Inhibition of receptor-mediated endocytosis immunoglobulin G by enterocytes isolated from the neonatal rat jejunum

The effect of inhibitors of respiration (NaN3 and DNP), glycolysis (2DG, IAA and NaF) and the microtubular-microfilament system (colchicine and cytochalasin B) on the uptake of rat immunoglobulin G (IgG) by enterocytes isolated from the neonatal rat gut has been assessed. After a 1 hour incubation, NaN3, and DNP had significantly reduced IgG uptake by between 32% and 35% of the control, IAA and 2DG were less effective and NaF, colchicine and cytochalasin B had no effect at all. The findings show that IgG is internalised by isolated enterocytes in vitro and that this internalisation is under metabolic control, that inhibitors of respiration are more effective in blocking uptake than inhibitors of glycolysis.     

Protein binding to brush borders of enterocytes from the jejunum of the neonatal rat

The specific binding of IgG to jejunal brush borders was greatest at acidic pH, at neutral pH no specific binding occurred. Specific binding declined with age-no specific binding occurred in borders from 20-and 24-day-old animals. There was no specific binding of IgG to borders from ileal enterocytes. Human transferrin and bovine serum albumin did not bind specifically to borders. The affinity of binding (-Ka) and the receptors site numbers per border estimated for rat IgG were 18.64 X 10(6) M-1 to 3.53 X 10(6) sites; for human IgG, 25.06 X 10(6) M-1 to 3.30 X 10(6) sites; for bovine IgG, 10.48 X 10(6) M-1 to 2.11 X 10(6) sites and for sheep IgG, 7.26 X 10(6) M-1 to 2.34 X 10(6) sites.     

Control of the flux in the arginine pathway of Neurospora crassa. The flux from citrulline to arginine.

The arginine pathway is a complex one, having many branch points and effector interactions. In order to assess the quantitative role of the various mechanisms that influence the flux in the pathway, the system was divided experimentally into two moieties by the introduction of a genetic block abolishing ornithine carbamoyltransferase activity. This normally produces citrulline from ornithine within the mitochondria. The endogenous citrulline supply was replaced by citrulline in the growth medium, and control of the influx rate was achieved by using glycine or histidine as uptake inhibitors. By modulating the influx rate over a large range of values, the importance of such factors as reversibility, saturation, inhibition and induction in affecting the flux and the sizes of intermediate pools between citrulline and arginine was assessed. The role of expansion fluxes as important controls in the exponentially growing system was established.     

Immobilization and treatment of Streptococcus faecalis for the continuous conversion of arginine into citrulline.

Citrulline is one of the steps of the arginine dihydrolase system of Streptococcus faecalis. We have shown that the bacteria, immobilized in polyacrylamide gel and treated with Cetyl trimethyl ammonium bromide (CTAB) or heat, were able to convert arginine to citrulline. Used continuously in a column reactor, the entrapped cells have a stable enzymatic activity for at least 30 days at 45 degrees C.     

Interaction of Leu-enkephalin with isolated enterocytes from guinea pig: binding to specific receptors and stimulation of cAMP accumulation

The specific binding of Leu-enkephalin and the stimulatory effect of the peptide on cAMP accumulation have been assessed in isolated enterocytes of guinea pig. The binding was reversible as well as time and temperature dependent. Two classes of binding sites could be defined: a class with a relatively high affinity (Kd = 0.7 microM) that represented 1% of total binding capacity, and another class with low affinity (Kd = 55.5 microM). The stimulation of cAMP accumulation was also shown to depend on time and temperature and was potentiated by a phosphodiesterase inhibitor. Half-maximal stimulation of cAMP accumulation was observed at 119 microM and maximal stimulation (27-fold basal level) at 300 microM Leu-enkephalin. Both steps of the interaction were not modified by Na+ but exhibited a high specificity since modification in the structure of Leu-enkephalin resulted in an important loss of binding affinity and stimulatory activity.     

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.     

Selective, high conversion of D-glucose to 5-keto-D-gluoconate by Gluconobacter suboxydans

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

An in silico model of enterocytic glutamine to citrulline conversion pathway

Enterocyte is one of the main sites of amino acids metabolism and particularly of the citrulline biosynthesis. Working at the cellular scale and applying ordinary differential equations (ODEs) formalism, we have built a mathematical model of the enterocytic glutamine to citrulline conversion in the fasting state. This model enables us to test different physiopathological scenarios of enzyme activity loss. Results from two different approaches were compared: a standard approach (KA) based on the Michaelis–Menten assumptions and an association–dissociation approach (VH) based on the kinetic mass action law. For both approaches, ODEs system was numerically solved using Mathematica?. In both cases, the model correctly predicts the physiological plasma citrulline steady-state, but the two approaches present clear differences for metabolites of enzymes having a complex mechanism, challenging the validity of the KA approach in such cases. When physiopathological scenarios of enzyme activity loss are simulated, both approaches predict a very sharp transition from the physiological citrulline plasma level to the lack of its production: the concentration profiles of these simulations show a clear threshold of which characteristics vary with the involved enzyme. Moreover, amongst all enzymes included in the model, the ornithine aminotransferase (OAT) shows the highest sensitivity in the system whatever the approach used. This model points out the limits of the Michaelis–Menten approach to model complex enzyme mechanisms. It highlights the key role of OAT in the studied citrulline synthesis pathway and also suggests an order of magnitude about the optimal ratio of enzyme concentrations in this pathway.     

Stimulation by D-glucose of mitochondrial oxidative events in islet cells.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.