Properties and regulation of a trans-plasma membrane redox system in rat liver

1. Regulation of the reduction of ferricyanide by the isolated perfused rat liver was studied. 2. The rate of reduction was dependent on the rate of supply of ferricyanide and independent of perfusate oxygen concentration. 3. The effect of pH was also examined; the rate of reduction was optimal at pH 7.4 and was inhibited to a greater extent by pH values below 7.4 than those above 7.4. 4. The effects of substrates on the rate of ferricyanide reduction was assessed. Reductants of the cytosolic and mitochondrial NADH/NAD⁺ couple were tested. 2-Hydroxybutyrate (10mm), lactate (10mm), glycerol (10mm) and ethanol (10mm) each had no effect. Dihydroxyacetone (10mm) stimulated the rate. 5. Dehydroascorbate (1mm), stimulated the rate of ferricyanide reduction; the stimulation did not appear to be attributable to the production of reduced substances that were excreted to reduce extracellular ferricyanide. 6. The effects of glucagon and cyclic AMP on the rate of ferricyanide reduction were examined. Glucagon inhibited the rate by approx. 30% and half-maximal inhibition occurred at 0.1 nm, corresponding to the concentration at which half-maximal stimulation of glucose release occurred. Cyclic AMP stimulated glucose release but had no significant effect on the rate of ferricyanide reduction. It is concluded that the trans-plasma membrane redox system of liver that reduces extracellular ferricyanide is regulated by glucagon. The rate is also altered by the substrate dihydroxyacetone. The effect of glucagon may be direct as it cannot be mimicked by cyclic AMP and it occurs directly following exposure to the hormone.     

Development of a digital infrared video camera system for recording and remote capturing

A digital infrared video camera system was developed for recording and remote capturing. The components are described in detail. Based on 2 years of experience within a wild boar (Sus scrofa) project, the system gave proof of its practicability, reliability and efficiency.     

Use of a membrane oxygenator with small volumes of perfusion for correction of functional changes in respiration disorders

Experiments on 11 dogs under hypoventilation hypoxia (a decrease in the respiratory minute volume by 40-50%) were made to study the efficacy of membrane oxygenation using a membrane Sever-OMP oxygenator of the blood under the conditions of minor perfusion (14-17% of the minute volume of circulation). The animals of the main series (7 dogs) with a veno-venous connection of the membrane oxygenator (MO) tolerated hypoxia quite well for 2 hours. The control animals died. The conclusion is made that membrane oxygenation with small volumes of perfusion (with the MO connected according to Seldinger) can be used in conjunction with artificial ventilation where the latter one is not effective enough.     

Defining the molecular components of calcium transport regulation in a reconstituted membrane system

Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to determine accurately not only the PLB effects on K(Ca) (Ca concentration at half-maximal activity) of the Ca-ATPase, but also the effects on V(max) (maximal activity). Wild-type PLB (WT-PLB) and two gain-of-function mutants, N27A-PLB and I40A-PLB, showed not only the previously reported increase in K(Ca), but also an increase in V(max). Specifically, V(max) increases linearly with the intramembrane PLB concentration, and is approximately doubled when the sample composition approaches that of cardiac SR. Upon phosphorylation of PLB at Ser-16, the K(Ca) effects were almost completely reversed for WT- and N27A-PLB but were only partially reversed for I40A-PLB. Phosphorylation induced a V(max) increase for WT-PLB, and a V(max) decrease for N27A- and I40A-PLB. We conclude that PLB and PLB phosphorylation affect V(max) as well as K(Ca), and that the magnitude of both effects is sensitive to the PLB concentration in the membrane.     

The adaptive regulation of amino acid transport system A is associated to changes in ATA2 expression

The activity of transport system A for neutral amino acids is adaptively stimulated upon amino acid starvation. In cultured human fibroblasts this treatment causes an increase in the expression of the ATA2 system A transporter gene. ATA2 mRNA increase and transport stimulation are suppressed by system A substrates, but they are unaffected by other amino acids. Supplementation of amino acid-starved cells with substrates of system A causes a decrease in both ATA2 mRNA and system A transport activity. These results suggest a direct relationship between ATA2 expression and system A transport activity.     

Development of a versatile computer integrated control system for bioprocess controls

A general approach is described for the implementation of a networked multi-unit computer integrated control system. The use of data acquisition hardware and graphical programming tools alleviates tedious programming and maintains potency and flexibility. One application of the control system, the control of a mammalian cell perfusion culture based on a key nutrient glucose concentration, was demonstrated. The control system offers customized user interface for all process control parameters and allows the flexibility for continued improvement and implementation of new tailored functions. The temperature, pH, dissolved oxygen and glucose level were accurately controlled. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of a supported emulsion liquid membrane system for propionic acid separation in a microgravity environment

Perstractive fermentation is a good way to increase the productivity of bioreactors. UsingPropionibacteria as the model system, the feasibility of using supported emulsion liquid membrane (SELM) for perstractive fermentation is assessed in this study. Five industrial solvents were considered as the solvent for preparing the SELM. The more polar a solvent is, the higher the partition coefficient. However, toxicity of a solvent also increases with its polarity. CO-1055 (industrial decanol/octanol blend) has the highest partition coefficient toward propionic acid among the solvents that has no molecular toxicity towardPropionibacteria. A preliminary extraction study was conducted using tetradecane as solvent in a hydrophobic hollow fiber contactor. The result confirmed that SELM eliminates the equilibrium limitation of conventional liquid-liquid extraction, and allows the use of a non-toxic solvent with low partition coefficient.     

The blowfly salivary gland - a model system for analyzing the regulation of plasma membrane V-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are heteromultimeric proteins that use the energy of ATP hydrolysis for the electrogenic transport of protons across membranes. They are common to all eukaryotic cells and are located in the plasma membrane or in membranes of acid organelles. In many insect epithelia, V-ATPase molecules reside in large numbers in the apical plasma membrane and create an electrochemical proton gradient that is used for the acidification or alkalinization of the extracellular space, the secretion or reabsorption of ions and fluids, the import of nutrients, and diverse other cellular activities. Here, we summarize our results on the functions and regulation of V-ATPase in the tubular salivary gland of the blowfly Calliphora vicina. In this gland, V-ATPase activity energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). Because of particular morphological and physiological features, the blowfly salivary glands are a superior and exemplary system for the analysis of the intracellular signaling pathways and mechanisms that modulate V-ATPase activity and solute transport in an insect epithelium.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

An adaptive on-line control feed rate system in a laboratory scale bakers yeast process

Summary A new control policy for the on-line optimization of the nutrient supply in bakers yeast process is proposed. A feed rate corresponding to minimal substrate uptake time was shown to be optimal for cell yield and specific growth rate. Cultivation results of baker's yeast are presented.Nomenclature c glucose concentration in wort (mol.l^–1) - C total glucose used (mol) - c_e ethanol concentration in wort (mg.l^–1) - c_p glucose concentration in fresh medium (mol.l^–1) - dt/dc glucose consumption time (sec.mol^–1) - F substrate feed rate (litre.hr^–1) - q_c glucose uptake rate (mol.hr^–1) - Q_c specific glucose uptake rate (moll.g^–1.hr^–1) - qO₂ oxygen uptake rate (mol.hr^–1) - QO₂ specific oxygen uptake rate (mol.g^–1.hr^–1) - r_x productivity (g.l^–1.hr^–1) - t time (hr) - x biomass concentration (g.l^–1) - X total biomass (g) - Y_x/c cell yield (g.g^–1): (g.mol^–1) - Y_o/c consumed oxygen to glucose ratio (mol.mol^–1)     

Basic module for an adaptive control system based on neurone information processing

The design of such devices as robotic aids for handicapped people, powered prostheses and manipulative aids such as page turners would benefit from the use of an adaptive control system. Much recent work on adaptive networks has been based on simplified models of the information processing capabilities of neurones. Neurones are now known to be capable of association learning and memory and this study incorporates these features in a neurone model. A single neuronal input system, the NMDA-type glutamate receptor, is modelled by deriving finite difference equations from its reaction dynamics so that the concentration of several molecules in the receptor can be plotted as a function of time. The model shows association learning taking place at the glutamate receptor. A whole neurone with ten glutamate receptor regions is also modelled and shows that a neurone should be capable of recognizing patterns of inputs. As the neurone model is complicated and slow to run, a much simplified form of the model is described which embodies the basic features of neurone information processing in a simple algorithm.     

Development of a novel mammalian display system for selection of antibodies against membrane proteins

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 10⁹ variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (K_D) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.