The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

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)     

The effect of short-term fasting and a single meal on protein synthesis and oxygen consumption in cod,Gadus morhua

Summary Rates of protein synthesis and oxygen consumption ( O₂) in cod were compared in both fasted and refed animals. During a 14-day fast both protein synthesis and respiration rates fell to stable values after 6 days. When a meal of whole sandeel at 6% body weight was fed to fish fasted for 6 days, protein synthesis and ( O₂) increased to a maximum at between 12 and 18 h after feeding. Peak ( O₂) was about twice the pre-feeding values, while whole animal protein synthesis increased four-fold. There were differences between tissues in the timing of maximum protein synthesis; the liver and stomach responded faster than the remainder of the body. Maximum protein synthesis rates in the liver and stomach occurred at 6 h after feeding, at which time their calculated contribution to total ( O₂) was 11%. Similar calculations suggested that the integrated increment in whole animal protein synthesis contributed between 23% and 44% of the post-prandial increase in ( O₂). It was concluded that protein synthesis is an important contributor to increased ( O₂) after feeding in cod.Abbreviations A _s absolute rate of protein synthesis - ASDA apparent specific dynamic action - ATP adenosine triphosphate - k _s fractional rate of protein synthesis - k _s/RNA amount of protein synthesized per unit RNA - ( O₂) oxygen consumption - PCA perchloric acid - RNA ribonucleic acid     

Modification of 3,5-diiodo-4-hydroxybenzoic acid (DIHB) activity and stimulation of ethylene production by small concentrations of oxygen in the root environment

The apical 2 cm of seedling roots of oilseed rape (Brassica napus L., cv. Primor) produced more ethylene than adjacent, older tissue. Treatment with 5 × 10^–3 mol m^–3 3,5-diiodo4-hydroxybenzoic acid (DIHB), a presumed inhibitor of ethylene action, failed to stimulate root extension. Larger concentrations were inhibitory. Ethylene, applied as ethephon decreased root extension but DIHB (5 × 10^–3 mol m^–3) partially overcame this effect. Oxygen concentrations below that present in air also inhibited root extension but this was not ameliorated by DIHB.Roots of barley seedlings (Hordeum vulgare L., cv. Midas) evolved ethylene more slowly than roots of oilseed rape. DIHB (10^–3–10^–2 mol m^–3) stimulated root extension in the absence of ethephon. Ethephon alone retarded root extension but DIHB partially overcame this inhibition. Small concentrations of oxygen also inhibited root extension but DIHB failed to ameliorate the effect even though the slow growth of oxygen-deficient roots (3–5% oxygen) was associated with abnormally fast rates of endogenous ethylene production.Extension growth in different oxygen concentrations was more closely associated with rates of oxygen consumption than with the amount of ethylene produced. Thus respiration rather than ethylene appeared to limit root extension under oxygen deficiency. This may explain why DIHB was unable to offset this form of environmental stress.     

The role of energy-spilling reactions in the growth ofKlebsiella aerogenes NCTC 418 in aerobic chemostat culture

When cell-saturating amounts of glucose and phosphate were added to steady state cultures ofKlebsiella aerogenes that were, respectively, glucose-and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must posses effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved.Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and there-fore expressed a higherY _o value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of maintenance and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment.The maintenance rates of oxygen consumption ofK. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source.The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures ofK. aerogenes is discussed.     

Estimation of specific growth rate from agitation speed in DO-stat culture

Summary A simple and effective method to estimate the specific growth rate estimation has been developed based on the observation of time changes in the agitation speed in dissolved oxygen(DO)-stat cultures of Brevibacterium ketoglutamicum. The estimation was compared with that using carbon dioxide evolution rate (CER). Estimated values of specific growth rates by both methods agreed well with the data directly calculated from cell concentration change although the use of agitation speed gave a slightly better result than CER.Nomenclature CER Carbon dioxide evolution rate (mmol/sec) - OUR Oxygen uptake rate (mmol/sec) - OTR Oxygen transfer rate (mmol/sec) - RPM Agitation speed (rev./min) - C* Saturated dissolved oxygen concentration (mmol/L) - Dissolved oxygen concentration (mmol/L) - k Time index - k _L a' Mass transfer coefficient (sec^-1) - Y _X/O2 Cellular yield based on oxygen consumed (g-cell/mmol O₂) - Specific growth rate (hr^-1) - Constant - t Fermentation time - t Sampling time for RPM and CER measurements     

Effect of oxygen on substrate utilization for nitrogen fixation and growth in Frankia spp.

Frankia, the actinomycete partner in the nitrogenfixing symbiosis of certain woody non-legumes, has been shown to fix nitrogen in pure culture under aerobic conditions. The sensitivity of in vivo nitrogen-fixation (acetylene reduction) to oxygen tension in the gas phase was measured in short-term assays with two Frankia isolates designated ARI3 and CcI3. The carbon source utilized had an effect on the optimum O₂ concentration for acetylene reduction. Cells utilizing an organic acid, e.g., propionate or pyruvate had maximum nitrogenase activity at an oxygen concentration of 15 to 20%. In contrast, cells respiring a sugar, e.g., trehalose or glucose, or endogenous reserves (glycogen or trehalose) had maximum acetylene reduction activity at 5 to 10% in the gas phase. Oxygen uptake kinetics showed that respiration in vesicle-containing cells utilizing trehalose had a biphasic response to oxygen concentration with a diffusion limited component at oxygen concentrations of 20 M to more than 300 M. These results suggested that trehalose was oxidized in the vesicles as well as in the vegetative hyphae. Oxygen concentration also had an effect on the trehalose-supported growth of cells (non nitrogenfixing, [⁺NH₄Cl]). Cells grown with 5–10% O₂ in the gas phase had a doubling time approximately half those grown with 20% O₂ (atmospheric). Propionate-grown cells showed similar growth rates at the two oxygen tensions, and grew faster (almost 2x) than the trehalose cells at 5–10% O₂. Trehalose also supported approximately 40% lower rates of oxygen uptake than propionate in vesicle-containing cells.     

Gas exchange and blood gases of Puna teal (Anas versicolor puna) embryos in the Peruvian Andes

Oxygen consumption, air cell gases, hematology, blood gases and pH of Puna teal (Anas versicolor puna) embryos were measured at the altitude at which the eggs were laid (4150 m) in the Peruvian Andes. In contrast to the metabolic depression described by other studies on avian embryos incubated above 3700 m, O₂ consumption of Puna teal embryos was higher than even that of some lowland avian embryos at equivalent body masses. Air cell O₂ tensions dropped from about 80 toor in eggs with small embryos to about 45 toor in eggs containing a 14-g embryo; simultaneously air cell CO₂ tension rose from virtually negligible amounts to around 26 torr. Arterial and venous O₂ tensions (32–38 and 10–12 toor, respectively, in 12- to 14-g embryos) were lower than described previously in similarly-sized lowland wild avian embryos or chicken embryos incubated in shells with restricted gas exchange. The difference between air cell and arterial O₂ tensions dropped significantly during incubation to a minimum of 11 torr, the lowest value recorded in any avian egg. Blood pH (mean 7.49) did not vary significantly during incubation. Hemoglobin concentration and hematocrits rose steadily throughout incubation to 11.5 g · 100 ml^-1 and 39.9%, respectively, in 14-g embryos.Abbreviations PO₂ partial pressure gradient of O₂ - BM body mass - D diffusion coefficient - G gas conductance (cm³·s^-1·torr^-1) - conductance to water vapor - IP internal pipping of embryos - P _ACO₂ partial pressure of carbon dioxide in air cell - P _AO₂ partial pressure of oxygen in air cell - P _aCO₂ partial pressure of carbon dioxide in arterial blood - P _aCO₂ partial pressure of oxygen in arteries - P _H barometric pressure (torr) - PCO₂ partial pressure of carbon dioxide - P _IO₂ partial pressure in ambiant air - PO₂ partial pressure of oxygen - P _VCO₂ venous carbon dioxide partial pressure - P _VO₂ mixed venous oxygen partial pressure - SE standard error - VO ₂ oxygen consumption     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Cytokinin inhibition of respiration by cells and mitochondria of soybean,Glycine max (L.) Merrill

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 M, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O₂ consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 M, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.Abbreviations BAP 6-benzylaminopurine - IPA 6-(²-isopentenyl)aminopurine     

Concurrent measurements of oxygen- and carbon-dioxide exchange during lightflecks inAlocasia macrorrhiza (L.) G. Don

Oxygen and CO₂ exchange were measured concurrently in leaves of shade-grownAlocasia macrorrhiza (L.) G. Don during lightflecks consisting of short periods of high photon flux density (PFD) superimposed on a low-PFD background illumination. Oxygen exchange was measured with a zirconium-oxide ceramic cell in an atmosphere containing 1 600 bar O₂ and 350 bar CO₂. Following an increase in PFD from 10 to 500 mol photons·m^-2·s^-1, O₂ evolution immediately increased to a maximum rate that was about twice as high as the highest CO₂-exchange rates that were observed. Oxygen evolution then decreased over the next 5–10 s to rates equal to the much more slowly increasing rates of CO₂ uptake. When the PFD was decreased at the end of a lightfleck, O₂ evolution decreased nearly instantaneously to the low-PFD rate while CO₂ fixation continued at an elevated rate for about 20 s. When PFD during the lightfleck was at a level that was limiting for steady-state CO₂ exchange, then the O₂-evolution rate was constant during the lightfleck. This observed pattern of O₂ evolution during lightflecks indicated that the maximum rate of electron transport exceeded the maximum rate of CO₂ fixation in these leaves. In noninduced leaves, rates of O₂ evolution for the first fraction of a second were about as high as rates in fully induced leaves, indicating that O₂ evolution and the electron-transport chain are not directly affected by the leaf's induction state. Severalfold differences between induced and noninduced leaves in O₂ evolution during a lightfleck were seen for lightflecks longer than a few seconds where the rate of O₂ evolution appeared to be limited by the utilization of reducing power in CO₂ fixation.Abbreviation PFD photon flux density (of photosynthetically active radiation)     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Respiratory activity and growth kinetics of Candida yeasts related to carbon sources and available energy

Summary Three yeasts of the genus Candida (Candida intermedia, candida lipolytica and Candida tropicalis) were cultivated batchwise on three different carbon sources: glucose, acetate, and hexadecane. Growth curves, oxygen uptake rates, CO₂ evolution rates and the amount of oxygen required for biomass production were determined. The data were compared and discussed from the point of maximum specific growth rate, maximum oxygen uptake rate, carbon conversion into CO₂ and biomass, consumption of oxygen and available energy for cell synthesis. The results indicated a relationship between _m m, Y_s, Y_O, and for different carbon sources. Y_O and were in the same order of magnitude for acetate (0.58 and 0.38 respectively) and hexadecane (0.45 and 0.40 respectively). These values were remarkably lower than those for glucose (1.26 and 0.54 respectively).Symbols av e^– Available electrons per mol of substrate (dimensionless) - E_av Energy available per mol of substrate (dimensionless) - C_d Dissimilated carbon (%) - m Maximum specific rate of oxygen uptake (mMO₂ h^–1 g^–1) - RQ CO₂ evolved per O₂ consumed - mol. wt. Molecular weight - Y_ATP Biomass mass yield based on mol of ATP generated (g) - Biomass mass yield based on available energy (g) - Y_M Biomass mass yield based on mol of organic substrate (g) - Y_O Biomass mass yield based on oxygen consumed (gg^–1) - 1/Y_O Oxygen consumed for one gram of biomass produced (gg^–1) - Y_s Biomass mass yield based on organic substrate (dimensionless) - b Reductance degree of biomass (equiv. available electrons/g atom carbon) - s Reductance degree of organic substrate (equiv. available electrons/g atom carbon) - Fraction of energy in organic substrate which is converted to biomass - b Weight fraction carbon in biomass (dimensionless) - s Weight fraction carbon in organic substrate (dimensionless) - _m Maximum specific growth rate (h^–1)     

Chemolithoutotrophic growth of Thiothrix ramosa

Thiothrix has been shown for the first time to be able to grow chemolithoautotrophically with thiosulphate or carbon disulphide as sole energy substrate. Thiosulphate served as the growth-limiting substrate for Thiothrix ramosa in chemostat culture. Maximum growth yield (Y_max) from yields at growth rates between 0.029–0.075 h^-1 was 4.0 g protein/mol thiosulphate oxidized. The key enzyme of the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, was present in these cells, as were rhodanese, adenylyl sulphate (APS) reductase and sulphur-oxidizing enzyme. Thiosulphate-grown cells oxidized thiosulphate, sulphide, tetrathionate and carbon disulphide. Oxidation kinetics for sulphide, thiosulphate and tetrathionate were biphasic: oxygen consumption during the fast first phase of oxidation indicated oxidation of sulphide, and the sulphane moieties of thiosulphate and tetrathionate, to elemental sulphur, before further oxidation to sulphate. Kinetic constants for these four substrates were determined. T. ramosa also grew mixotrophically in batch culture on lactate with a number of organic sulphur compounds: carbon disulphide, methanethiol and diethyl sulphide. Substituted thiophenes were also used as sole substrates. The metabolic versatility of T. ramosa is thus much greater than previously realised.     

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

Summary Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly--hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.Abbrevations ^cO₂,L concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t) - ^cH₂,L concentration of hydrogen in the liquid phase - ^cCO₂,L concentration of carbon dioxide in the liquid phase - S limiting substrate, concentration of - X total biomass, concentration of; total cell dry weight - P product; PHB, concentration of - R rest biomass: X-P, concentration of - ^rX dX/dt growth rate - ^rP dP/dt rate of PHB synthesis - ^rR dR/dt rate of rest biomass production - ^r0 ^dcO₂,L/dt rate of oxygen consumption - X dX/dt·1/X=r_X·1/X specific growth rate - P dP/dt·1/P=r_P·1/P specific rate of product formation - R dR/dt·1/R=r_R·1/R specific rate of rest biomass formation - r₀/R specific respiration rate     

Expression of the kynurenine enzymes in macrophages and microglial cells: regulation by immune modulators

Summary The regulation of the expression of indoleamine 2,3-dioxygenase (IDO) was studied in cloned murine macrophages (MT2) and microglial (N11) cells. Both cell lines express IDO and inducible nitric oxide synthase activity after interferon- (IFN-) stimulation. The regulation of IDO expression appears to differ in the two cell lines. Nitric oxide (NO) production negatively modulates the expression of IDO activity in IFN--primed macrophages, thereby indicating a cross-talk between the kynurenine and nitridergic pathways in these cells. Conversely, this down-regulation of IDO activity by NO does not occour in microglial cells. A differential regulation of IDO expression in the two cell lines was also observed with LPS and picolinic acid. Together with previous findings, these results indicate the existence of marked differences in the regulation of the expression of the kynurenine pathway enzymes between macrophages and microglial cells.Abbreviation used IFN- interferon- - IDO indoleamine 2,3-dioxygenase - NO nitric oxide - iNOS inducible nitric oxide synthase - NAME N-())-nitro-L-arginine methyl ester - SMTC S-methyl-L-thiocitrulline - BNI 3-bromo-7-nitroindazole - PIC picolinic acid - IL interleukin     

Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines

We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - BTX -bungarotoxin - BSA bovine serum albumin - FITC Fluoroscein isothiocyanate - G418 geneticin-418 - hpi hours post-infection - ie-1 immediate early 1 gene - nAChR nicotinic acetylcholine receptor alpha subunit - Sf Spodoptera frugiperda - tPA tissue plasminogen activator     

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     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.