983 resultados para hyaluronan synthase 2


Relevância:

30.00% 30.00%

Publicador:

Resumo:

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Abstract: Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons. Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase. Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

ZusammenfassungDie ATP-Synthase koppelt im Energiestoffwechsel der Zellen den Protonentransport über die biologische Membran mit der Synthese des energiespeichernden Moleküls ATP aus ADP und Phosphat. ATP-Synthasen bestehen aus 2 Subkomplexen, wobei der katalytische F1-Teil von der membranständigen Domäne abgelöst werden kann und nur zur ATP-Hydrolyse fähig ist. Der hochkooperative Reaktionsmechanismus der dreizentrigen ATP-Synthasen ist weitgehend unklar.Im Rahmen dieser Arbeit wurde der ATP-Synthasekomplex und ihr wasserlösliches katalytisches F1-Fragment aus Micrococcus luteus in präparativem Maßstab mittels chromatographischer Trennmethoden isoliert. Die Überprüfung der Funktionalität beider Enzyme erfolgte mit enzymatischen Methoden. Durch zeitaufgelöste Röntgenkleinwinkelstreuung wurde die Strukturdynamik der arbeitenden ATP-Synthase und ihres F1-Fragmentes aus Micrococcus luteus im Laufe des ATP-Hydrolysezyklus untersucht. Diese Methode diente zum Nachweis weiträumiger Konformationsänderungen innerhalb der arbeitenden Enzyme unter nativen physiologischen Bedingungen. Die zeitaufgelösten Streuexperimente fanden an der ESRF (Europäische Synchrotronstrahlungsquelle) in Grenoble (F) statt. Dort wurden für beide Enzyme im Laufe des ATP-Hydrolysezykus molekulare Bewegungen nachgewiesen. Als Referenz zu den zeitaufgelösten Messungen dienten statische Messungen zur Strukturuntersuchung der Proteine am schwächeren DESY. Anhand dieser Strukturdaten wurden Molekülmodelle der F1-ATPase und ATP-Synthase aus Micrococcus luteus konstruiert. Das Molekülmodell der F1-ATPase war die Grundlage zur Modellierung einzelner Teilschritte des ATP-Hydrolysezyklus bei 20°C. Die experimentellen Daten wurden mit einer Kippbewegung der membranseitigen Domäne der katalytischen b-Untereinheiten der F1-ATPase während des ATP-Hydrolysezyklus interpretiert.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Tiefes Wissen über den Ceramid Stoffwechsel ist rudimentär für das Verständnis der Haut-Pathophysiologie (z.B. für atopische Dermatitis oder Psoriasis ) und unabdingbar für gezielte Therapieansätze. Wenn die zwei wichtigen Barriere Funktionen, gegen transepidermalen Wasserverlust und Pathogene Invasionen undicht werden, sind bestimmte Barriere Komponenten wie z.B. Ceramide stark verändert. In Haut und Hoden führt die Deletion der Ceramid-Synthase 3 zu einem Arrest der epidermalen Reifung und der Spermatogenese, welches ihre Bedeutung für eine intakte Barriere heraushebt. Sphingosin (So), ein Abbauprodukt von Cer, wurde als antimikrobielles Mittel identifiziert. So konnte das Wachstum von Candida albicans hemmen und die Invasion von Pathogenen in tiefere Hautschichten verringern, wodurch ihre mögliche Rolle in der Therapie von Hauterkrankungen gezeigt wurde. Auch eine neue Klasse von Ceramiden, die 1-O-acylceramide, wurde entdeckt. 1-O-acylceramide könnten zu einer funktionellen Wasserdurchlässigkeit Barriere beitragen, da sie zu den hydrophobesten der epidermalen Cers gehören. Die neutrale Glucosylceramidase scheint topologisch mit der 1-Oacylceramid Produktion verbunden zu sein, sowie die Enzyme der Diacylglycerol O-Acyltransferase-2 (DGAT2) Familie eine Rolle dabei spielen könnten. Die Identifizierung der für die 1-O-acylceramid Synthese verantwortlichen Enzyme wir Gegenstand weiterer Forschung sein, jedoch zeigten Untersuchungen an Mäusen, defizient für die saure Ceramidase (Farber-Krankheit), dass Makrophagen ein weiterer potenzieller Produktionsort sein könnten.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

INTRODUCTION: N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. CASE REPORT: We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. CONCLUSION: NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

This study investigates the influence of 17β-estradiol (E2) on nitric oxide (NO) production in endothelial cell cultures and the effect of topical E2 on the survival of skin flap transplants in a rat model. Human umbilical vein endothelial cells were treated with three different E2 concentrations and nitrite (NO2) concentrations, as well as endothelial nitric oxide synthase (eNOS) protein expressions were analyzed. In vivo, random-pattern skin flaps were raised in female Wistar rats 14 days following ovariectomy and treated with placebo ointment (group 1), E2 as gel (group 2), and E2 via plaster (group 3). Flap perfusion, survival, and NO2 levels were measured on postoperative day 7. In vitro, E2 treatment increased NO2 concentration in cell supernatant and eNOS expression in cell lysates (p < 0.05). In vivo, E2 treated (gel and plaster groups) demonstrated significantly increased skin flap survival compared to the placebo group (p < 0.05). E2 plaster-treated animals exhibited higher NO2 blood levels than placebo (p < 0.05) paralleling the in vitro observations. E2 increases NO production in endothelial cells via eNOS activation. Topical E2 application can significantly increase survival of ischemically challenged skin flaps in a rat model and may augment wound healing in other ischemic situations via activation of NO production.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

BACKGROUND: nitric oxide (NO) plays an important role in the regulation of cardiovascular and glucose homeostasis. Mice lacking the gene encoding the neuronal isoform of nitric oxide synthase (nNOS) are insulin-resistant, but the underlying mechanism is unknown. nNOS is expressed in skeletal muscle tissue where it may regulate glucose uptake. Alternatively, nNOS driven NO synthesis may facilitate skeletal muscle perfusion and substrate delivery. Finally, nNOS dependent NO in the central nervous system may facilitate glucose disposal by decreasing sympathetic nerve activity. METHODS: in nNOS null and control mice, we studied whole body glucose uptake and skeletal muscle blood flow during hyperinsulinaemic clamp studies in vivo and glucose uptake in skeletal muscle preparations in vitro. We also examined the effects of alpha-adrenergic blockade (phentolamine) on glucose uptake during the clamp studies. RESULTS: as expected, the glucose infusion rate during clamping was roughly 15 percent lower in nNOS null than in control mice (89 (17) vs 101 (12) [-22 to -2]). Insulin stimulation of muscle blood flow in vivo, and intrinsic muscle glucose uptake in vitro, were comparable in the two groups. Phentolamine, which had no effect in the wild-type mice, normalised the insulin sensitivity in the mice lacking the nNOS gene. CONCLUSIONS: insulin resistance in nNOS null mice was not related to defective insulin stimulation of skeletal muscle perfusion and substrate delivery or insulin signaling in the skeletal muscle cell, but to a sympathetic alpha-adrenergic mechanism.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Listeria monocytogenes (LM) is a Gram-positive facultative intracellular bacterium that causes fatal meningoencephalitis in humans and ruminants. A current paradigm predicts that intracellular bacteria are controlled by nitric oxide (NO) whose synthesis is catalyzed by inducible nitric oxide synthase (iNOS). The ability of macrophages (Mphi) to express iNOS shows extreme interspecies variability. Here the expression of iNOS and synthesis of NO was studied in listeric encephalitis of cattle, sheep, and goats. iNOS was expressed by a subset of Mphi in cerebral microabscesses in all three species. The level of iNOS expression and the density of cells per lesion expressing iNOS was highest in cattle, intermediate in sheep, and lowest in goats. The accumulation of nitrotyrosine (NT), an indicator of local NO synthesis, was observed in lesions of cattle but not in those of small ruminants. The density of iNOS-expressing cells in lesions was inversely correlated with the number of bacteria. No species differences were observed in regard to reactive oxygen intermediate (ROI) production by stimulated granulocytes, using the flow cytometric dihydrorhodamine-123 (DHR) method indicating ROI generation. Thus, the marked species differences in iNOS expression, NT accumulation, and LM content in lesions of ruminants with listeric encephalitis are explained by different amounts of ROI produced. It suggests that variations in the ability of Mphi to synthesize NO are of pathophysiological significance in listeriosis.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.