Immunolocalization of a new intestinal antiproliferative factor in human intestinal epithelial cells.

A new intestinal antiproliferative factor (IAF) with an approximate molecular weight of 120 kDa has been purified from the human small intestine. This factor blocks the progression of human colon adenocarcinoma cells HT-29 from the G1 to the S phase. IAF, specific of the lower part of the digestive tract, was detected rather late in mouse embryonic development.

Purification of a new intestinal anti-proliferative factor from normal human small intestine.

Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies.

Inhibition of calcium influx during hypoxia/reoxygenation in primary cultured rat hepatocytes.

Calcium has been demonstrated to play an important role in hepatocyte damage during ischemia/reperfusion phases. Calcium influx was determined in primary cultured rat hepatocytes submitted to a succession of warm hypoxia and reoxygenation phases in the presence of diltiazem, gallopamil and a Na+/H+ antiport inhibitor, HOE-694. Only diltiazem significantly inhibited calcium influx with higher potency after reoxygenation than after hypoxia only, suggesting a complex mechanism of action of diltiazem which could act on different physiological functions involved in Ca2+ invasion of hepatocytes after hypoxic insult.

Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation.

Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of 'mature' final, brush border lactase-phlorizin hydrolase.

Thyroid hormone regulation of the Na+/glucose cotransporter SGLT1 in Caco-2 cells.

The expression of the Na+/glucose cotransporter (SGLT1) in response to thyroid hormone [3,5,3'-tri-iodo-l-thyronine (T3)] was investigated in the enterocytic model cell line Caco-2/TC7. In differentiated cells, T3 treatment induces an average 10-fold increase in glucose consumption as well as a T3 dose-dependent increase in SGLT1 mRNA abundance. Only cells grown on glucose-containing media, but not on the non-metabolizable glucose analogue alpha-methylglucose (AMG), could respond to T3-treatment.

Renal neutral alpha-D-glucosidase has no role in transport of D-glucose derived from maltose hydrolysis.

To reinvestigate the "hydrolase-related transport" concept, neutral alpha-D-glucosidase, a membrane-bound disaccharidase of renal proximal tubule, was first purified from brush-border membranes and then asymmetrically reincorporated into egg phosphatidylcholine vesicles. Proteolytic treatments and immunotitration studies demonstrated that this enzyme was integrated in native and artificial membrane vesicles with a similar topology. The uptake of free glucose and glucose produced by maltose hydrolysis was studied using 1) proteoliposomes containing integrated neutral alpha-D-glucosidase, in combination with other membrane proteins, and 2) proteoliposomes containing only the other membrane proteins but incubated in a medium containing neutral alpha-D-glucosidase in its hydrophilic form.

Effect of cross-linkers on the structure and function of pig-renal sodium-glucose cotransporters after papain treatment.

Kidney brush-border membranes contain two sodium-dependent glucose transporters, one with low and one with high affinity for phlorizin, the specific inhibitor of these transporters. Using Scatchard analysis of phlorizin binding and Western blotting with specific antibodies against these transporters, we demonstrate in this study that although both transporters were proteolysed by papain treatment, only the high-affinity phlorizin-binding sites were decreased. Papain treatment followed by cross-linking with homobifunctional disuccinimidyl tartarate restored only the structure of the low-affinity phlorizin-binding protein (approx.

Do pancreatic proteases play a role in processing prolactase and/or in the postweaning decline of lactase?

To assess the role of pancreatic proteases in the proteolytic processing and in the postweaning decline of lactase-phlorizin hydrolase (LPH), we have determined lactase activity and the different LPH forms in postweaned rats in which a jejunal loop was excluded from contact with pancreatic secretions by a jejunal bypass procedure. As a control for the absence of pancreatic proteases, pro-sucrase-isomaltase (proSI), which is known to be split by pancreatic proteases into heterodimeric SI, was used. Nearly all proLPH was processed to mature LPH, indistinguishable from LPH isolated from control animals.

Potassium uptake and water content in hepatocytes isolated from rat livers preserved in Euro-Collins and UW solutions and after transplantation.

Isolated hepatocytes from the rat were used to assess the maintenance of liver cell function in relation to the composition of the preservation medium. After separation by collagenase, they were incubated in Krebs-Ringer-Bicarbonate medium (KRB), Euro-Collins (EC), or University of Wisconsin (UW) solutions. Potassium influx, cell volume, and transaminase release were measured in cells freshly separated from control livers or from livers preserved in vitro up to 12 h in these media or having undergone orthotopic liver transplantation (OLT).

Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%).

Oligomeric structure of the sodium-dependent phlorizin binding protein from kidney brush-border membranes.

Immunodetection of solubilized kidney brush-border proteins on Western blots using antibodies against the 70 kDa phlorizin binding component of sodium-glucose cotransporter allows to identify an additional protein band with apparent molecular mass of 120 kDa in the presence of reducing agent dithiothreitol. Antibodies specifically eluted from the 70 kDa protein still recognize the 120 kDa protein on Western blot. The lack of dissociation of the 120 kDa protein from native brush borders or Triton X-100 extract in the presence of dithiothreitol can be improved by an extended incubation at 25 degrees C; this protein is full dissociated when purified by electroelution from polyacrylamide gel and gives two subunits with apparent molecular masses of 70 and 60 kDa by Coomassie staining and Western blot analysis.

Effect of human native low-density and high-density lipoproteins on prostaglandin production by mouse macrophage cell line P388D1: possible implications in pathogenesis of atherosclerosis.

Macrophages have been shown to play a key-role in the development of atherosclerotic lesions. Monocyte attraction and activation in the arterial wall lead to foam cell formation, cholesterol accumulation and secretion of inflammation mediators. Among macrophage secretions, prostacyclin and thromboxane are prostaglandins involved in the regulation of coagulation and vascular permeability.

Immunological recognition of sodium/D-glucose cotransporter from renal brush border membranes by polyclonal antibodies.

Antisera prepared in rabbit to a D-glucose-inhibitable phlorizin binding component of the pig kidney brush border membrane precipitated more than 90 percent of the D-glucose-inhibitable phlorizin binding activity from a Triton extract. These antibodies also stimulated D-glucose uptake by native brush border membranes at low D-glucose concentrations (1 mM) and inhibited it at higher D-glucose concentrations. Immunoblotting was used to locate polypeptide subunits of the glucose transporter in polyacrylamide gels of proteins extracted from the brush border membranes.

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.

Use of mild detergent gel electrophoresis for isolation and characterization of the kidney brush border D-glucose transporter.

Gradient gel electrophoresis was performed under mild detergent conditions to separate pig kidney brush border membrane proteins and to identify the smallest functional molecular protein entity of the D-glucose transporter. The various protein bands obtained from the nondenaturing gel system in a semipreparative scale were eluted by electrodialysis. These proteins were then reintegrated into proteoliposomes and tested for D-glucose-inhibitable [3H]phlorizin binding.

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles.

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled D-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and D-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues.

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography.

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of D-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards D-glucose compared to other sugars tested was shown as well as the main features of D-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of D-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated.

Influence of sodium ions on detergent solubilization of pig brush border D-glucose transport system for reconstitution experiments.

The D-glucose uptake by liposomes resulting from the association of egg lecithins with solubilized membrane proteins was measured in order to assess their sodium dependent D-glucose transport activity. Membrane proteins were extracted by Triton X-100 solubilization of pig kidney brush border membrane vesicles, which were suspended either in KCl medium or in NaCl medium. When measured by equilibrium isotope exchange procedure in sodium conditions, the D-glucose uptake by sodium detergent extract associated to liposomes occurred with a higher velocity than that obtained with liposomes reconstituted from potassium detergent extract.

Glucose transport by horse kidney brush borders. I.--Transport properties of brush border membrane closed vesicles.

Brush border membranes isolated from horse kidney cortex as closed right-side out vesicles show selective permeability when analyzed on sucrose and dextran gradients. These vesicles can actively accumulate D-glucose. The preservation of the glucose transport system is demonstrated by the following features: (a) the uptake and release rates of D-glucose are higher in the presence of a sodium gradient, showing that D-glucose transport is a sodium-dependent process; (b) this transport, specific for the D-isomer, is inhibited by phlorizin; (c) the D-glucose transport system is saturable; (d) no inhibition of D-glucose transport is found with C-mannose; (e) the D-glucose uptake is sensitive to osmotic variations.

[The length of polyadenylic acid tracts of Sindbis virus RNA: the possible existence of two synthetic mechanisms].

The poly (A) tracts of RNA's from viral particles and from replicative complex belong to two classes of 180 and of 60 nucleotides in length. Those of the mRNA are homogenous and around 280 nucleotides in length. The results presented here strongly suggest the possibility of a copying mechanism in the first case and of a posttranscriptional addition in the latter.