Both regions process memories, have similar cytoarchitectural properties, and are important neurogenic foci in adults. Lizards show striking levels of widespread neurogenesis in adulthood and can regenerate entire cortical areas after injury. Nitric oxide (NO) is an important regulatory factor of mammalian neurogenesis and hippocampal function. However, little is known about its role in nonmammalian neurogenesis. Here, we analyzed the distribution, morphology, and dendritic complexity (Neurolucida reconstructions) of NO-producing neurons through NADPH diaphorase (NADPHd) activity, and how they compare with the distribution of doublecortin-positive (DCX+) neurons in the hippocampal formation of the neotropical lizard Tropidurus hispidus. NADPHd-positive (NADPHd+) neurons in the dorsomedial cortex (DMC; putatively homologous to mammalian CA3) were more numerous and complex than the ones in the medial cortex (MC; putatively homologous to the dentate gyrus). We found that NADPHd+ DMC neurons send long projections into the MC. Interestingly, in the MC, NADPHd+ neurons existed in 2 patterns: small somata with low intensity of staining in the outer layer and large somata with high intensity of staining in the deep layer, a pattern similar to the mammalian cortex. Additionally, NADPHd+ neurons were absent in the granular cell layer of the MC. In contrast, DCX+ neurons were scarce in the DMC but highly numerous in the MC, particularly in the granular cell layer. We hypothesize that NO-producing neurons in the DMC provide important input to proliferating/migrating neurons in the highly neurogenic MC.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

Nitrosative Stress in the Rat Retina at the Onset of Streptozotocin-Induced Diabetes

Background/Aims. Nitric oxide is a multifunctional molecule that can modify proteins via nitrosylation; it can also initiate signaling cascades through the activation of soluble guanylate cyclase. Diabetic retinopathy is the leading cause of blindness, but its pathogenesis is unknown. Multiple mechanisms including oxidative-nitrosative stress have been implicated. Our main goal was to find significant changes in nitric oxide (NO) levels and determine their association with nitrosative stress in the rat retina at the onset of diabetes. Methods: Diabetes was induced by a single intraperitoneal administration of streptozotocin. The possible nitric oxide effects on the rat retina were evaluated by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a specific marker for NO-producing neurons, detected by histochemistry performed on whole retinas and retina sections. Immunohistochemistry was also performed on retina sections for iNOS, 3-nitrotyrosine (3-NT) and glial fibrillary acidic protein (GFAP). Retinal nitric oxide levels were assessed by measuring total nitrate/nitrite concentrations. Retinal cGMP levels were determined by radioimmunoassay. Western blots for nitrotyrosine (3-NT) and oxidized proteins were performed. Results: In the present study, we found increased activity of NADPH-diaphorase and iNOS immunoreactivity in the rat retina at the onset of diabetes; this increase correlated with a remarkable increase in NO levels as early as 7 days after the onset of diabetes. However, cGMP levels were not modified by diabetes, suggesting that NO did not activate its signaling cascade. Even so, Western blots revealed a progressive increase in nitrated proteins at 7 days after diabetes induction. Likewise, positive nitrotyrosine immunolabeling was observed in the photoreceptor layer, ganglion cell layer, inner nuclear layer and some Müller cell processes in the retinas of diabetic rats. In addition, levels of oxidized proteins were increased in the retina early after diabetes induction; these levels were reduced by the administration of L-NAME. In addition, stress in Müller cells was determined by immunoreactivity to the glial fibrillary acidic protein. Conclusions: Our findings indicated the occurrence of nitrosative stress at the onset of diabetes in the rat retina and emphasized the role of NO in retinal function and the pathogenesis of retinopathy.

Decreased vascular supply leads to higher numbers of NADPH diaphorase amacrine cells in the degenerating mouse retina

Decreased vascular supply leads to higher numbers of NADPH diaphorase amacrine cells in the degenerating mouse retina

Diaphragm Muscle Contraction Decrease in a Mouse Model of Ovalbumin-Induced Allergic Airway Inflammation

Objective: We investigated diaphragm contractile and inflammatory properties of mice with OVA sensitization and challenge. Methods: BALB/c mice were sensitized to OVA by intraperitoneal (i.p.) injection at 0 and 7 days, and challenged with aerosolized OVA on 21, 22, and 23 days (O/O group). Budesonide/Formoterol combination was inhaled on days 21, 22, and 23 before OVA challenge on those same days (O/OC group). Control mice were sensitized and challenged with an aerosolized saline (O-group). The diaphragm contractile and inflammatory properties were measured on day 24. NOS activity in the diaphragm muscle was evaluated by NADPH diaphorase staining. IL-4 and IL-13 levels of BALF, as well as lung tissue and diaphragm muscle homogenates were measured by ELISA. Results: Force-frequency (F/f) curves of O/O and O/OC shifted downward in comparison with O- (p<0.05). NADPH diaphorase staining results of O/O and O/OC showed a significantly higher density compared with O-. The IL-4 level of diaphragm muscle homogenates increased significantly in the O/O compared with the O- and O/OC. Conclusions: OVA sensitization and challenge decreased diaphragm muscle contraction, increased NOS activity, IL-4 levels of diaphragm in a mouse model. Budesonide/Formoterol combination could protect diaphragm muscle weakness and inflammation. According to the traditional concept of the contemporary Immunology, neither autoimmune diseases nor allergic diseases can be cured completely. Nevertheless, a fortunate coincidence led me to discovery of a novel concept that eliminations of the causes of these diseases are possible. In other words, combinations of pathogenic antibodies with responsible cells, namely, cytolytic T lymphocytes in cases of autoimmune diseases and mast cells in cases of allergic diseases, can be decomposed by replacing the pathogenic antibodies with non-specific antibodies. In more detail, intradermal injections with a non-specific antigen preparation induce production of non-specific antibodies in the body of the patient. Repetitions of the injections bring about an accumulation of them. Accumulated non-specific antibodies will occupy most of the receptors on the surface of responsible cells. When the accumulation reaches the sufficient level, virtually no pathogenic antibodies would remain on the receptors. That is, no causes of the diseases remain. Naturally, where there is no cause, there is no disease. Details are demonstrated elsewhere.