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BACKGROUND: Platelet derived extracellular vesicles (EVs) display a pro-coagulant phenotype and are generated throughout platelet concentrate (PC) storage. Cold storage (CS) of PCs is thought to provide a superior haemostatic advantage over room temperature (RT) storage and could prolong the storage time. However, the effect of storage conditions on EV generation and PC function is unknown. We investigated EV production under CS and RT conditions and assessed whether these EVs exhibited a more pro-coagulant phenotype in model experiments. MATERIALS AND METHODS: Buffy-coat-derived PCs in a platelet additive solution (PAS) to plasma ratio of approximately 65:35 were stored at RT (22 ± 2°C) or CS (4 ± 2°C) for a prolonged storage duration of 20 days. Impedance aggregometry assessed platelet function. EVs were isolated throughout storage and quantified using nanoparticle tracking analysis. EVs were applied to a coagulation assay to assess the impact on fibrin clot formation and lysis. RESULTS: CS produced significantly larger EVs from day 4 onwards. EV concentration was significantly increased in CS compared to RT from day 15. EVs, regardless of storage, significantly reduced time to clot formation and maximum optical density measured compared to the no EV control. Clot formation was proportionate to the number of EV applied but was not statistically different across storage conditions when corrected for EV number. CONCLUSION: EVs in CS and RT units showed similar clot formation capacity. However, the higher number of larger EVs generated in CS compared to RT suggests PC units derived from CS conditions may overall exhibit a haemostatically superior capacity compared to RT storage.
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Vesículas Extracelulares , Fibrina , Humanos , Plaquetas , Coagulação Sanguínea , Criopreservação , Preservação de SangueRESUMO
The coronavirus, COVID-19 pandemic spread across the globe in 2020, with an initial high case mortality in those requiring intensive care treatment due to serious complication. A vaccine programme was quickly developed and currently the UK is one of highest double vaccinated and boosted countries in the world. Despite tremendous efforts by the UK, new cases of COVID-19 are still occurring, due to viral mutation. A major problem associated with COVID-19 is the large a-symptomatic spread within the population. Little investigation into the a-symptomatic population has been carried out and therefore we pose that the residual effects of a-symptomatic infection is still largely unknown. Prior to mass vaccination, a multi-phased single cohort study of IgM and IgG COVID-19 antibody prevalence and the associated haemostatic changes were assessed in a Welsh cohort of 739 participants, at three time points. Positive antibody participants with age and gender matched negative antibody controls were assessed at 0, 3 and 6 months. Antibody positive females appeared to have lower antibody responses in comparison to their a-symptomatic male counterparts. Despite this initial testing showed a unique significant increase in TRAP-6-induced platelet aggregation, prothrombin time (PT) and clot initiation time. Despite coagulation parameters beginning to return to normal at 3 months, significant decreases are observed in both haemoglobin and haematocrit levels. The production of extracellular vesicles (EV) was also determined in this study. Although the overall number of EV does not change throughout the study, at the initial 0 months' time point a significant increase in the percentage of circulating pro-coagulant platelet derived EV is seen, which does not appear to be related to the extent of platelet activation in the subject. We conclude that early, but reversible changes in haemostatic pathways within the a-symptomatic, female, antibody positive COVID-19 individuals are present. These changes may be key in identifying a period of pro-coagulative risk for a-symptomatic female patients.
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COVID-19 , Hemostáticos , Estudos de Coortes , Feminino , Humanos , Imunoglobulina G , Masculino , Pandemias/prevenção & controle , SARS-CoV-2RESUMO
With the commercialization and increasing availability of Unmanned Aerial Vehicles (UAVs) multiple rotor copters have expanded rapidly in plant phenotyping studies with their ability to provide clear, high resolution images. As such, the traditional bottleneck of plant phenotyping has shifted from data collection to data processing. Fortunately, the necessarily controlled and repetitive design of plant phenotyping allows for the development of semi-automatic computer processing tools that may sufficiently reduce the time spent in data extraction. Here we present a comparison of UAV and field based high throughput plant phenotyping (HTPP) using the free, open-source image analysis software FIJI (Fiji is just ImageJ) using RGB (conventional digital cameras), multispectral and thermal aerial imagery in combination with a matching suite of ground sensors in a study of two hybrids and one conventional barely variety with ten different nitrogen treatments, combining different fertilization levels and application schedules. A detailed correlation network for physiological traits and exploration of the data comparing between treatments and varieties provided insights into crop performance under different management scenarios. Multivariate regression models explained 77.8, 71.6, and 82.7% of the variance in yield from aerial, ground, and combined data sets, respectively.
RESUMO
Syngenta is one of the major agrochemical companies with enormous breadth of technologies in Crop Protection, Seeds and Seed Care. Through an exceptionally broad product range and research investment, we are not only able to provide the grower with integrated offers now but also truly innovative and transformative technologies in the future. In this commentary Syngenta scientists give their views on the key wheat pathogen Zymoseptoria tritici from its business importance in Europe, the way we screen new Z. tritici fungicides, the way we monitor the evolution of fungicide resistance and breed for Z. tritici resistance. These four points are continuously revisited and adapted during the development of new fungicides, and academic collaborations are critically important to stay at the fore front of developments in cell biology, physiology and genetic research.
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Ascomicetos/efeitos dos fármacos , Cruzamento , Resistência à Doença/genética , Fungicidas Industriais/isolamento & purificação , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Triticum/microbiologia , Europa (Continente) , Triticum/genéticaRESUMO
This article outlines a new hypothesis that illustrates the potential role of the stomach (and subsequent chemical reactions involving nitrite therein) in modifying thienopyridines, such as clopidogrel. Gastric modification of thienopyridines can occur before standard accepted biotransformation pathways ensue. We hypothesised that thienopyridines expose the free thiol group once acidified (by the stomach) before biotransformation into active metabolites, and in the presence of nitrite (from saliva and the stomach) to form nitrosothiol derivatives (Thienopyridine induced-SNO formation). We have performed in vitro studies with each of the thienopyridines tablets/compounds confirming direct Th-SNO formation from the parent (inactive) drug by the following mechanism. Thienopyridine-SH + H(+ (Stomach)) + [Formula: see text] â Thienopyridine-SNO + H2O Thienopyridine-SNO (an S-nitrosothiol molecule) would have the potential to participate in all the reactions expected of native nitric oxide (NO) with added benefit that the NO "moiety" is protected, transportable and largely preserved from further reactive metabolism. All these biochemical steps are present in humans and could occur prior to enzymatic biotransformation.
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Mucosa Gástrica/metabolismo , Nitrogênio/metabolismo , Inibidores da Agregação Plaquetária/química , Piridinas/química , S-Nitrosotióis/metabolismo , Compostos de Sulfidrila/metabolismo , Síndrome Coronariana Aguda/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Clopidogrel , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Nitratos/química , Óxido Nítrico/química , Oxigênio/química , Inibidores da Agregação Plaquetária/efeitos adversos , Receptores Purinérgicos P2Y12/metabolismo , Ticlopidina/análogos & derivados , Ticlopidina/químicaRESUMO
STUDY QUESTION: Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers. WHAT IS KNOWN ALREADY: Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear. STUDY DESIGN, SIZE, DURATION: Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2)). PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray. MAIN RESULTS AND THE ROLE OF CHANCE: PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05). LIMITATIONS, REASON FOR CAUTION: Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.
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Anexina A5/sangue , Plaquetas/metabolismo , Síndrome do Ovário Policístico/metabolismo , Adulto , Estudos de Casos e Controles , Micropartículas Derivadas de Células/metabolismo , Feminino , Humanos , Resistência à Insulina , Fatores de RiscoRESUMO
OBJECTIVE: To assess circulating biochemical indices of endothelial function and nitro-oxidative stress in women with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. POPULATION: Seventeen women with PCOS and eighteen age- and body mass index-matched healthy volunteers. METHODS: Nitric oxide (NO) metabolite levels were assessed by chemiluminescence. Electron paramagnetic resonance spectroscopy with spin trapping was used to assess oxidative stress ex vivo and in vitro. Antioxidant capacity was measured using oxygen radical absorbance. MAIN OUTCOME MEASURES: Biochemical indices of endothelial function, including NO metabolites, lipid-derived radicals and antioxidant capacity. RESULTS: Plasma NO metabolites were similar in the two groups (nitrite: 257±116 nmol/l [PCOS], 261±135 nmol/l [controls] P=0.93; nitrate: 27±7 µmol/l [PCOS], 26±6 µmol/l [controls] P=0.89). Alkoxyl free radicals (lipid-derived) were detected as the dominant species, but levels were not different between women with PCOS and controls whether measured directly ex vivo (median 7.2 [range 0.17-16.73]e6 arbitrary units [a.u.] and 7.2 [1.7-11.9]e6 a.u., respectively, P=0.57) or when stimulated in vitro to test radical generation capacity (1.23 [0.3-5.62]e7 a.u. and 1.1 [0.48-15.7]e7 a.u. respectively, P=0.71). In regression analysis, visceral fat area was independently associated with in vitro oxidative potential (ß=0.6, P=0.002). Total plasma antioxidant capacity (94±30% [PCOS], 79±24% [controls], P=0.09) and plasma hydroperoxides (7.5±4 µmol/l [PCOS], 6.7±5 µmol/l [controls], P=0.21) were not different between groups. However, lipophilic antioxidant capacity was lower in women with PCOS compared with controls (92±32 and 125±48%, respectively, P=0.02). CONCLUSIONS: Young overweight women with PCOS display a reduced lipophilic antioxidant capacity compared with healthy volunteers, but no change in circulating free radicals or nitro-oxidative stress.
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Endotélio/metabolismo , Peróxidos Lipídicos/sangue , Óxido Nítrico/sangue , Obesidade/sangue , Estresse Oxidativo , Síndrome do Ovário Policístico/sangue , Espécies Reativas de Oxigênio/sangue , Adolescente , Adulto , Glicemia , Índice de Massa Corporal , Estudos de Casos e Controles , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Humanos , Resistência à Insulina , Gordura Intra-Abdominal , Medições Luminescentes , Pessoa de Meia-Idade , Obesidade/complicações , Sobrepeso/sangue , Sobrepeso/complicações , Síndrome do Ovário Policístico/complicações , Análise de Regressão , Gordura Subcutânea , Adulto JovemRESUMO
Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)-based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP-based wheat markers have been made available via the use of next-generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial-scale breeding programmes. To identify exome-based co-dominant SNP-based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re-sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co-dominant. Validation of a subset of these putative co-dominant markers confirmed that 96% were true polymorphisms and 65% were co-dominant SNP assays. The new co-dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web-based database to facilitate their use on genotyping programmes worldwide.
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Exoma/genética , Polimorfismo de Nucleotídeo Único , Triticum/genética , Mapeamento Cromossômico , PoliploidiaRESUMO
OBJECTIVE: Pharmacological activation of the nuclear receptor PPARγ is linked to numerous beneficial effects in the contexts of inflammation, lipid homeostasis, Type-2 Diabetes (T2D) and atherosclerosis. These beneficial effects include priming of circulating monocytes for differentiation towards an 'alternative' anti-inflammatory M2 macrophage phenotype. As we have recently shown that participation in low-intensity exercise increases PPARγ expression and activity in leukocytes from previously sedentary individuals, we aimed to elucidate whether low-intensity exercise elicited a pattern of gene expression similar to that reported for M2 monocyte-macrophage differentiation. METHODS: 17 sedentary individuals undertook an 8-week low-intensity exercise programme (walking 10,000steps/day, three times/week). Changes in expression of PPARs and the PPARγ co-activators PGC-1α and PGC-1ß; Th2 (IL-4; IL-10) and Th1 (IL-6) cytokines; and markers for the M2 (AMAC1, CD14, MR, IL-4) and the 'classical' pro-inflammatory M1 (MCP-1, TNFα, IL-6) phenotypes, were determined using RT-PCR (to assess leukocyte mRNA expression) and ELISA (to assess plasma cytokine levels). RESULTS: Exercise was associated with upregulation of M2 markers, PGC-1α and PGC-1ß, and with downregulation of M1 markers. Moreover, plasma levels of Th2 cytokines increased after exercise, while those of Th1 cytokines decreased. However, other PPARs (PPARα; PPARß/δ) did not undergo marked exercise-induced activation or upregulation. Thus, participation in low-intensity exercise may prime monocytes for differentiation towards an M2 macrophage phenotype via PPARγ/PGC-1α/ß. CONCLUSION: Given the similarities between these effects and pharmacologically induced M2 polarisation, we propose that exercise-induced PPARγ/PGC-1α/ß-mediated M2 polarisation may constitute a novel anti-inflammatory benefit of low-intensity exercise.
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Exercício Físico , Regulação da Expressão Gênica , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Leucócitos/metabolismo , PPAR gama/metabolismo , Células Th2/citologia , Adulto , Diferenciação Celular , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
BACKGROUND AND PURPOSE: It is postulated that nitrite requires reduction to nitric oxide in order to exert its relaxant effect upon isolated hypoxic vessels. Herein, we evaluate the relative contribution of nitric oxide and characterize the downstream mechanisms of nitrite-induced vasorelaxation. EXPERIMENTAL APPROACH: Aortic rings were treated with pharmacological agents and exposed to hypoxia (<1% O(2)). Following pre-constriction, nitrite (10 microM final) was added to appropriate baths; isometric tension was recorded throughout. KEY RESULTS: Nitrite (under hypoxic conditions at physiological pH) is capable of exerting physiological effects that cannot be completely inhibited by the inhibitor of soluble guanylate cyclase (sGC), 1H [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or a nitric oxide scavenger (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide). Simultaneous blockade of both sGC and cyclooxygenase (COX) completely inhibited the response to nitrite. With regard to the nitric oxide-dependent component, we confirm that aldehyde oxidase, but not xanthine oxidase or endothelial nitric oxide synthase, was important for the actions of nitrite in our model. CONCLUSIONS AND IMPLICATIONS: Nitric oxide generated from nitrite is not exclusively responsible for the physiological actions observed in isolated hypoxic vessels. Nitrite operates via different pathways dependent on the presence or absence of endothelium to produce vasorelaxation. In intact vessels, both sGC and COX enzymes appear to be important. Irrespective of this difference in relaxation mechanism, nitrite is capable of producing the same maximum relaxation, regardless of the presence of endothelium. Having investigated possible nitrite reduction sites, we confirm that aldehyde oxidase is important for the actions of nitrite.
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Óxido Nítrico/fisiologia , Nitritos/metabolismo , Vasodilatação/fisiologia , Vasodilatadores/metabolismo , Animais , Aorta Torácica/fisiologia , Hipóxia Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Técnicas In Vitro , Masculino , Relaxamento Muscular/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Coelhos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase Solúvel , Vasodilatação/efeitos dos fármacosRESUMO
We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood-brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia-mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O(2)) and following 6 h passive exposure to hypoxia (12% O(2)). Blood flow velocity in the middle cerebral artery (MCAv) and mean arterial blood pressure (MAP) were measured for determination of CA following calculation of transfer function analysis and rate of regulation (RoR). Nine subjects developed clinical AMS (AMS+) and were more hypoxaemic relative to subjects without AMS (AMS-). A more marked increase in the venous concentration of the ascorbate radical (A(*-)), lipid hydroperoxides (LOOH) and increased susceptibility of low-density lipoprotein (LDL) to oxidation was observed during hypoxia in AMS+ (P < 0.05 versus AMS-). Despite a general decline in total nitric oxide (NO) in hypoxia (P < 0.05 versus normoxia), the normoxic baseline plasma and red blood cell (RBC) NO metabolite pool was lower in AMS+ with normalization observed during hypoxia (P < 0.05 versus AMS-). CA was selectively impaired in AMS+ as indicated both by an increase in the low-frequency (0.07-0.20 Hz) transfer function gain and decrease in RoR (P < 0.05 versus AMS-). However, there was no evidence for cerebral hyper-perfusion, BBB disruption or neuronal-parenchymal damage as indicated by a lack of change in MCAv, S100beta and neuron-specific enolase. In conclusion, these findings suggest that AMS is associated with altered redox homeostasis and disordered CA independent of barrier disruption.
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Doença da Altitude/sangue , Radicais Livres/sangue , Doença Aguda , Adulto , Doença da Altitude/fisiopatologia , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Barreira Hematoencefálica/fisiologia , Encéfalo/fisiopatologia , Circulação Cerebrovascular , Cefaleia/fisiopatologia , Homeostase , Humanos , Hipóxia/sangue , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Hipóxia Encefálica/sangue , Hipóxia Encefálica/fisiopatologia , Masculino , Estresse Oxidativo , Adulto JovemRESUMO
We previously identified an action of nitric oxide (NO) within the nucleus tractus solitarii (NTS) that attenuates the cardiac component of the baroreceptor reflex. In the present study we have tested the hypothesis that angiotensin II (AngII), acting on angiotensin type 1 receptors (AT1R), can release NO within the NTS and that its actions are mediated by soluble guanylate cyclase (sGC). Utilising cryogenic electron paramagnetic resonance (EPR), we have detected NO release in brainstem samples following AngII, but not saline, microinjections into the NTS. In these experiments, we confirmed that both AngII and a NO donor (diethylamine NONOate) in the NTS both depressed the baroreflex bradycardia. In additional studies, we showed that the latter effects were both sensitive to blockade of sGC using 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). To initiate studies to resolve the cellular source of NO released by angiotensin II in the NTS, we performed immunohistochemical/electron microscopy studies on the distribution of AT1R. We found AT1R located on NTS neurones and blood vessels. Since a rise in intracellular calcium [Ca]i levels is prerequisite for nNOS activation, we imaged responses in [Ca]i in NTS neurones during exposure to AngII in vitro using confocal microscopy. Our data indicate a paucity of neurones showing changes in [Ca]i when exposed to AngII (200 nM). We suggest that AngII-induced release of NO is from non-neuronal sites. With the presence of AT1R on blood vessel endothelial cells we propose that AngII released NO in the NTS is due to activation of endothelial nitric oxide synthase located within the endothelium. The present study supports the novel concept that AngII can trigger NO release in the NTS by a mechanism of vascular-neuronal signalling that affects central neuronal networks regulating cardiovascular function.
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Angiotensina II/farmacologia , Óxido Nítrico/metabolismo , Núcleo Solitário/efeitos dos fármacos , Núcleo Solitário/enzimologia , Animais , Barorreflexo/efeitos dos fármacos , Interações Medicamentosas , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/farmacologia , Hidrazinas/farmacologia , Imuno-Histoquímica/métodos , Técnicas In Vitro , Masculino , Microscopia Imunoeletrônica/métodos , Doadores de Óxido Nítrico/farmacologia , Óxidos de Nitrogênio/farmacologia , Oxidiazóis/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/ultraestrutura , Núcleo Solitário/metabolismo , Núcleo Solitário/ultraestruturaRESUMO
AIMS/HYPOTHESIS: Reduced bioavailability of endothelium-derived nitric oxide is implicated in diabetic macrovascular and microvascular disease. In patients with diabetes, we hypothesised that protein glycosylation can alter nitric oxide binding affinity of haemoglobin and plasma proteins, hence reducing nitric oxide availability and causing an alteration in nitric oxide metabolism. METHODS: Binding of nitric oxide to haemoglobin was studied across a range of glycosylation levels in vitro (HbA(1c) 5.9 to 9.8%). In clinical studies nitrate, nitrite, nitrosyl haemoglobin and plasma nitrosothiols were measured in venous blood from 23 patients with uncomplicated Type I (insulin-dependent) diabetes mellitus and 17 non-diabetic control subjects. Samples were analysed at baseline and after nitric oxide was added ex vivo. RESULTS: Nitric oxide-haemoglobin binding was increased at a HbA(1c) greater than 8.5% compared with 5.9% (p<0.01). Basal nitrosyl haemoglobin was higher in diabetic patients compared with the control subjects (0.59+/-0.12 micro mol/l vs 0.24+/-0.12 micro mol/l, p<0.05). Plasma nitrosothiols, and nitrite and nitrate (NOx) concentrations were similar in diabetic patients compared with the control subjects (7.64+/-0.79 micro mol/l vs 5.93+/-0.75 micro mol/l, 13.98+/-2.44 micro mol/l vs 12.44+/-2.15 micro mol/l, respectively). In blood from diabetic patients, added nitric oxide was metabolised preferentially to nitrosyl haemoglobin and plasma nitrosothiols, with a twofold increase in nitrosyl haemoglobin observed across all concentrations of nitric oxide (p<0.05). These preferential increases correlated positively with HbA(1c). CONCLUSION/INTERPRETATION: Nitrosyl haemoglobin is increased in patients with Type I diabetes. Preferential metabolism to nitrosyl haemoglobin and nitrosothiols occurs after increases in nitric oxide. Our results show an accentuated association between nitric oxide and glycosylated proteins, especially deoxygenated haem. An altered metabolic fate of nitric oxide could influence microvascular regulation and tissue perfusion.
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Diabetes Mellitus Tipo 1/sangue , Hemoglobinas Glicadas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacocinética , Adulto , Idade de Início , Disponibilidade Biológica , Glicemia/metabolismo , Coleta de Amostras Sanguíneas/métodos , Colesterol/sangue , Espectroscopia de Ressonância de Spin Eletrônica , Glicosilação , Hemoglobinas/metabolismo , Humanos , Cinética , Nitratos/sangue , Óxido Nítrico/sangue , Nitritos/sangue , Triglicerídeos/sangueRESUMO
Recent developments of EPR instrumentation that allow the use of large tissue samples or whole animals and the ability to image spatially resolved EPR signals has led to novel applications of EPR spectroscopy in vivo. Utilising a 1 GHz EPR spectrometer with a 3.4-cm birdcage resonator, it was possible to detect and measure nitric oxide and oxygen in the livers of mice with lipopolysaccharide (LPS)-induced septic shock. Nitric oxide was detected as the nitric oxide (NO) complex of Fe-diethyldithiocarbamic acid (Fe-DETC) while pO2 was measured from the EPR linewidth of the oxygen-sensitive coal material 'gloxy'. LPS treatment stimulated the production of nitric oxide in the liver and the general circulation and the oxygenation of liver tissue was decreased. Selective placement of the EPR probes allowed images of nitric oxide and oxygen to be obtained in the liver. The spectral and spatial information obtained with this technique will allow improved understanding of the pathophysiology of such diseases.
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Óxido Nítrico/análise , Oxigênio/análise , Choque Séptico/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Up to three gibberellin (GA) 20-oxidase genes have now been cloned from several species including Arabidopsis, bean (Phaseolus vulgaris), and potato (Solanum tuberosum). In each case the GA 20-oxidase genes exhibit different patterns of tissue expression. We have performed extensive northern analysis on one of the potato GA 20-oxidase genes (StGA20ox1), which is the only one that shows significant transcript levels in leaves. We show that levels of StGA20ox1 transcript are elevated in transgenic antisense plants that have reduced levels of phytochrome B (PHYB) compared with wild-type plants, implicating PHYB in the control of GA biosynthesis. We show that StGA20ox1 transcript levels vary in leaves of different age throughout the plant and cycle throughout the day, furthermore they are up-regulated by light and down-regulated in the dark. The degree of the response to the light-on signal is similar in potato plants deficient in phytochrome A or PHYB and wild-type plants. The induction of StGA20ox1 by blue light raises the possibility that a blue light receptor may be involved in the control of this gene by light.
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Luz , Oxigenases de Função Mista/genética , Células Fotorreceptoras , Fitocromo/metabolismo , Solanum tuberosum/genética , Fatores de Transcrição , Elementos Antissenso (Genética) , Northern Blotting , Regulação da Expressão Gênica de Plantas , Giberelinas/biossíntese , Giberelinas/metabolismo , Oxigenases de Função Mista/metabolismo , Fitocromo B , Reguladores de Crescimento de Plantas/biossíntese , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismoRESUMO
Chronic human exposure to low levels of inorganic arsenic increases the incidence of vascular diseases and specific cancers. Exposure of endothelial cells to environmentally relevant concentrations of arsenic trioxide (arsenite) induces oxidant formation, activates the transcription factor NF-kappaB, and increases DNA synthesis (Barchowsky et al., Free Radic. Biol. Med. 21, 783-790, 1996). We show, in the current study, that arsenite induces concentration-dependent cell proliferation or death in primary porcine aortic endothelial cells. Low concentrations caused cell proliferation and were associated with increased superoxide and H(2)O(2) accumulation, cSrc activity, H(2)O(2)-dependent tyrosine phosphorylation, and NF-kappaB-dependent transcription. These concentrations were insufficient to activate MAP kinases. However, the MAP kinases, extracellular signal-regulated kinase and p38, were activated in response to levels of arsenite that caused cell death. These data suggest that arsenite-induced oxidant accumulation and subsequent activation of tyrosine phosphorylation represent a MAPK-independent pathway for phenotypic change and proliferation in vascular cells.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Morte Celular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Óxidos/farmacologia , Animais , Anticorpos/imunologia , Aorta/fisiologia , Trióxido de Arsênio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes , Detecção de Spin , Suínos , Transfecção/efeitos dos fármacos , Tirosina/metabolismoRESUMO
We describe a technique that utilizes electron paramagnetic resonance (EPR) to measure NO(*) and pO(2) directly, and non-invasively, from tissue in vivo. Diethyldithiocarbamate (DETC) was injected with iron so as to complex with NO(*) in the tissue. Gloxy (an oxygen-sensitive, paramagnetic material) was also implanted into the tissue of interest (brain or liver). Because the signals arising from gloxy and NO-Fe-(DETC)(2) did not overlap, they could be monitored and measured simultaneously in vivo. The gloxy was not responsive to NO(*) and/or DETC. As model systems we either injected SNP (an NO(*) donor) into animals and monitored NO(*) and pO(2) simultaneously from brain, or endotoxin (lipopolysaccharide; LPS) was injected in order to induce a septic episode and NO(*) and pO(2) measured from liver. We found a close correlation between levels of SNP-derived NO(*) and brain pO(2) in vivo. During sepsis, liver pO(2) decreased dramatically at 300-360 min after endotoxin injection, and this coincided with decreases in mean arterial blood pressure and increased tissue NO(*) detected. These studies demonstrate the potential usefulness of this technique for making direct in vivo measurements of NO(*) and pO(2) simultaneously from tissue.
Assuntos
Química Encefálica , Espectroscopia de Ressonância de Spin Eletrônica , Fígado/química , Óxido Nítrico/análise , Oxigênio/análise , Animais , Carbono/química , Hipóxia Celular , Ditiocarb/análogos & derivados , Endotoxemia/metabolismo , Compostos Ferrosos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Doadores de Óxido Nítrico , Nitroprussiato , Especificidade de Órgãos , Pressão Parcial , VasodilataçãoAssuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias Mamárias Animais/tratamento farmacológico , Metaloendopeptidases/antagonistas & inibidores , Oxigênio/análise , Fenilalanina/análogos & derivados , Tiofenos/uso terapêutico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Feminino , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/fisiopatologia , Camundongos , Camundongos Endogâmicos C3H , Oximetria/métodos , Fenilalanina/uso terapêuticoRESUMO
Elevated levels of arsenite, the trivalent form of arsenic, in drinking water correlates with increased vascular disease and vessel remodeling. Previous studies from this laboratory demonstrated that environmentally relevant concentrations of arsenite caused oxidant-dependent increases in nuclear transcription factor levels in cultured porcine vascular endothelial cells. The current studies characterized the reactive species generated in these cells exposed to levels of arsenite that initiate cell signaling. These exposures did not deplete 5'-triphosphate, nor did they affect basal or bradykinin-stimulated intracellular free Ca2+ levels, indicating that they were not lethal. Electron paramagnetic resonance (EPR) spectroscopy, including spin trapping with carboxy-PTIO (cPTIO), demonstrated that 5 microM or less of arsenite did not increase *NO levels over a 30-min period relative to *NO release stimulated by bradykinin. However, these same levels of arsenite rapidly increased both oxygen consumption and superoxide formation, as measured by EPR oximetry and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), respectively. Pretreatment of the cells with DPI, apocynin, or superoxide dismutase abolished arsenite-stimulated DMPO-OH adduct formation. Finally arsenite increased extracellular accumulation of H2O2, measured as oxidation of homovanillic acid, with the same time and dose dependence, as seen for superoxide formation. These data suggest that superoxide and H2O2 are the predominant reactive species produced by endothelial cells after arsenite exposures that stimulate cell signaling and activate transcription factors.
Assuntos
Arsenitos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta , Arsenitos/administração & dosagem , Benzoatos , Cálcio/metabolismo , Células Cultivadas , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Peróxido de Hidrogênio/metabolismo , Imidazóis , Óxidos de Nitrogênio/metabolismo , Consumo de Oxigênio , Marcadores de Spin , Superóxidos/metabolismo , SuínosRESUMO
Gene expression is known to change in response to UV-B radiation. In this paper, we have investigated three factors in Arabidopsis leaves that are likely to influence these changes: development, protective pigments and the 'chloroplast signal'. During late leaf development the major change in pigment composition, after exposure to UV-B radiation, is an increase in UV-absorbing pigments. Chl and Chl a/b ratio do not change substantially. Similarly Chl fluorescence is not altered. In contrast, RNA transcripts for photosynthetic proteins are reduced more in older leaves than in young leaves. To determine the role of flavonoids in UV-B protection, plants of Arabidopsis mutant tt-5, which have reduced flavonoids and sinapic esters, were exposed to UV-B and RNA transcript levels determined. The tt-mutants were more sensitive to UV-B radiation than wild-type. To examine the role of the chloroplast signal in regulating UV-B-induced changes in gene expression, Arabidopsis gun mutants (genome uncoupled) have been used. The results show that UV-B-induced down-regulation still takes place in gun mutants and strongly suggests that the chloroplast signal is not required. Overall, this study clearly demonstrates that UV-B-induced changes in gene expression are influenced by both developmental and cellular factors but not chloroplastic factors.