Quantitative increases of extracellular vesicles in prolonged cold storage of platelets increases the potential to enhance fibrin clot formation.

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.

Zymoseptoria tritici: A major threat to wheat production, integrated approaches to control.

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.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

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.

Detailed characterisation of circulatory nitric oxide and free radical indices--is there evidence for abnormal cardiovascular homeostasis in young women with polycystic ovary syndrome?

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.

Discovery and development of exome-based, co-dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.).

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.

Early but reversible haemostatic changes in a-symptomatic females expressing COVID-19 antibodies.

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.

Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function.

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.

Comparative UAV and Field Phenotyping to Assess Yield and Nitrogen Use Efficiency in Hybrid and Conventional Barley.

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.

Detection of angiotensin II mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (EPR) spectroscopy.

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.

Binding of endotoxin to macrophages; interactions of spin-labelled saccharide residues.

The molecular mechanisms of endotoxin action are poorly understood. A prerequisite to cellular activation by this agent must be interaction (binding) with the plasma membrane. In this study we have investigated the role of the polysaccharide region of endotoxin (LPS) in binding to macrophages and macrophage-like cell lines. The LPS molecules, from Escherichia coli O111.B4, J5 and the lipid-A, were spin labelled with 2,2,6,6-tetramethylpiperidine-N-oxyl] (Tempo) free radical in their sugar residues, and examined by electron spin resonance spectroscopy. This is the first report of the synthesis of spin-labelled endotoxins. Measurement of the rotational correlation times (Tc) indicated that the saccharide resides do not bind to membrane surface structures and suggests that the binding of LPS to macrophages is mediated by the lipid acyl chains. Anti-sera to LPS from E. coli O111.B4 was effective in binding to the polysaccharide of the same LPS bound to the cell surface.

Superoxide production by phagocytosing macrophages in relation to the intracellular distribution of oxygen.

We simultaneously measured the concentration of oxygen ([O2]) within the phagosomal and extracellular compartments of macrophages. By combining electron paramagnetic resonance (EPR) oximetry techniques with that of spin-trapping, we found that a significant difference in oxygen concentration ([O2]) exists between these two compartments and we were able to monitor (1) how [O2] in the extracellular compartment and the rate of mitochondrial consumption affected this difference in [O2], and (2) to what extent this gradient of [O2] influenced production of reactive oxygen species by phagosomes. Under conditions where the [O2] in the inflowing gas was high (210 microM; air), the [O2] in the extracellular and phagosomal compartments was 180 and 141 microM, respectively. This was sufficient to maintain maximum superoxide production in these cells. When extracellular [O2] was reduced to 84 or 36 microM, the [O2] in phagosomes within the cells (31.7 and 7.7 microM, respectively) was too low to maintain superoxide production by the NADPH-oxidase system within the phagosomes. The [O2] in the extracellular compartments of these samples, however, was always sufficient to maintain superoxide production by phagosomes at the cell surface. Our findings suggest that the distribution of oxygen surrounding and within macrophages can influence their ability to perform microbicidal and tumoricidal functions, even at an [O2] in the media that appears to be adequate.

Somatomedin-C binding to cultured human fibroblasts is dependent on donor age and culture density.

Cultured fibroblasts have been used extensively to study age-related changes in the cellular response to serum stimulation. Since somatomedin-C (Sm-C) is an important growth factor in serum, we determined if there were age-related changes in Sm-C fibroblast receptor number or affinity and if culture density influenced these changes. Skin fibroblasts were obtained from six normal donors in three separate age groups and tested for their capacity to bind Sm-C. Sparse cultures (10-15K cells/well) derived from fetal donors had an affinity for Sm-C that was 4.7-fold greater than that of cultures derived from elderly (74-96 yr old) donors [9.4 +/- 0.2 (+/- SD) compared to 2.0 +/- 0.2 X 10(10) M-1]). When grown to high density (60-100K cells/well), the affinity of the fetal cultures was significantly reduced to 2.2 +/- 0.2 X 10(10) M-1 (P less than 0.001) and was not significantly different from the affinity of high density elderly donor cultures (2.3 +/- 0.4 X 10(10) M-1). Intermediate age donors (3-14 yr old) also had a significant reduction in receptor affinity with increasing density. Fetal donor cultures showed no density-dependent changes in receptor number. Fetal donor cells at low density had 5.2 +/- 1.0 X 10(4) receptors/cell compared to 5.9 +/- 0.6 X 10(4) receptors/cell in the high density cultures. In contrast, cells derived from donors aged 3-14 yr had 12.0 +/- 1.6 X 10(4) receptors/cell at 15K cells/well and 5.1 +/- 0.6 X 10(4) at 80K cells/well (P less than 0.05). Cultures from elderly donors had significantly greater mean receptor numbers per cell compared to fetal donor cells at four of five densities tested and had a significantly lower receptor number per cell with increasing culture density 25.2 +/- 1.2 X 10(4) (10-15K cells/well) compared to 5.2 +/- 0.2 X 10(4) (60-100K) cells/well. Thus, increasing donor age at low culture density was associated with an increase in receptor number per cell and a decrease in receptor affinity. At high culture densities, these differences were not detected. These changes in Sm-C receptor number and affinity at low density could lead to donor age-related changes in the cellular response to Sm-C.

The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study.

We have studied the effects of bacterial endotoxin on the oxygen consumption of a variety of target cells, and found that the rate of utilization of oxygen by treated cells was decreased in a time- and dose-dependent manner. Precise EPR measurement of oxygen concentrations enabled us to demonstrate that this effect was linked to mitochondrial dysfunction and was particular to each cell type. Such detailed knowledge on oxygen utilization by viable whole cells and the varied effects of endotoxin are as yet undocumented. Oxygen consumption was shown to decrease quite markedly in CHO cells and kidney cells from the cortex region. Cells from the kidney medulla region had lower baseline consumption and were stimulated to increased levels of oxygen consumption on addition of similar doses of endotoxin. Macrophages exhibited a dual response in that in addition to inhibiting mitochondrial oxygen consumption, endotoxin pretreatment primed these cells to exhibit an enhanced oxidative burst on stimulation with Zymosan. These results show that endotoxin has a direct effect on normal cellular oxygen consumption and is an important parameter that must be considered when following the early effects on cells and tissues during the septic syndrome.

Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite.

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.

Endotoxin-induced changes in intrarenal pO2, measured by in vivo electron paramagnetic resonance oximetry and magnetic resonance imaging.

Electron Paramagnetic Resonance (EPR) oximetry was used to measure tissue oxygen tension (pO2-partial pressure of oxygen) simultaneously in the kidney cortex and outer medulla in vivo in mice. pO2 in the cortex region was higher compared to that in the outer medulla. An intravenous injection of endotoxin resulted in a sharp drop in pO2 in the cortex and an increase in the medulla region, resulting in a transient period of equal pO2 in both regions. In control kidneys, functional Magnetic Resonance (MR) images showed the cortex region to have high signal intensity (T2*-weighted images), indicating that this region was well supplied with oxygenated hemoglobin, whereas the outer medulla showed low signal intensity. After administration of endotoxin, we observed an immediate increase in signal intensity in the outer medulla region, reflecting an increased level of oxygenated blood in this region. Pretreatment of mice with NG-monomethyl-L-arginine prevented both the changes in tissue pO2 and distribution of oxygenated hemoglobin, suggesting that localized production of nitric oxide has a critical role to play in renal medullary hemodynamics. In combining in vivo EPR with MR images of kidneys, we demonstrate the usefulness of these techniques for monitoring renal pO2 and changes in the distribution of oxygen.

Separation and enrichment of the active component of carbon based paramagnetic materials for use in EPR oximetry.

Carbon based paramagnetic materials are frequently used for EPR oximetry, especially in vivo, but the EPR spectra of these materials often have more than one paramagnetic center and/or relatively low signal intensity. To determine whether the multi-components of carbon based materials could be separated and enriched in the active component, we used density gradient centrifugation to separate the materials into several fractions. We studied two types of coals, gloxy and Pocahontas, and found these materials to have large density distribution. The separated density fractions had very different EPR spectra and intensities. The active component from the coal material had a more homogeneous EPR signal and significantly increased EPR signal intensity, whereas for India ink, only slight changes were observed. This result can be very useful in the development of better probes for EPR oximetry.

Applications of in vivo electron paramagnetic resonance (EPR) spectroscopy: measurements of pO2 and NO in endotoxin shock.

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.

Direct detection of tissue nitric oxide in septic mice.

Although nitric oxide (NO) is a central mediator during endotoxin-induced sepsis, direct detection of tissue NO in vivo, has until recently been difficult, and techniques have relied on indirect measurement of bi-products in blood or invasive technology. We have utilized electron paramagnetic resonance (EPR) in conjunction with the spin-trapping technique to detect NO directly, and non-invasively, from the tissue of septic mice. Relative signal intensity arising from NO complexed with iron and diethyldithiocarbamate (DETC) measured directly from the liver and kidney of mice given endotoxin was maximal at 6 hours post endotoxin. We failed to detect an EPR signal from mice given pyrogen-free saline. The quality of the EPR signal obtained (high signal to noise ratio of 15:1) using our experimental set-up and L-band EPR hardware was such that we were able to establish a time course of NO production in tissue following endotoxin, and measurement of NO from other organs (kidney and spleen). Our EPR results probably reflected NO arising from inducible NO-synthase enzymes as a result of endotoxin stimulation. This technique was extended to experiments in which we first implanted an oxygen sensitive material (gloxy) into the liver of mice, and then monitored NO production following endotoxin. Due to the fact that the EPR spectrum from gloxy and that of NO-Fe-(DETC)2 do not overlap, we were able to monitor NO production and pO2 simultaneously in tissue, in real time.

Are there significant gradients of pO2 in cells?

It is widely recognized that the intracellular oxygen tension (pO2) plays an important role in cellular function and metabolism. In experimental and theoretical consideration involving the role of the pO2 the values that are used usually are those for the pO2 at the exterior of cells, because these values can be more readily measured. Such an approach is based on the assumption that the intracellular pO2 is very similar to the extracellular pO2 because oxygen freely diffuses across cell membranes and within cells, at a rate that is similar to that in the extracellular media. For the past several years we have been developing and applying electron paramagnetic resonance (EPR) techniques to test directly whether there are intracellular gradients of pO2 and if so, where they occur and what factors determine them. We previously reported significant gradients between the average pO2 in the intracellular and extracellular compartments in cell suspensions (Glockner 1989). More recently we developed techniques that enabled us to measure simultaneously the concentration of oxygen within a specific compartment, the phagosomes of activated macrophages, and the extracellular compartment and found gradients of up to 48 microM under some conditions (James 1995). The precise mechanism of the intracellular-extracellular oxygen gradient remains uncertain. The possibilities include that the diffusion of oxygen is not as free as assumed (e.g. that the cell membrane can act as a barrier) and the occurrence of active transport of O2 out of the cells. We report here on the use of a simple theoretical approach to evaluate the values of three key parameters which might account for the observed intracellular-extracellular oxygen measurements: (1) oxygen consumption; (2) the diffusion coefficient of oxygen in the cytosol; (3) the solubility of oxygen in the cytosol. We used two different models for the relationship between the oxygen consuming compartment (assumed to be primarily the mitochondria) and the intracellular compartment in which the measurements were made (especially phagosomes): uniform and non-uniform distribution of the mitochondria. Using these models and consensus values from the literature, we were unable to account for the experimentally observed differences in pO2 between the intracellular and extracellular compartments. Also we found that with the variation of any one parameter we could not plausibly account for the measurements made in the phagosomal and extracellular compartments. There are at least three logical possibilities to account for these results: 1) this methodology is erroneous and/or produces artifacts in the system resulting in invalid results; 2) the observation of a gradient in oxygen concentration between these two compartments arises from significant simultaneous variations of more than one of the critical parameters which are used conventionally to calculate potential gradients in pO2; 3) there is another factor not considered in the model which accounts for the observation (e.g. active transport; significantly higher than expected barriers to oxygen diffusion in the membrane).

A new mechanism of action of thienopyridine antiplatelet drugs - a role for gastric nitrosthiol metabolism?

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] ↔ 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.