A comparison of oxygenation methods fro high-density perfusion culture of animal cells

A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble ( approximately 5 mm in diameter), micron-sized bubble ( approximately 80 mum in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium. (c) 1993 John Wiley & Sons, Inc.     

Effects of ecological engineered oxygenation on the bacterial community structure in an anoxic fjord in western Sweden

Oxygen-depleted bodies of water are becoming increasingly common in marine ecosystems. Solutions to reverse this trend are needed and under development, for example, by the Baltic deep-water OXygenation (BOX) project. In the framework of this project, the Swedish Byfjord was chosen for a pilot study, investigating the effects of an engineered oxygenation on long-term anoxic bottom waters. The strong stratification of the water column of the Byfjord was broken up by pumping surface water into the deeper layers, triggering several inflows of oxygen-rich water and increasing oxygen levels in the lower water column and the benthic zone up to 110 μmol l⁻¹.We used molecular ecologic methods to study changes in bacterial community structure in response to the oxygenation in the Byfjord. Water column samples from before, during and after the oxygenation as well as from two nearby control fjords were analyzed. Our results showed a strong shift in bacterial community composition when the bottom water in the Byfjord became oxic. Initially dominant indicator species for oxygen minimum zones such as members of the SUP05 clade declined in abundance during the oxygenation event and nearly vanished after the oxygenation was accomplished. In contrast, aerobic species like SAR11 that initially were restricted to surface waters could later be detected deep into the water column. Overall, the bacterial community in the formerly anoxic bottom waters changed to a community structure similar to those found in oxic waters, showing that an engineered oxygenation of a large body of anoxic marine water is possible and emulates that of a natural oxygenation event.     

A NEW INCUBATION TECHNIQUE FOR THE STUDY OF SYNAPTOSOMAL TRANSPORT

A simple, inexpensive method is described for the incubation and study of transport in synaptosomes. This method avoids the many potential sources of error inherent in the methods currently in use. The transport of two sugars and two amino acids has been studied, using this system, and the resulting data is compared to similar studies already published by others. A steady-state was achieved and maintained for 60 min independent of tissue concentration, except in the case of γ-aminobutyric acid. An analysis of the entry kinetics of 2-deoxy-d -glucose has been performed, the results indicating that the present method offers the advantage of substantially increasing oxygenation and hence, efficiency of the uptake. Using α-aminoisobutyric acid, analysis of substrate flux within the system was performed, illustrating the utility of the method. The specific advantages offered by the technique over other methods in use are discussed. It is concluded that adequate oxygenation of the synaptosome during study is essential to reliable results and can easily be achieved by means of the methodology described in this report.     

Microbial energetics and stoichiometry for biodegradation of aromatic compounds involving oxygenation reactions

Oxygenation reactions significantly alter the energy and electron flows and, consequently, the overall stoichiometry for the microbial utilization of aromatic compounds. Oxygenation reactions do not yield a net release of electrons, but require an input of electrons to reduce oxygen molecules. The biodegradation pathway of phenanthrene as a model compound was analyzed to determine the impact of oxygenation reactions on overall stoichiometry using the half-reaction method. For individual oxygenation reactions, the half-reaction method for analyzing the electron and energy flows must be modified, because the reactions do not release electrons for synthesis or energy generation. Coupling the oxygenation reaction to subsequent reaction steps provides a net electron release for the coupled reactions. Modeling results indicate that oxygenation reactions increase the oxygen requirement and reduce the cell yield, compared to the conventional mineralization represented by hydroxylation reactions in place of oxygenations. The computed yields considering oxygenation reactions conform better to empirical yields reported in the literature than do yields computed by the hydroxylation single-step methods. The coupled-reaction model also is consistent with information about the ways in which micro-organisms that degrade aromatics accumulate intermediates, regulate degradation genes, and organize enzyme clusters.     

Intermittent electrical stimulation redistributes pressure and promotes tissue oxygenation in loaded muscles of individuals with spinal cord injury

Deep tissue injury (DTI) is a severe form of pressure ulcer that originates at the bone-muscle interface. It results from mechanical damage and ischemic injury due to unrelieved pressure. Currently, there are no established clinical methods to detect the formation of DTI. Moreover, despite the many recommended methods for preventing pressure ulcers, none so far has significantly reduced the incidence of DTI. The goal of this study was to assess the effectiveness of a new electrical stimulation-based intervention, termed intermittent electrical stimulation (IES), in ameliorating the factors leading to DTI in individuals with compromised mobility and sensation. Specifically, we sought to determine whether IES-induced contractions in the gluteal muscles can 1) reduce pressure in tissue surrounding bony prominences susceptible to the development of DTI and 2) increase oxygenation in deep tissue. Experiments were conducted in individuals with spinal cord injury, and two paradigms of IES were utilized to induce contractions in the gluteus maximus muscles of the seated participants. Changes in surface pressure around the ischial tuberosities were assessed using a pressure-sensing mattress, and changes in deep tissue oxygenation were indirectly assessed using T?*-weighted magnetic resonance imaging (MRI) techniques. Both IES paradigms significantly reduced pressure around the bony prominences in the buttocks by an average of 10-26% (P < 0.05). Furthermore, both IES paradigms induced significant increases in T?* signal intensity (SI), indicating significant increases in tissue oxygenation, which were sustained for the duration of each 10-min trial (P < 0.05). Maximal increases in SI ranged from 2-3.3% (arbitrary units). Direct measurements of oxygenation in adult rats revealed that IES produces up to a 100% increase in tissue oxygenation. The results suggest that IES directly targets factors contributing to the development of DTI in people with reduced mobility and sensation and may therefore be an effective method for the prevention of deep pressure ulcers.     

Factors defining the rate of oxygen uptake by the red blood cell

Using numerical methods, the initial rates of oxygen uptake by the red blood cell have been computed. The methods accommodate both a water layer and membrane which may act as diffusive impedance to gas influx. The differential solubilities of the gas in these two layers have also been incorporated in the methods. The presence of a 0.50–0.65 μm deoxygenated water layer has been calculated to simulate the experimental results by Roughton (1959). Experimental studies of CO and NO uptake by the red cell could also be simulated. Although a membrane-only model with given parameters can also account for the observed rates of oxygenation of the red cell (Weingardenet al., submitted for publication), the additional incorporation of differential solubilities of oxygen in the different layers of the RBC yields results that indicate a three layer model to be more plausible. Using a thin layer-red cell oxygenation system, the rates of oxygenation were determined for red cells surrounded by a 4.2 μm deoxygenated water layer. The rates were found to compare favorably to the results of the theoretical model.     

Yield prediction and stoichiometry of multi-step biodegradation reactions involving oxygenation

Microorganisms can initiate the degradation of organic compounds by oxygenation reactions that require the investment of energy and electrons. This diversion of energy and electrons away from synthesis reactions leads to decreased overall cell yields. A thermodynamic method was developed that improves the accuracy of cell yield prediction for compounds degraded through pathways involving oxygenation reactions. This method predicts yields and stoichiometry for each step in the biodegradation pathway, thus enabling modeling a multi-step biodegradation process in which oxygenations occur and intermediates may persist. EDTA and benzene biodegradation are presented as examples. The method compares favorably with other yield prediction methods while providing additional information of yields for intermediates produced in the degradation pathway.     

Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia

Oxygen supply and diffusion into tissues are necessary for survival. The oxygen partial pressure (pO(2)), which is a key component of the physiological state of an organ, results from the balance between oxygen delivery and its consumption. In mammals, oxygen is transported by red blood cells circulating in a well-organized vasculature. Oxygen delivery is dependent on the metabolic requirements and functional status of each organ. Consequently, in a physiological condition, organ and tissue are characterized by their own unique 'tissue normoxia' or 'physioxia' status. Tissue oxygenation is severely disturbed during pathological conditions such as cancer, diabetes, coronary heart disease, stroke, etc., which are associated with decrease in pO(2), i.e. 'hypoxia'. In this review, we present an array of methods currently used for assessing tissue oxygenation. We show that hypoxia is marked during tumour development and has strong consequences for oxygenation and its influence upon chemotherapy efficiency. Then we compare this to physiological pO(2) values of human organs. Finally we evaluate consequences of physioxia on cell activity and its molecular modulations. More importantly we emphasize the discrepancy between in vivo and in vitro tissue and cells oxygen status which can have detrimental effects on experimental outcome. It appears that the values corresponding to the physioxia are ranging between 11% and 1% O(2) whereas current in vitro experimentations are usually performed in 19.95% O(2), an artificial context as far as oxygen balance is concerned. It is important to realize that most of the experiments performed in so-called normoxia might be dangerously misleading.     

A kinetic description of dioxygen motion within alpha- and beta-subunits of human hemoglobin in the R-state: geminate and bimolecular stages of the oxygenation reaction

Laser flash photolysis technique is used to study human hemoglobin (HbA) oxygenation. Monomolecular geminate oxygenation of triliganded R-state HbA molecules is described by a function of three exponentials. Geminate oxygenation of the alpha-subunit within R-state HbA is characterized by two components with time constants of 0.14 and 1 ns, while geminate oxygenation of the beta-subunit within HbA is characterized by two components with time constants of 1 and approximately 30 ns. Bimolecular oxygenation of triliganded R-state HbA molecules is described by a biexponential law. Two observed rate constants are assigned to oxygenation of the alpha- and beta-subunit within HbA. The bimolecular association rate constants for O(2) rebinding with the alpha- and beta-subunit within triliganded R-state HbA are k(alpha) = 18.8 +/- 1.3 (microM x s)(-1) and k(beta) = 52 +/- 4 (microM x s)(-1), respectively. The apparent quantum yields of photodissociation of the beta- and alpha-subunit within completely oxygenated R-state HbA differ from each other by a factor of 3.6 and are equal to 0.041 +/- 0.004 and 0.0114 +/- 0.0012, respectively. The apparent quantum yield of photodissociation of completely oxygenated R-state HbA is equal to 0.026 +/- 0.003.     

Response of glycine and glycyl-L-valine uptake parametrs across in vitro everted sacs of chicken gut to variations in oxygenation rate and fasting

Energy-dependent accumulation of glycine and glycyl-L-valine within the small intestinal mucosa in a chicken model of in vitro local oxygenation of the small intestinal preparation was studied. It has been shown that the most effective bilateral oxygenation significantly increase accumulation of glycyl-L-valine in the proximal segment as compared to that under oxygenation only from serosal surface both in the fed and 24-hour fasted chickens, whereas in other segments these differences was less apparent. This may be due to increased H+/ peptide cotransporter expression in the proximal segment. Thus the bilateral oxygenation probably may turn on an additional amount of already existing (but non-functional during serosal oxygenation) H+/ peptide co-transporters. Moreover, low glycine transporter expression may be the reason why supplemental oxygen (bilateral oxygenation) has no effect on glycine accumulation in the distal segment of fed chickens. A 48-hour fasting decreases glycyl-L-valine accumulation in the proximal (and medial) segments, possibly due to progressive decrease in villus height. It is concluded that: a) the accumulation rate of glycine was greater when presented as the glycyl-L-valine than when presented as the equivalent amount of free amino acid; b) the rates of accumulation of glycyl-L-valine are highest in the proximal segment, decrease in the medial segment and are the lowest in the distal segment; c) the serosal oxygenation is less effective than the mucosal and bilateral oxygenation, which markedly stimulates accumulation of nutrients in the intestinal mucosa; d) a 24-hour fasting increases glycyl-L-valine accumulation in the proximal segment only, while glycine uptake was increased in the distal segment.     

A computational model that links non-periodic vasomotion to enhanced oxygenation in skeletal muscle

We propose a model of a capillary network in which chaotic capillary activity is crucial for efficient oxygenation of a muscle fiber. Tissue oxygenation by microcirculation is controlled by a complex pattern of opening and closing of precapillary sphincters, a phenomenon known as vasomotion. We model the individual precapillary sphincter as a non-linear oscillator that exhibits perfectly periodic vasomotion in isolation. The precapillary sphincters surrounding an active fiber are considered as a network; specific modes of interaction within this network result in complex patterns of vasomotion. In our model, efficient oxygenation of the fiber depends crucially on the mode of interaction among the vasomotions of the individual capillaries. Network interactions that lead to chaotic vasomotion are found to be essential for meeting the tissue oxygen demands precisely. Interactions that cause regular rhythmic patterns of vasomotion fail to meet oxygenation demands accurately.     

A plausible sequence of the conformational changes of hemoglobin induced by oxygenation.

Recent experimental data of oxygen equilibrium constants of human adult hemoglobin, which are measured over a wide range of oxygen pressures, are analyzed successfully from the viewpoint that the change in the molecular structure of hemoglobin induced by oxygenation is considered individually at each stage of oxygenation. Then, a simple phenomenological rule, which explains quantitatively the values of the four Adair constants with only three parameters, is found for hemoglobin under normal physiological conditions. The temperature dependence of these parameters suggests a sequence of the conformational changes such that until the third stage of oxygenation the conformational changes occur within the deoxy quaternary structure and at the fourth stage of oxygenation the deoxy quaternary structure is altered to the oxy one. The effects of pH and phosphate compounds on the Adair constants are discussed, and a possible modification and extension of the rule is suggested. The connection between the rule and the molecular structures of deoxy- and oxyhemoglobin is also discussed.     

The effect of modification of sulfhydryl groups in soybean lipoxygenase-1

Soybean lipoxygenase-1 was found to contain five free sulfhydryl groups and no disulfide bridges. Three sulfhydryl groups react readily with methylmercuric halides. This modification results in significant changes of the catalytic properties of the enzyme. Comparison of modified and native lipoxygenase-1 shows the following: 1. The catalytic constant of the oxygenation of linoleic acid is reduced by approximately 50%, whereas the affinity towards linoleic acid remains unaltered. 2. At high concentrations of substrate and low concentrations of enzyme the kinetic lag phase in the oxygenation is considerably longer. 3. The regio- and stereospecificities of the oxygenation are significantly lower. 4. Besides hydroperoxides, oxo-octadecadienoic acids (4%) are formed during the oxygenation. 5. The cooxidation capacity is considerably enhanced. Treatment of methylmercury-modified lipoxygenase-1 with NaHS results in the complete recovery of the sulfhydryl groups and of the catalytic properties.     

Enzymatic sulphur oxygenation reactions

Sulphur is a key constituent in a wide variety of biologically important compounds, ranging from amino acids and coenzymes to antibiotics and pesticides. In analogy with the more widely studied metabolism of aromatic or aliphatic hydrocarbons and amines, the intial step in metabolism of sulphur compounds is commonly oxygenation on sulphur. While sulphur oxygenation in vivo has been known for many years, it is only within the past decade that many of the enzymes responsible have been identified, and molecularlevel details have become available. This review focuses on the molecular aspects of enzymatic sulphur oxygenation, and considers mono and dioxygenases active on inorganic sulphur, organic thiols, thioethers, thioesters and thiones. Information from very diverse areas of the literature is brought together, and the implications of sulphur oxygenation reactions to drug design, as well as to environmental and toxicological areas, are mentioned.     

OxymiRs in cutaneous development,wound repair and regeneration

The state of tissue oxygenation is widely recognized as a major microenvironmental cue that is known to regulate the expression of coding genes. Recent works have extended that knowledge to demonstrate that the state of tissue oxygenation may potently regulate the expression of microRNAs (miRs). Collectively, such miRs that are implicated in defining biological outcomes in response to a change in the state of tissue oxygenation may be referred to as oxymiRs. Broadly, oxymiRs may be categorized into three groups: (A) the existence (expression and/or turnover) of which is directly influenced by changes in the state of tissue oxygenation; (B) the existence of which is indirectly (e.g. oxygen-sensitive proteins, metabolites, pH, etc.) influenced by changes in the state of tissue oxygenation; and (C) those that modify biological outcomes to changes in the state of tissue oxygenation by targeting oxygen sensing pathways. This work represents the first review of how oxymiRs may regulate development, repair and regeneration. Currently known oxymiRs may affect the functioning of a large number of coding genes which have hitherto fore never been linked to oxygen sensing. Many of such target genes have been validated and that number is steadily growing. Taken together, our understanding of oxymiRs has vastly expanded the implications of changes in the state of tissue oxygenation. This emerging paradigm has major implications in untangling the complexities underlying diseases associated with ischemia and related hypoxic insult such as chronic wounds.     

Isoflavanoids of dalbergia Oliveri

The heartwood of Dalbergia oliveri has yielded 11 natural products of which two are new 3-phenylcoumarins. The structures of the extractives have been examined by physical methods and in addition the assigned structures have been confirmed by synthesis. The oxygenation pattern relating the structures of chalcone, isoflavone, pterocarpans, 3-arylcoumarins, coumestones and the isoflavan and isoflavanone suggests a common biosynthetic origin.     

(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2–(R)-naproxen and COX-2–(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.     

Dynamics of erythrocyte oxygenation in vitro

The process of blood oxygenation in vitro was described theoretically. It was established that the dynamics of the oxygenation degree of different blood samples can be obtained from the certain universal time function by a change of the time scale. A special device was created for experimental investigation of oxygenation dynamics. The experimental data are in agreement with the theory.     

Quantitative description of blood oxygenation process in oxygenator for extracorporal circulation

Based on conceptions and assumptions concerning the blood oxygenation process, some fundamental quantitative relations for red blood corpuscle oxygenation and blood oxygenation kinetics are presented. A distribution function is introduced expressing the probability density for the occurrence of a red blood cell with a specific oxygen content. By means of a kinetic equation deduced the distribution function is connected with spatial distribution of oxygen pressure and with blood flow rate. For the given initial conditions the kinetic equation is solved for a one-dimensional case, and this solution is applied to a generalized oxygenator in a stationary case. The generalized oxygenator presents a system of through-flow elements in which blood flows and contacts oxygen. Each of the through-flow elements is characterized by length, blood flow rate, probability of red blood corpuscle entry and by a quantity depending on oxygen pressure. Results obtained for the generalized oxygenator are then applied to a disc oxygenator with certain presumptions concerning blood oxygen saturation at the system's output expressed in dependence on geometry and performance conditions. Stress is laid upon the influence of blood flow in the oxygenator, on oxygenation; and two extreme cases are compared—series and parallel types of disc oxygenator.