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Biomedical subjects

P E James

Publications and source records attributed to P E James.

At least 19 recordsLinked to original sources
PubMed2006

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.

Angiotensin II↗
PubMed2002

Abnormal metabolic fate of nitric oxide in Type I diabetes mellitus.

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.

Adult↗
PubMed2001

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.

Animals↗
PubMed2000

Regulation of transcript levels of a potato gibberellin 20-oxidase gene by light and phytochrome B.

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.

Antisense Elements (Genetics)↗
PubMed1999

Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways.

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.

Animals↗
PubMed1999

Simultaneous measurement of NO(*) and PO(2) from tissue by in vivo EPR.

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.

Animals↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Carbon↗
PubMed1998

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.

Animals↗
PubMed1998

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).

Animals↗
PubMed1998

Factors affecting UV-B-induced changes in Arabidopsis thaliana L. gene expression: the role of development, protective pigments and the chloroplast signal.

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.

Arabidopsis↗
PubMed1997

Gloxy: an oxygen-sensitive coal for accurate measurement of low oxygen tensions in biological systems.

This paper describes the characteristics of a new oxygen sensitive, paramagnetic material that has some significant advantages for measurements of tissue pO2 by in vivo EPR. This paramagnetic component of Welsh coal, termed "gloxy" was found to have valuable EPR features that allow accurate measurement of low oxygen tensions in vivo; these include large oxygen-dependent changes in linewidth, a high number of paramagnetic spin centers (resulting in high signal amplitude), and stability in tissue allowing repeated pO2 measurements to be made in vivo with high precision. Renal pO2 was measured deep in the medulla region of isolated perfused kidneys and found to be lower than that in the cortex (1.7 +/- 0.05 and 7.1 +/- 0.3 mm Hg, respectively). The quality of the EPR signal obtained from the renal outer medulla and also from tumors in mice was such that the pO2 measurements were obtained with a precision of +/-3% of the measured pO2 (Kidney: 1.7 +/- 0.05 mmHg; Tumor: 1.37 +/- 0.04 mmHg). In vitro tests on the viability of cells and in vivo studies using Gloxy demonstrate the stability and inertness of this oxygen-sensitive material.

Animals↗