The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Below-halocline oxygen consumption in the Kattegat

Sediment and seston oxygen consumption rates below the sharp halocline in the south-eastern part of the shallow Kattegat were measured and compared to calculated rates of carbon addition through the halocline. The mean rate of decrease in deep-water oxygen concentrations between March and September 1988 was 1.0 ml O₂ M^–3 h^–1. Measurements of benthic oxygen uptake using laboratory-incubated sediment cores from depths 30 m gave a mean value of 7.8 ml O₂ m^–2 h^–1. Below-halocline water (from 20 m, 30 m and 1 m above bottom) incubated in bottles showed oxygen consumption rates varying from 0.5 ml O₂ m ^–3 h^–1 in March to 2.8 ml O₂ M^–3 h^-1 in late August. The sum of benthic and deep-water oxygen consumption was equivalent to a mean oxygen decrease rate of 1.7 ml O₂ m^–3 h^–1 below the halocline. Of the total oxygen consumption below the halocline 65% was due to oxygen up-take in the water and 35% was due to benthic oxygen consumption. The sum of oxygen consumption measured in sediment cores and in bottles corresponds to a carbon utilisation of 80.1 g C m^–2 (respiratory quotient (RQ), assumed 1.0 and 1.4 for water and sediment, respectively), while the decrease in deep-water oxygen concentration was equivalent to 43.0 g C m^–2 (RQ assumed = 1.0). Using published values for the external N loading (including deep-water supply), ¹⁵NO₃-uptake, ¹⁴CO₂-uptake in combination with % ¹⁵NO₃-uptake of total ¹⁵N-uptake (nitrate, ammonia and urea) and a Redfield C/N ratio of 6.6, rates of carbon addition (new or export production) through the halocline were calculated to 31.9, 46.7 and 36.3 g C m^–2, respectively, with a mean value of 38.3 g C m^–2 for the 8 month period March–September. This is somewhat less than the value (50.5 g C m^–2) calculated from a published empirical relationship between total and export production. The fact that the calculated carbon addition through the halocline was appreciably less than the carbon equivalent of the measured below-halocline respiration may be an effect of sediment focusing (horizontal transport of sedimenting material to deeper areas), since the bottom area below the halocline is much smaller than the total area of the Kattegat. A lower observed decrease in the oxygen concentration below the halocline compared to the sum of measured sediment and deep-water oxygen consumption on the other hand indicates oxygen supply to below-halocline waters through advection and/or vertical entrainment.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

The effect of low oxygen uptake rate on the fatty acid profile of the oleaginous yeast Apiotrichum curvatum

Summary The effect of limiting the available oxygen on the fatty acid profile of Apiotrichum curvatum ATCC 20509 during growth on sulphuric acid casein whey was studied. At oxygen uptake rates (OUR) lower than 7 mmol O₂/l per hour, applied during the oil accumulating phase of the fermentation, a decrease in total unsaturated fatty acids was observed. It was possible to decrease the unsaturated fatty acids (oleate from 55% to 41% and linoleate from 9% to 3%) by limiting the OUR of the culture to <3 mmol O₂/l per hour. However at this low OUR, a lower oil coefficient (a measure of the efficiency of lactose substrate conversion to oil) was recorded. Furthermore the fermentation time was increased. An OUR of 5 mmol O₂/l per hour appeared to be the limit below which adverse effects on oil yields and increased fermentation times occurred. At this OUR, the accumulated oil contained 45% oleate and 5% linoleate. These effects were demonstrated in a 20-l air-lift fermentor and confirmed in a scaled down 500-l industrial type bubble column fermentor. Offprint requests to: R. J. Davies     

Community structure and photosynthetic activity of epilithon from a highly acidic (pH≤2) mountain stream in Patagonia,Argentina

We explored a benthic community living on stones in an acidic (pH2) stream of active volcanic origin from Patagonia, Argentina, by combining in situ measurements (temperature, pH, conductivity, dissolved oxygen), photosynthesis of intact biofilms (measured with microsensors by the light–dark shift method), pure-culture experiments on isolated algae, and confocal laser scanning microscopy on the biofilms. The epilithon of the Agrio River was dominated (99% of total biomass) by one species: Gloeochrysis (Chrysophyceae). This species was observed as brown, mucilaginous, 200-m-thick films on stones, growing in clumps in a dense matrix of fungal hyphae, bacteria, and inorganic particles held together by extracellular polymeric substances. Gloeochrysis was isolated and cultivated. The photosynthetic rate measured at saturation irradiance was 120 mol oxygen (mg chlorophyll a)^–1h^–1 under laboratory conditions, and the saturation rate of photosynthesis by carbon dioxide was 90 mol oxygen (mg chlorophyll a)^–1 h^–1 for oxygen evolution. Photosynthetic activity of the biofilm was light-dependent and saturated above 200 mol photons m^–2 s^–1. In the dark, the stone surface became anoxic. Our data suggest that primary production in the Agrio River was not limited by light, carbon, or phosphorus but instead, nitrogen-limited.     

Light respiration in <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Respiration and photosynthesis are two important processes in microalgal growth that occur simultaneously in the light. To know the rates of both processes, at least one of them has to be measured. To be able to measure the rate of light respiration of Chlorella sorokiniana, the measurement of oxygen uptake must be fast, preferably in the order of minutes. We measured the immediate post-illumination respiratory O₂ uptake rate (OUR) in situ, using fiber-optic oxygen microsensors, and a small and simple extension of the cultivation system. This method enables rapid and frequent measurements without disturbing the cultivation and growth of the microalgae. Two batch experiments were performed with C. sorokiniana in a short light-path photobioreactor, and the OUR was measured at different time points. The net oxygen production rate (net OPR) was measured online. Adding the OUR and net OPR gives the gross oxygen production rate (gross OPR), which is a measure for the oxygen evolution by photosynthesis. The gross OPR was 35–40% higher than the net OPR for both experiments. The respiration rate is known to be related to the growth rate, and it is suggested that faster algal growth leads to a higher energy (ATP) requirement, and as such, respiratory activity increases. This hypothesis is supported by our results, as the specific OUR is highest in the beginning of the batch culture when the specific growth rate is highest. In addition, the specific OUR decreases toward the end of the experiments until it reaches a stable value of around 0.3 mmol O₂ h⁻¹ g⁻¹. This value for the specific OUR is equal to the maintenance requirement of C. sorokiniana as determined in an independent study of (Zijffers et al. 2010 (in press)). This suggests that respiration could fulfill the maintenance requirements of the microalgal cells.     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

Methane and oxygen dynamics in a shallow floodplain lake: the significance of periodic stratification

Temperature, dissolved oxygen and dissolved methane profiles were measured during autumn and summer, in a shallow floodplain lake in south-eastern Australia to determine the effects of water-column stability on methane and oxygen dynamics. The water column was well mixed in autumn. Strong thermal stratification developed in the late afternoon in summer, with top-to-bottom temperature differences of up to 6 °C. Methane concentrations in surface waters varied over a daily cycle by an 18-fold range in summer, but only by a 2-fold range in autumn. The implication of short-term temporal variation is that static chambers deployed on the water surface for short times (less than a day) in summer will significantly underestimate the diffusive component of methane emissions across the water–atmosphere interface. There was a marked diel variation in dissolved oxygen concentrations in summer, with the highest oxygen values (commonly 5–8 mg l^–1) occurring in the surface waters in late afternoon; the bottom waters were then devoid of oxygen (< 0.2 mg l^–1). Because of high respiratory demands, even the surface water layers could be nearly anoxic by morning in summer. The concentration of dissolved oxygen in the surface waters was always less than the equilibrium value. When the water column became thermally stratified in summer, the dissolved oxygen and methane maxima were spatially separated, and planktonic methanotrophy would be limited to a moving zone, at variable depth, in the water column. In summer the whole-wetland rates of oxygen production and respiration, calculated from long-term (5 h) shifts in dissolved oxygen concentrations over a diel period, were approximately 6–10 and 3–6 mmol m^–3 h^–1, respectively. These values correspond to net and gross primary production rates of 0.7–1.2 and 1.0–1.9 g C m^–3 day^–1, respectively.     

The energetics of starvation and growth after refeeding in juveniles of three cyprinid species

Synopsis Experiments were conducted to monitor changes in body mass and metabolic energy expenditure before, during, and after periods of starvation in juveniles of three species of cyprinids: Leuciscus cephalus, Chalcalburnus chalcoides mento, and Scardinius erythrophthalmus. During the starvation period all fish lost weight at about the same rate and the total amount of oxygen consumed during an experimental period of 20 h was about 40% lower in the starved than in the fed groups. Upon refeeding, both mass specific maintenance; and routine rates of metabolism as well as relative growth rates increased rapidly, the peaks of these increases being directly proportional to the length of the starvation period. Maximum compensatory growth was observed after four weeks of starvation in C. chalcoides and S. erythrophthalmus, with relative growth rates reaching 30% d^-1 during the first measuring interval after refeeding. The pattern of time-dependent compensatory growth displayed by these fish is similar to the responses of a colonial hydroid in which the rate of catch-up growth increased with the amount of stress to which the animals had been exposed. The exact cost of compensatory growth cannot be calculated because oxygen consumption and growth were not measured simultaneously. However, on the basis of data and calculations reported by Wieser & Medgyesy (1990) it appears that compensatory growth, if fuelled by the metabolic power indicated by our measurements of oxygen consumption, would have to be about twice as efficient as normal growth in the related species Rutilus rutilus.     

Evidence for net uptake of GABA into mouse astrocytes in primary cultures—Its sodium dependence and potassium independence

Content of GABA was measured in cultured, normal astrocytes (from the brain cortex of newborn mice) together with the effect of nonradioactive GABA on the efflux of labeled GABA from cells previously loaded with [¹⁴C]GABA. An increase of external GABA concentration from 0 to 25 M evoked a rise of the GABA content in the cells to a level which was approximately 50 times that of the incubation medium. Neither 200 nor 2000 M nonradioactive GABA had any effect on the rate of release of radioactivity from cells loaded with [¹⁴C]GABA. Both the high tissue/medium ratio and the lack of a GABA-induced enhancement of the release of radioactivity indicate that the previously observed high-affinity uptake of GABA in cultured astrocytes represents a net uptake and not a homoexchange with endogenous GABA. This uptake is sodium dependent but was found to be unaffected in potassium-free media; the quantitative correlation between GABA transport and sodium transport differed from that reported for synaptosomes.     

Light-oriented locomotion in certain myxobacter species

Summary Euglena gracilis, strain Z, was grown in synchronous culture. Carbon source used was either dl-lactic acid (L) or a mixture of l-glutamic and dl-malic acids (GM). Synchronisation was obtained by transfering the cells in the exponential growth phase, at regular intervals—each 3 days—to a fresh medium.Respiration (measured during a whole cell cycle, 12 h) was 20±6 l/H/10⁶ cells on (GM) and 46±7 l/H/10⁶ cells on (L) medium. At the same time as the increased rate of oxygen uptake on lactate medium, we observed the appearance of a giant chondriome in the cells. On glutamate—malate containing medium the size of mitochondria was normal.

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Les micrographies électroniques ont été réalisées par Mr. C. Mattei, Technicien CNRS.     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

Patterns of routine swimming and metabolic rate in juvenile cyyprinids at three temperatures: analysis with a respirometer-activity-monitoring system

Summary The spontaneous swimming activity and oxygen consumption of juvenile roach (Rutilus rutilus (L.)), were monitored for 48–72 h at 8, 15 and 20°C and a photoperiod (L:D) of 11:13. At low levels of activity metabolic cost is constant and thus corresponds to the standard rate of metabolism (low cost activity). At higher levels of activity metabolic cost increases in proportion with the degree of activity. The slope for this high cost activity indicates an expenditure of 0.4–1.3 mol O₂·g^-1·h^-1 per arbitrary activity unit at the three experimental temperatures. Extrapolation of this relationship to zero activity would underestimate the measured standard metabolic rate. High cost activity occurred in only one experiment out of three at 8°C, low cost activity hardly at all at 20°C, whereas at 15°C both forms of activity were present in five of the seven experiments conducted. Thus, not only the intensity but also the pattern of activity is affected by environmental temperature.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction of cytochrome P4501A (CYP1A1) enzyme activity is one of the best-studied direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds and has been shown to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons (PAH) in different experimental animal species as well as in humans. TCDD has also been shown to modulate cytokine gene expression in human keratinocytes, including IL-1, TGF- and TFG-₂. In the present studies, the aim was to determine whether different cellular targets of human origin differed in susceptibility to TCDD as measured by CYP1A1 activity and mRNA expression, and whether cytokine gene induction/suppression correlated with TCDD susceptibility. Human airway epithelial cells, alveolar macrophages (AM), peripheral blood monocytes and lymphocytes (PBL) were exposed to 10^-10–10^-7 mol/L TCDD. CYP1A1 enzyme activity was determined by ethoxyresorufin-O-deethylase (EROD) activity, mRNA expression of CYP1A1 was measured by semiquantitative PCR assay. The secretion and/or gene expression of specific cytokines, including IL-6, IL-8, and IL-1 were also examined. Overall, there was a clear correlation between TCDD-induced enzyme activity and CYP1A1 mRNA levels, which were dose-dependently increased in the bronchoepithelial cells and PBL. The human airway epithelial cells (BEAS-S6 cell line and primary cells) appeared to be the most inducible cellular target, with up to 50-fold increases at 10^-8 mol/L TCDD with an EC₅₀ of 3×10^-11 mol/L TCDD. The pokeweed mitogen-activated peripheral blood lymphocytes revealed approximately 5-fold less capacity in CYP1A1 activity, with high interindividual variabilities (EC₅₀ 3×10^-9 mol/L TCDD). In contrast, CYP1A1 enzyme activity in both AM and purified peripheral blood monocytes, which were costimulated with LPS and/or GM-CSF, could not be detected. CYP1A1 mRNA levels, however, were detectable and only marginally enhanced in response to TCDD. The ability of all these cells to express and produce the proinflammatory cytokines IL-6 and IL-8 was neither enhanced nor impaired by TCDD. These results indicate that cell types found in human lung and peripheral blood vary in susceptibility to TCDD, with the lung epithelium being highly susceptible and the alveolar macrophage being nonsusceptible. However, expression and production of specific cytokines such as IL-6 and IL-8, which may potentiate inflammatory processes and/or work as mitogens, does not appear to be influenced by TCDD.     

Theoretical assessment of alternative mechanisms for non-photochemical quenching of PS II fluorescence in barley leaves

The components of non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves have been quantified by a combination of relaxation kinetics analysis and 77 K fluorescence measurements (Walters RG and Horton P 1991). Analysis of the behaviour of chlorophyll fluorescence parameters and oxygen evolution at low light (when only state transitions — measured as qN_t — are present) and at high light (when only photoinhibition — measured as qN_i — is increasing) showed that the parameter qN_t represents quenching processes located in the antenna and that qN_i measures quenching processes located in the reaction centre but which operate significantly only when those centres are closed. The theoretical predictions of a variety of models describing possible mechanisms for high-energy-state quenching, measured as the residual quenching, qN_e, were then tested against the experimental data for both fluorescence quenching and quantum yield of oxygen evolution. Only one model was found to agree with these data, one in which antennae exist in two states, efficient in either energy transfer or energy dissipation, and in which those photosynthetic units in a dissipative state are unable to exchange energy with non-dissipative units.Abbreviations: F_o, F_m room-temperature chlorophyll fluorescence yield with all centres open, closed - F_v variable fluorescence yield - LHC II light-harvesting chlorophyll-protein complex of PS II - PS I, PS II Photosystem I, II - P700, P680 primary donor in Photosystem I, II - Q_A primary electron acceptor of PS II - P_max maximum quantum yield of oxygen evolution - qN coefficient of non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i coefficient of non-photochemical quenching due to high-energy-state, state transition, photoinhibition - qO coefficient of quenching of dark level fluorescence - qP coefficient of photochemical quenching of variable fluorescence - _P intrinsic quantum yield of open PS II reaction centres = _s/qP - _{PS 2} quantum yield of PS = qP × F_v/F_m - _S quantum yield of oxygen evolution = rate of oxygen evolution/light intensity     

Generation of superoxide-radical by the NADH: Ubiquinone oxidoreductase of heart mitochondria

Besides major NADH-, succinate-, and other substrate oxidase reactions resulting in four-electron reduction of oxygen to water, the mitochondrial respiratory chain catalyzes one-electron reduction of oxygen to superoxide radical followed by formation of hydrogen peroxide. In this paper the superoxide generation by Complex I in tightly coupled bovine heart submitochondrial particles is quantitatively characterized.The rate of superoxide formation during -controlled respiration with succinate depends linearly on oxygen concentration and contributes approximately 0.4% of the overall oxidase activity at saturating (0.25 mM) oxygen. The major part of one-electron oxygen reduction during succinate oxidation (80%) proceeds via Complex I at the expense of its -dependent reduction (reverse electron transfer). At saturating NADH the rate of formation is substantially smaller than that with succinate as the substrate. In contrast to NADH oxidase,the rate-substrate concentration dependence for the superoxide production shows a maximum at low (50 µM)concentrations of NADH. NAD⁺ and NADH inhibit the succinate-supported superoxide generation. Deactivation of Complex I results in almost complete loss of its NADH-ubiquinone reductase activity and in increase in NADH-dependent superoxide generation. A model is proposed according to which complex I has two redox active nucleotide binding sites.One site (F) serves as an entry for the NADH oxidation and the other one (R) serves as an exit during either the succinate-supported NAD⁺ reduction or superoxide generation or NADH-ferricyanide reductase reaction.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 150–159.Original Russian Text Copyright © 2005 by Vinogradov, Grivennikova.This revised version was published online in April 2005 with corrections to the post codes.