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Oxygen tension in a murine tumor: a combined EPR and MRI study.

The effects of fusinite, a new agent for the measurement of the concentration of oxygen in vivo by EPR, on MRI images have been studied. There was little effect on spin-echo T1-weighted images, but the fusinite resulted in large effects on T2-weighted images. Especially large effects could be observed when using spoiled gradient echo sequences (SPGR). The observed measurements of oxygen by EPR corresponded to the relative vascularity at the site of the fusinite both histologically and by MRI studies of vascularity using Gd-DTPA as a contrast agent. We conclude that by using the effects of fusinite on magnetic susceptibility, it can be located accurately and noninvasively with MRI and thereby the value of the use of fusinite to measure concentration of oxygen in vivo is enhanced.

Animals↗

Hemodynamic oxygen transport and 2,3-diphosphoglycerate changes after transfusion of patients in acute respiratory failure.

The goals of management of patients with respiratory failure include improving arterial oxygenation with PEEP and red cell transfusion to maintain oxygen carrying capacity, both of which contribute to improving tissue oxygen delivery. However, standard CPD-stored blood is rapidly depleted of 2,3 diphosphoglycerate (2,3 DPG) and ATP, with resultant inadequacy of the red cell oxygen transport function. In 15 patients requiring mechanical ventilation with PEEP whose initial Hct less than or equal to 35%, we studied the effect of transfusion of 7 ml/kg of CPD-stored packed red blood cells on hemodynamic and oxygen delivery variables, pulmonary venous admixture (QA/QT), and erythrocytic P50, 2,3 DPG and ATP concentrations. Hemodynamics were not significantly altered by transfusion. 2,3 DPG decreased significantly from 14.5 +/- 1.1 to 13.1 +/- 1.5 mcmol/g Hb (mean +/- SD, p less than 0.05). There was no significant change in P50 or ATP. QA/QT rose significantly, from 20.1 +/- 7.8 to 28.9 +/- 12.3% (mean +/- SD, p less than 0.02). In our patients, an increase in arterial oxygen content obtained by transfusion was not followed by any associated decrease in cardiac work, as implied by solution of equations for oxygen delivery and oxygen consumption. The rise in QA/QT is undesirable in patients requiring PEEP, since it complicates management of their mechanical ventilatory support.

2,3-Diphosphoglycerate↗

Involvement of singlet oxygen in chloroaluminum phthalocyanine tetrasulfonate-mediated photoenhancement of lipid peroxidation in rat epidermal microsomes.

In recent studies chloroaluminum phthalocyanine tetrasulfonate (AlPCTS) has been shown to be an effective photosensitizer for the tumor necrosis in a modality known as photodynamic therapy, but the mechanism of photodynamic effect of AlPCTS is poorly understood. In this study, in vitro incubation of rat epidermal microsomes with AlPCTS followed by exposure to red light (approximately 675 nm) resulted in an increase in ADP/iron-supported lipid peroxidation, a measure of membrane damage. This photodestructive effect was found to be dependent on both the duration of light exposure and the dose of AlPCTS. Studies employing various quenchers of reactive oxygen species revealed that scavengers of singlet oxygen (histidine, 2,5-dimethylfuran, beta-carotene and sodium azide) afforded substantial protection (up to 90%) of photoenhancement whereas the scavengers of hydrogen peroxide (catalase), superoxide anion (superoxide dismutase), and hydroxyl radical (sodium benzoate, mannitol and ethanol) were ineffective in this regard. Our data indicate that AlPCTS-mediated photodestruction mainly involves a type II reaction via singlet oxygen formation and suggest that the latter could play a significant role in the tumor necrosis evoked by AlPCTS and light.

Animals↗

Arctic life adaptation--II. The function of musk ox (Ovibos muschatos) hemoglobin.

1. The hemoglobin system from musk ox (Ovibos muschatos) has been characterized from the functional point of view with special regard to the effect of organic phosphates and temperature. 2. The results are similar to those previously obtained in the case of reindeer and confirm that hemoglobins from arctic animals may display very low enthalpy change for the reaction with oxygen. 3. This finding is considered an example of molecular adaptation of respiratory pigments to extreme environmental conditions.

Adaptation, Physiological↗

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P.

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.

Base Sequence↗

Oxygen-dependent protection of radiation lung damage in mice by WR 2721.

The modification of early and late radiation damage to the mouse lung by oxygen and WR 2721 has been studied by measurement of breathing rate, lethality, pleural fluid and hydroxyproline content. Protection by hypoxia and sensitization by hyperoxia of early radiation pneumonitis were demonstrated. There was a tendency for the protective effect of WR 2721 to decrease as the breathed oxygen concentration was raised above normal levels. WR 2721 protection of the late damage was higher (PF = 1.6-1.65) than was seen for early pneumonitis (PF = 1.3-1.35) when either breathing rate or lethality were used. Protection factors (PF) gained from measurements of pleural fluid at a year after treatment were similar to those for other endpoints of late damage (PF = 1.7). In contrast, the measurement of fibrosis through determination of lung hydroxyproline at 1 year gave a somewhat lower protection factor for WR 2721. In the same experiments the degree of epilation on the dorsal thorax was scored at 6 weeks. One hundred per cent oxygen gave enhancement (dose enhancement factor (DEF) = 1.2), 9 per cent oxygen reduced damage (DEF less than 0.7) and WR 2721 gave PF values in excess of 1.4 at all oxygen concentrations used. This showed that the radiation response of hair follicles was more sensitive to WR 2721 or to changes of oxygen than the lung. The results presented indicate a competitive interaction between WR 2721 and oxygen for the same injury site causing a shift in the oxygen K curve to higher oxygen concentrations. The validity of applying functional or survival measurements to assess the extent of pulmonary fibrosis is discussed.

Amifostine↗

Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate.

To investigate DNA damage induced by Pb2+ and its prevention by scavengers, we determined DNA strand breakage and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in DNA using plasmid relaxation assay and HPLC with electrochemical detection, respectively. Lead acetate induced DNA strand breakage in 10 mM of Hepes buffer, pH 6.8, in a time- and dose-dependent manner. Compared with lead, zinc acetate did not significantly induce DNA breakage. The singlet oxygen scavengers NaN3 and 2,2,6,6-tetramethyl-4-piperidone (TEMP) inhibited lead-induced DNA breakage more efficiently than the hydroxyl radical scavengers mannitol and DMPO. Deuterium oxide (D2O), a singlet oxygen enhancer, potentiated lead-induced DNA breakage. At low ratios to Pb2+, NADPH, glutathione, and 2-mercaptoethanol enhanced lead-induced DNA breakage, whereas high ratios of these agents protected it. Catalase and superoxide dismutase (SOD) did not protect DNA breaks induced by Pb2+. Lead-induced DNA breakage was markedly enhanced by H2O2, and this induction was inhibited by NaN3, TEMP, EDTA, catalase, BSA, and glutathione. In contrast, mannitol and SOD potentiated Pb2+/H2O2-induced DNA breaks. The results indicate that singlet oxygen, lead, and H2O2 are all involved in the reaction system, whereas hydroxyl radical and superoxide did not. Lead could cause a small amount of 8-OHdG formation in calf thymus DNA and dose-dependently induced the formation of this adduct in the presence of H2O2. Singlet oxygen scavengers were more effective than hydroxyl radical scavengers in protection from lead/H2O2-induced 8-OHdG adducts. Taken together, these results suggest that lead may induce DNA damage through a Fenton-like reaction and that singlet oxygen is the principal species involved.

8-Hydroxy-2'-Deoxyguanosine↗

Myocardial oxygen tension and relative capillary density in isolated perfused rat hearts.

Oxygen plays an important role in cardiac function. Many methods have been applied to measure tissue oxygen tension (PO2). Electron paramagnetic resonance (EPR) oximetry appears to have some significant advantages for use in the beating heart. This study presents the serial measurement of myocardial PO2 by EPR oximetry in the isolated crystalloid perfused heart during changes of influent PO2, coronary flow rate, oxygen consumption and end-diastolic pressure. Baseline myocardial PO2 was 198 +/- 12 mmHg (mean +/- S.E.). Myocardial PO2 increased as expected with increased delivery (concentration or flow) or decreased consumption. With increasing flow rate, myocardial PO2 increased in a sigmoid fashion. A critical flow or pressure was reached when myocardial PO2 rapidly increased to a higher level. Increased left ventricular end-diastolic pressure caused local vascular compression and resulted in a decrease of myocardial PO2. Myocardial capillary density in the intact contracting heart was calculated to be 2300 +/- 110/mm2, using local myocardial PO2 and a cylindrical model for oxygen diffusion in tissue. Relative capillary density did not change with mild to moderate hypoxia, increased with increasing flow and increasing oxygen consumption and decreased with elevated diastolic pressure. We conclude that the application of EPR oximetry with LiPc to the isolated heart provides accurate and dynamic evaluation of local myocardial PO2 in the contracting heart. Using various models of oxygen delivery and diffusion in tissue, these data may also be used to serially follow capillary density.

Animals↗

A regulated environmental perfusion system for the study of anoxic or hypoxic cultured neurons using microfluorescence imaging and electrophysiology.

We describe the design and performance of a newly developed regulated environmental perfusion system (REPS). This system allows study of the effects of anoxia or hypoxia in cultured cells at physiological temperature, without the use of oxygen-scavenging compounds or metabolic inhibitors. The REPS incorporates a "canoe-shaped" flow-through chamber with access from above to allow positioning of pipettes for patch-clamp, microinjection, rapid-application perfusion, or microprobes for monitoring physical parameters. The combination of laminar flow and complete washout of perfusate within the chamber, and the use of a gas-tight perfusate delivery system and pressurized reservoirs containing media with pre-stabilized oxygen tensions (pO2 values) allow rapid production of accurate perfusate pO2 within the chamber. Perfusate pO2 in the chamber declined monoexponentially with time constants of </= 20 s to stable, pre-determined levels of 0 or 2 kPa (15 Torr). Shielding the gas/liquid interface of the chamber with an argon curtain only minimally decreased time constants at flow rates >/= 2 ml/min. The perfusion chamber of the REPS is easily mounted on the stage of an inverted microscope, for use with fluorescence imaging or electrophysiological studies of cultured cells. In tests with cultured rat cortical neurons, intracellular calcium concentration increased exponentially to values exceeding 1 microM during 10 min of anoxic insult, and returned to baseline values within 1 min after restoring normoxia.

Animals↗

Oxygen binding properties of hemoglobin from the white rhinoceros (beta 2-GLU) and the tapir.

The beta-chain of rhinoceros hemoglobin contains glutamic acid at position beta 2, and important site for the binding of organic phosphates. We have investigated the oxygen binding properties of this hemoglobin and its interaction with ATP, 2,3-diphosphoglycerate, CO2 and chloride. The results show that the presence of GLU at position beta 2 nearly abolishes the effect of organic phosphates and CO2, whereas the oxygen-linked binding of chloride is not affected. Thus rhinoceros hemoglobin has only protons and chloride anions as major allosteric effectors for the control of its oxygen affinity. From the results obtained with hemoglobin solutions it can be calculated that the blood oxygen affinity of the rhinoceros must be rather high with a P50 of about 20 torr at pH 7.4 and 37 degrees C, which conforms with observations obtained for other large mammals.

Animals↗

Synthesis and photophysical properties of a new cationic water-soluble Zn phthalocyanine.

The synthesis of tetrakis (1,1-dimethyl-2-trimethylammonium)ethylphthalocyaninato Zn(II) tetraiodide 2--a new cationic phthalocyanine--is reported as well as its photophysical properties in water, relevant for the use of this dye as photodynamic sensitizer. The hydrophobicity of the macrocycle leads to strong aggregation which lowers the fluorescence and singlet oxygen quantum yields of aqueous solutions of 2. Photophysical parameters agree with those of other Zn phthalocyanines when correction is performed for the fraction of light absorbed by the monomer.

Cations↗

[The effect of dimephosphon, sermion and piracetam on the reactivity of the cerebral vessels, on local cerebral blood flow and on oxygen tension in brain tissue].

The paper gives the results of experimental and clinical studies of the nootropic agents dimephosphone, sermion, and piracetam on local cerebral circulation, cerebrovascular responsiveness and O2 tension. The vasoactivity of the parameters studied has been shown to be displayed by their ability to normalize cerebrovascular responsiveness and it is associated with decreased brain tissue oxygen consumption when dimephosphone is applied and with its increased one when sermion and piracetam are used.

Adolescent↗

Implantable glucose sensors: choosing the appropriate sensing strategy.

The special requirements for implantable glucose sensors which differ from laboratory analysers and in vitro probes include continuous operation without drift, compatibility with in vivo body conditions, electrical and toxicological safety and patient acceptability. We have studied the effect of oxygen tension, operating temperature and pH, and the stability of various potentially implantable amperometric glucose sensors so as to aid the choice of the technologies most suitable for in vivo application.

Biosensing Techniques↗

Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide.

Effects of nitric oxide (NO) and NO generating agents, on the electron transport system of mitochondria were examined in a study of the mechanism and physiological importance of NO in energy metabolism. In the presence of various substrates, uncoupled respiration was inhibited by NO in manner which was both dose- and oxygen tension-dependent. Simultaneously measuring changes in cytochrome absorption spectra and respiration showed that the site of action of NO is cytochrome oxidase. Similar inhibition was also brought about by 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO donor. Electron paramagnetic resonance (EPR) analysis revealed that inhibition of uncoupled respiration occurred only during the presence of NO in the reaction mixture. The inhibitory effect of NO was increased significantly by lowering the concentration of mitochondrial protein. No appreciable inhibition of respiration was observed in the presence of 3-morpholinosydnonimine (SIN-1), a peroxynitrite anion (ONOO-) generating reagent, but inhibition did occur in the presence of superoxide dismutase (SOD). These results indicate that NO reversibly interacts with mitochondria at complex IV thereby inhibiting respiration particularly under physiologically low oxygen tension and that de novo generated ONOO may have no significant effect under the present experimental conditions.

Animals↗

Effects of changes in the concentrations of substrate and oxygen on the perfused rabbit heart.

Rabbit hearts were perfused with bicarbonate-buffered fluid containing 1.0 mM glucose and 1.5 mM lactate. Pairs of redox substrates were measured in the perfusion fluid and the heart after perfusate concentrations of beta-hydroxybutyrate (beta-HB) and oxygen were varied. The perfusate lactate to pyruvate ratio was shown to accurately represent the tissue ratio. The intracellular redox state varied independently of changes in substrate uptake. Increasing perfusate beta-HB concentration caused increased beta-HB uptake, decreased lactate uptake, and unchanged glucose uptake. Decreased perfusate oxygen tension caused inhibition of lactate and beta-HB uptakes and, when exteme, caused increased glucose uptake. The combination of a modest reduction in oxygen tension and increased perfusate beta-HB concentration caused a more reduced environment in the tissue than either change by itself. This combination of changes may have additive deleterious effects as suggested by clinical observations.

Animals↗

Sensitization of human colon adenocarcinoma cells (LoVo) to reactive oxygen species by a lysosomotropic compound.

The in situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer therapy. The present results show that multidrug-resistant human colon adenocarcinoma cells (LoVo) are significantly more sensitive than corresponding wild-type cells to hydrogen peroxide and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. Pre-treatment of the cells with N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a lysosomotropic compound, sensitized both cell lines to the subsequent exposure to spermine metabolites, as was evident from the decrease of cell survival by a log unit. The sensitizing effect was greater in the case of the multidrug-resistant cell line, an aspect of particular importance with respect to potential therapeutic applications of the method, since conventional cancer therapy suffers from the development of drug resistance. Cell viability was determined using a clonogenic assay. MDL 72527 (at 300 microM) produced numerous cytoplasmic vacuoles, presumably of lysosomal origin, after 6-h exposure, which decreased in size and number (in the presence of the drug) by 24 h and had almost disappeared completely at 48 h. Mitochondrial damage, as observed by transmission electron microscopy, seemed to correlate better with the cytotoxic effects of the treatment than the formation of vacuoles. We suggest that the release of lysosomal enzymes into the cytosol by MDL 72527 is the main reason for its sensitizing effect. It is known that lysosomotropic compounds, which release lysosomal enzymes, produce oxidative stress and apoptosis.

Adenocarcinoma↗

Direct determination of p-nitrophenyl substituent organophosphorus nerve agents using a recombinant Pseudomonas putida JS444-modified Clark oxygen electrode.

A microbial biosensor for rapid, sensitive, selective, and cost-effective determination of the total content of organophosphorus nerve agents with p-nitrophenyl substituent is reported. The biosensor consisted of genetically engineered PNP-degrader Pseudomonas putida JS444 expressing organophosphorus hydrolase (OPH) on its cell surface immobilized on a dissolved oxygen electrode. Surface-expressed OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, methyl parathion, and parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen. The oxygen consumption was measured and correlated to the concentration of organophosphates. The sensor signal and response time were optimized with 0.086 mg dry weight of cell and operating in 50 mM pH 7.5 citrate-phosphate buffer with 50 microM CoCl(2) at room temperature. When operated at optimized conditions, the biosensor measured as low as 55 ppb of paraoxon, 53 ppb of methyl parathion, and 58 ppb of parathion without interference from most phenolic compounds and other commonly used pesticides, such as atrazine, coumaphos, sutan, sevin, and diazinon. The operational life of the microbial biosensor was approximately 5 days when stored in the operating buffer at 4 degrees C.

Aryldialkylphosphatase↗