Adding two components of radiosensitization by oxygen

It has been shown, or inferred, in various contexts that radiosensitization of cells by oxygen is the sum of two (or more) components. If the component sensitivities conform with the Alper and Howard-Flanders equation their sum cannot also conform, but, in practice, even the most meticulous experimental techniques will fail to reveal lack of conformity unless one of the component K values is at least nine times the other. Thus, despite the many results that have demonstrated conformity with the equation, the existence of at least two components may well be a general phenomenon. The killing of cells by radiation is attributable to a summation of lesions in different structures; different K values for the contributing components are therefore to be expected, since neither oxygen nor its competitors are likely to be present in uniform concentration in all elements of the cell nucleus. Provided the components have intrinsic values of o.e.r. greater than one, their addition results in sensitivity that increases monotonically with PO2, approaching asymptotically to the overall o.e.r. which is a weighted average of the component o.e.r.s. In a curve plotted with PO2 on a linear scale a point of inflection can occur only if one component o.e.r. has a value less than one (i.e. oxygen is protective for that component), and then only if relationships between the other parameters satisfy certain conditions. In cases in which points of inflection in the sensitivity curve has been observed these are unlikely to be accounted for by the addition of two components. The analysis of the consequences of adding two components of oxygen sensitization could apply also to chemical sensitization of hypoxic cells.     

Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen

Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% ¹⁸O₂ and 80% N₂ indicates that one atom of ¹⁸O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing ¹⁸O₂ indicates that one atom of ¹⁸O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

     

Dimensionless criteria for estimating oxygen transfer in aeration systems

The effect of power consumption on oxygen transfer efficiency has been evaluated for both surface and subsurface aeration systems. Two new dimensionless criteria (numbers) have been proposed for this purpose. They are termed the efficiency criterion or T(o) number and the power consumption effect criterion or Av number. The analysis presented in this article is based on data obtained on various full-scale aeration systems, and it shows that for each aerator there is an optimum power consumption at which maximum oxygen transfer efficiency results. The oxygen transfer efficiency has maximum values when the efficiency criterion (T(o) number) reaches an approximately constant value. The results have proven that when there are several aerators with identical diameters and the same rotational velocity but different geometrical shapes, the most efficient one will be (after having reached the maximum transfer efficiency) the one with the highest power consumption. For two aerators varying in diameter but with the same parameter as above (maximum transfer, equal rotational velocity), different efficiencies are obtained, the difference being a function of d(-0.5). The results of investigating subsurface aeration systems show that the efficiency of decreased.     

Studies of insufficient mixing in bioreactors: Effects of limiting oxygen concentrations and short term oxygen starvation on Penicillium chrysogenum

The effect of oxygen limitation on the respiration rate of Penicillium chrysogenum was studied. The results show that measurements of critical oxygen tensions within a process that on morphological or on physical grounds exhibits an inhomogenous structure are not likely to resemble the Monod model.In order to study the effects of short term oxygen starvation on the respiratory capacity of Penicillium chrysogenum, a two compartment fermenter was constructed. This fermenter consists of one well mixed aerobic part (CSTR) and one minor anaerobic part (CPFR). In the latter the circulation time as well as the volume can be varied. After passage of the whole cell culture volume through the anaerobic part, irreversible inhibition of the respiration was observed. This was caused by a circulation time of 5 and 10 min in the plug flow reactor and with a volume of 6% of the stirred tank reactor volume. However, circulation times of 1 and 2 min with an anaerobic zone of 1% of the stirred tank reactor volume did not give any irreversible effects on the respiratory capacity.This was compared with the results of the previously established model ln(1 — I _OUR//100)^–1 = kt [1]. The I _OUR is the percentage irreversible inhibition of the respiration, t is the anaerobic circulation time and k is a constant. The two compartment fermenter results agree with the earlier model at circulation times of 5 and 10 min, but not with the shorter times, and this suggests that a lag phase exists in the inactivation kinetics.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Model for oxygen transfer in a shake flask

The shake flask is a very common and useful tool for the study of submerged fermentations on a small scale. However, the oxygen supply may easily become a limiting factor. A model for the aeration in shake flasks is presented that enables one to predict whether in the course of an experiment the oxygen supply is becoming a growth-limiting factor or not. The results of measurements of the transfer coefficient of a cotton plug and the oxygen mass-transfer coefficient k_la are also given.     

Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX·chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX·chloride complex and a ternary MSOX·chloride·MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.     

Water induced dismutation of superoxide anion generates singlet molecular oxygen

Direct spectroscopic measurement of 1268 nm singlet oxygen emission from KO2 suspensions at room temperature in three non-protonic solvents--CCl4, Cl2FCCClF2, and C6F14 by the action of water is reported. The results clearly show that the singlet oxygen generation is due to a water induced reaction, and suggest that one role of the enzyme superoxide dismutase may be the protection of biological structures, for example, lipid membranes, from degradation by singlet oxygen.     

How patients use domiciliary oxygen.

Forty-five patients in Southampton who received domiciliary oxygen were visited at home to find out how they used and coped with their oxygen. Generally, the organisation and administration of supplies presented no problems, nearly all the apparatus complied with the drug tariff, and most patients coped well with the equipment. Only two patients were taking oxygen for prescribed periods; the others were taking it when necessary for symptomatic relief. No patient received oxygen for over five hours daily. Most patients thought that they were helped by oxygen, but only four said that it allowed them to increase their level of activity, and the overall benefit seemed slight. This was partly because oxygen was usually limited to one room, so patients used oxygen after rather than during exercise. The amount of oxygen consumed differed widely, ranging from three and a half cylinders a week in three patients to less than one cylinder in six months in 17 patients. The average yearly cost of oxygen per patient ranged from 500 pounds in patients consuming one cylinder or more per week, to 15 pounds in those consuming less than one cylinder in six months. The main cost of domiciliary oxygen is determined by the number of cylinder refills, so patients who use it infrequently are a relatively small drain on resources.     

Transient characteristics of oxygen probes with significant liquid film effects

Various experimental procedures for the determination of transient characteristics with significant liquid film effects were tested. A comparison between transient characteristics obtained experimentally and those calculated from rational models indicates that all procedures but one give highly inconsistent results. Recalculation of transient characteristics with no liquid film (easily measured in the gas phase) to that with liquid film (occurring in viscous liquids) is recommended as well as the selected experimental procedure which yields consistent results in the situations where the steady-state probe reading is decreased up to one-half due to the liquid film.     

Effect of amino acids and oxygen on chemotaxis in Escherichia coli

Adler, Julius (University of Wisconsin, Madison). Effect of amino acids and oxygen on chemotaxis in Escherichia coli. J. Bacteriol. 92:121-129. 1966.-Motile cells of Escherichia coli placed at one end of a capillary tube containing a mixture of the 20 amino acids commonly found in proteins migrate out into the tube in two bands. The bands are clearly visible to the naked eye, and they can also be demonstrated by microscopy, photography, and densitometry, and by assaying for bacteria throughout the tube. The occurrence of more than one band is not due to heterogeneity among the bacteria, since each band can be used over to give rise to two again. The first band uses all the oxygen to oxidize portions of one or more of the amino acids, including serine, and the second band consumes the residual serine anaerobically. The results are interpreted to mean that E. coli shows chemotaxis toward oxygen and serine. When no energy source is added to the medium, a band of bacteria still appears. It consumes all the oxygen to oxidize an endogenous energy source. The addition of any one of 10 oxidizable amino acids stimulates the rate of travel of this band. Alanine, an example that was studied in detail, supports such a band that consumes all the oxygen to oxidize a portion of the alanine. Serine, the only amino acid that this strain can use either aerobically or anaerobically when grown under the conditions used here, gives rise to two bands.     

Intraphagosomal oxygen in stimulated macrophages

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO₂ in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d₁₇-1-oxyl iodide (d-Cat₁)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl (¹⁵N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley-Liss, Inc.     

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

Binding of oxygen and carbon monoxide to arthropod hemocyanin: an allosteric analysis

The binding of oxygen and carbon monoxide to hemocyanin from the mangrove crab Scylla serrata and the lobster Homarus americanus has been studied by thin-layer optical absorption and front face fluorescence techniques. Three types of experiments were performed on subunit and oligomeric preparations of each hemocyanin: oxygen binding, carbon monoxide binding, and oxygen-carbon monoxide competition studies. The results obtained from the subunit preparations of dissociated oligomers from both hemocyanins show that the binding site can be ligated by either one oxygen or one carbon monoxide. The binding results obtained with the oligomeric samples of hemocyanin from both species cannot be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] since the data from the three types of binding experiments cannot be fit with a single set of binding constants. The MWC model has been extended by including a third allosteric form, and an analysis based on the three-state model is able to fit the data from the three types of experiments with the same set of binding constants. The comparison of the oxygen to carbon monoxide affinity ratios (kO2/kCO) indicates that the structure around the binding site of subunits in the T form oligomer is similar to that of the free subunits. The oligomeric forms of both these hemocyanins bind carbon monoxide with a weak but definite positive cooperativity. An analysis of the affinity ratios for the T, S, and R forms suggests that the high affinity of the R form results from a specific interaction between oxygen and binding site.     

Hydroxide instead of bicarbonate in the structure of the oxygen evolving complex

The energy diagram for the catalytic cycle of dioxygen evolution in photosystem II has been recomputed using a slightly different model than the one used previously, since the results showed an erroneous trend in the critical region from S(2) to S(3). By replacing the bicarbonate ligand, which was bridging between calcium and the outer manganese, by a hydroxide a significant improvement of the energy diagram is obtained. Most notably, the S(2) to S(3) transition is now exergonic as it should be. However, for the S(2)(0) state an artificial constraint is needed on the hydrogen bonding, indicating that there is still some problem with the model used. This hydroxide model was used to study the effects of replacing calcium with strontium, magnesium and cadmium. The computed results reproduce the results observed experimentally that magnesium and cadmium suppress water oxidation and that strontium slows it down. For both magnesium and cadmium the process stops already at S(2).     

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

Singlet oxygen is essential for neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.