Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.
- MeSH
- Agrobacterium enzymology genetics MeSH
- Genes, Bacterial MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Flavin Mononucleotide metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Crystallography, X-Ray MeSH
- Protein Structure, Quaternary MeSH
- Models, Molecular MeSH
- NADPH Dehydrogenase chemistry genetics metabolism MeSH
- Operon MeSH
- Oxidative Stress MeSH
- Oxidoreductases chemistry genetics metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.
- MeSH
- Microscopy, Electron MeSH
- Ferredoxins metabolism MeSH
- Photosystem I Protein Complex chemistry isolation & purification metabolism MeSH
- Hordeum chemistry enzymology radiation effects MeSH
- Plant Leaves chemistry enzymology radiation effects MeSH
- Models, Molecular MeSH
- NAD metabolism MeSH
- NADPH Dehydrogenase chemistry isolation & purification metabolism MeSH
- Native Polyacrylamide Gel Electrophoresis MeSH
- Oxidation-Reduction MeSH
- Light MeSH
- Light-Harvesting Protein Complexes chemistry isolation & purification metabolism MeSH
- Electron Transport MeSH
- Thylakoids metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Nitric oxide (NO) may play a role in the pathophysiology of excitotoxicity. It is also possible that increase in Ca²⁺ overload and NO-mediated events are involved in neuronal loss during excitotoxicity. Using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we have investigated the effects of melatonin on NADPH-d positive hippocampal neurons after kainic acid (KA) induced excitotoxicity in female rats of Wistar strain. Cytosolic Ca²⁺ (free calcium) in all the respective experimental groups was also studied. Kainic acid was administered, with a single dose of 10 mg/kg/bw (body weight) to the animals. KA treated rats were given melatonin at a dose of 20 mg/kg/bw (for 14 day). On the last day of treatment, animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anaesthesia. Cryostat sections (20 microm) were cut and stained for NADPH-d positive neurons. KA exposed animals showed a significantly increased number of NADPH-d positive neurons in the dorsal and ventral blade of the dentate gyrus (DG), hilus, CA1 and CA3 area of hippocampus, with a parallel increase in intracellular free Ca²⁺ ion concentration, as compared to the control group. KA + melatonin-treated animal groups showed reduced number of NADPH-d positive neurons in DG, hilus, CA1 and CA3 areas and a decline in cytosolic Ca²⁺ concentration, as compared to KA treated group. Our study suggests that the enhanced levels of cytosolic Ca²⁺ and nitric oxide (NO) play an important role in kainate induced excitotoxicity. Inhibition of NO production may be another means whereby melatonin can reduce oxidative damage and seems to play important role in neuroprotection.
- MeSH
- Excitatory Amino Acid Agonists pharmacology MeSH
- Behavior, Animal drug effects MeSH
- Hippocampus cytology drug effects MeSH
- Immunohistochemistry MeSH
- Rats MeSH
- Kainic Acid pharmacology MeSH
- Melatonin pharmacology MeSH
- NADPH Dehydrogenase metabolism MeSH
- Neurons drug effects physiology MeSH
- Nitric Oxide physiology MeSH
- Cell Count MeSH
- Rats, Wistar MeSH
- Free Radical Scavengers pharmacology MeSH
- Nitric Oxide Synthase metabolism MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Syndróm multiorgánovej dysfunkcie spôsobuje vo väčšine prípadov úmrtie pacienta, a preto si vyžaduje zvýšenú pozornosť. Spúšťajúcim faktorom a cieľovou štruktúrou patofyziologických dejov vedúcich k tomuto syndrómu je endotel čreva. Oxid dusnatý pôsobí ako nonadrenergný a noncholinergný neurotransmiter v hladkej svalovine tenkého čreva a hrá významnú úlohu pri takomto type poškodenia. Metodika: Experimentálne zvieratá, samce dospelých potkanov kmeňa Wistar boli rozdelené do troch skupín v závislosti od trvania reperfúzie po predchádzajúcej hodinovej ischémii. Výsledky sme porovnávali s kontrolnou skupinou zvierat bez ischemicko-reperfúzneho poškodenia. Vzorky jejuna sme analyzovali histochemickou metódou na stanovenie nikotínamidadeníndinukleotidfosfát diaforázovej aktivity, ktorá zodpovedá prítomnosti oxidu dusnatého v nervových štruktúrach. Výsledky: Aktivita nitrergických neurónov enterického nervového systému jejuna sa prudko znížila v skupine s hodinovou reperfúziou, následne sa v skupine potkanov po 24-hodinovej reperfúzii zvýšila a v skupine po 30-dňovej reperfúzii dosiahla hodnoty rovnaké, aké sme získali analýzou rezov jejuna kontrolnej skupiny. Záver: Z výsledkov vyplýva, že ischemicko-reperfúzne poškodenie výrazne ovplyvňuje neuróny enterického nervového systému znížením aktivity neurotransmiteru oxidu dusnatého hneď po ischemicko-reperfúznom poškodení. Následné zvýšenie jeho aktivity 24 hod po zásahu svedčí o kompenzačnej reakcii neurónov na poškodenie, ktorá sa pri udržaní kompenzačných mechanizmov ustáli do 30 dní po ischémii návratom do stavu, aký bol pred ischemicko-reperfúznym poškodením. Pri zlyhaní kompenzačných mechanizmov nastáva smrť do 1 týždňa po navodenej ischémii.
Even nowadays, multiple organ failure syndrome still causes high mortality, therefore it needs special attention. Endothel of the small intestine causes bacterial translocation as well as local and systemic inflammation, the main pathophysiological changes that lead to the development of the syndrome. Nitric oxide is a nonadrenergic and noncholinergic neurotransmitter in the intestinal smooth muscle and it plays an important role in the process of ischemic-reperfusion injury. Methods: Experimental animals, adult male Wistar rats, were divided into three groups according to the reperfusion period after previous ischemic episode lasting for one hour. The results were compared to a control group without experimental ischemic-reperfusion injury. Samples of the jejunum were histochemically analyzed in order to visualize the nicotinamide adenine dinucleotide phosphate diaphorase, which is a marker of the nitric oxide in the nerve structures. Results: The activity of the nitrergic neurons rapidly decreased in the group with one hour reperfusion; consequently, this activity gradually increased in the group with 24 hour reperfusion, and finally in the group with 30 days reperfusion there were no changes in the activity of the nitrergic neurons compared to the control group. Conclusion: These results indicated that the jejunal ischemic-reperfusion injury affected the neurons of the enteric nervous system and resulted in early decrease in the activity of the nitric oxide neurotransmitter one hour after the injury. Further gradual increase of its activity 24 hours after ischemic-reperfusion injury could be considered a result of the plasticity process. On day 30 after ischemic-reperfusion injury all the histochemical changes reached control levels. After the compensation mechanisms failure, death occurred within a week after elicited ischemia.
- Keywords
- ischemicko-reperfúzní poškození, inervace,
- MeSH
- Mesenteric Arteries pathology MeSH
- Adult MeSH
- Histocytochemistry MeSH
- Ischemia * metabolism MeSH
- Jejunum * chemistry innervation metabolism MeSH
- Rats MeSH
- NADPH Dehydrogenase metabolism MeSH
- Nitrergic Neurons * chemistry MeSH
- Rats, Wistar MeSH
- Reperfusion MeSH
- Intestinal Mucosa chemistry innervation metabolism MeSH
- Case-Control Studies MeSH
- Nitric Oxide Synthase chemistry MeSH
- Animals MeSH
- Check Tag
- Adult MeSH
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Lumbar Vertebrae MeSH
- Financing, Organized MeSH
- Histocytological Preparation Techniques MeSH
- Thoracic Vertebrae MeSH
- Lumbosacral Region MeSH
- Spinal Cord anatomy & histology cytology growth & development MeSH
- Microscopy MeSH
- NADPH Dehydrogenase MeSH
- Neurons MeSH
- Neurotransmitter Agents MeSH
- Nitrate Reductase (NADPH) MeSH
- Nitric Oxide metabolism MeSH
- Rats, Wistar MeSH
- Nitric Oxide Synthase MeSH
- Age Factors MeSH
- Frozen Sections MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
We used NADPH-diaphorase staining to study effects of magnesium pre-treatment during long-lasting hypoxia on the brain structure of rats. NADPH-diaphorase is an enzyme co-localized in neurons with NO-synthase that is responsible for NO synthesis. NO participates in hypoxic-ischaemic injury of the brain. Hypoxia was induced in consecutive days from the 2nd till the 11th day of postnatal life in a hypobaric chamber (for 8 hours per day). Magnesium was administered before each hypoxia exposition. At the age of 12 days, the animals were transcardially perfused with 4% buffered neutral paraformaldehyde under the deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in five hippocampal regions. In comparison to the control animals, intermittent hypoxia brought about higher density of NADPH-diaphorase positive neurons in all studied areas of the hippocampal structure: in CA1 and CA3 areas of the hippocampus and in hilus, in the dorsal and ventral blades of the dentate gyrus. Magnesium pre-treatment during hypoxia reduced number of NADPH-diaphorase positive neurons in all studied areas.
- MeSH
- Barotrauma metabolism MeSH
- Staining and Labeling methods utilization MeSH
- Animal Experimentation MeSH
- Financing, Organized MeSH
- Hippocampus metabolism physiopathology drug effects MeSH
- Magnesium diagnostic use MeSH
- Evidence-Based Medicine MeSH
- Hypoxia-Ischemia, Brain etiology metabolism physiopathology MeSH
- NADPH Dehydrogenase diagnostic use MeSH
- Nitrergic Neurons drug effects MeSH
- Nitric Oxide metabolism MeSH
- Rats, Wistar MeSH
- Statistics as Topic MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- MeSH
- Research Support as Topic MeSH
- Hippocampus enzymology physiology MeSH
- Magnesium administration & dosage pharmacology MeSH
- Fetal Hypoxia physiopathology MeSH
- Rats MeSH
- Hypoxia, Brain physiopathology MeSH
- NADPH Dehydrogenase MeSH
- Neuroprotective Agents MeSH
- Nitrergic Neurons MeSH
- Nitric Oxide antagonists & inhibitors MeSH
- Perinatology MeSH
- Nitric Oxide Synthase antagonists & inhibitors MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
Zápalový proces je nevyhnutnou odpoveďou na poškodenie rôzneho pôvodu, ktorý sa odohráva aj v centrálnom nervovom systéme (CNS). Hoci hromadiace sa údaje presvedčivo dokumentujú, že chronický zápalový proces je dôležitou súčasťou neurodegenratívnych chorôb, akými sú Alzheimerova choroba, Parkinsonova choroba, Huntingtonova choroba, menej je známe o akútnom zápalovom procese v CNS. Zatiaľ čo neurodegeneratívne choroby sú charakterizované neprítomnosťou významnej infiltrácie z krvi pochádzajúcich mononukleárnych buniek, prítomnosť mikroglie/makrofágov je typická pre akútnu neuroinflamáciu. V priebehu fokálnej, netraumatickej neuroinflamácie, ktorá je vyvolaná mikroinjekciou zymosanu do parenchýmu miechy, dochádza k miestnej aktivizácii mikroglie/makrofágov. V našej práci sme zisťovali vznik, rozsah a vývoj fokálnej lézie a priebeh aktivizácie mikroglie/makrofágov po stereotaxickej aplikácii zymosanu (100 nl) do bočných povrazcov miechy v akútnom období po podaní zymosanu (6 hod.–8 dní). Intenzívnu aktivitu NADPH-diaforázy v makrofágoch lézie sme sledovali 1–4 dni po zymosanovej injekcii, v ďalsích dňoch prežívania klesala. Naše výsledky potvrdzujú aktívnu úlohu mikroglie/makrofágov pri fokálnej netraumatickej neuroinflamácii a podporujú inovatívne názory na procesy akútnej neuroinflamácie v CNS.
Inflammation is an inevitable response on injury of different origin that takes place, as well, in the central nervous system (CNS). Although accumulating evidence suggests that chronic inflammation plays an important part in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, less is known about the processes of the acute CNS inflammation. While neurodegenerative diseases are characterized by the lack of the prominent infiltrates of blood-derived mononuclear cells the involvement of microglia/macrophages is typical for the acute neuroinflammation. Focal non-traumatic neuroinflammation produced by microinjection of zymosan to the parenchyma of spinal cord induces local activation of microglia/macrophages. In our study, we evaluated focal lesion progress and microglia/macrophages response following stereotaxic application of zymosan (100 nl) to the lateral funiculi of the spinal cord in acute post-injection time period (6 h–8 days). Intensive expression of NADPH-diaphorase in lesion macrophages was observed 1–4 days after zymosan injection, in the next days its activity declined. Our results confirm active role of microglia/macrophages in focal non-traumatic neuroinflammation and strengthen innovative view on processes of acute inflammation in CNS.
- MeSH
- Research Support as Topic MeSH
- Rats MeSH
- Macrophages enzymology MeSH
- Spinal Cord enzymology pathology MeSH
- Microglia immunology MeSH
- Models, Animal MeSH
- NADPH Dehydrogenase analysis MeSH
- Neurogenic Inflammation immunology pathology MeSH
- Animals MeSH
- Zymosan administration & dosage MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
