In vitro assembly of rat pancreas tubulin in the presence of taxol.

A rat pancreas supernatant was applied to an affinity column where colchicine analogues had been coupled to CNBr-Sepharose 4B, and subsequent elution with 0.35 M sodium chloride gave tubulin among other proteins. Incubation with 5 microM taxol, a natural plant product, resulted in the assembly of tubulin as checked by turbidimetry at 350 nm. Electron microscope observation of the structures obtained revealed (i) the presence of numerous microtubules with the same morphological parameters as brain microtubules, and (ii) that immunoreactive tubulin molecules were well-distributed along the microtubules as shown by the immunogold staining technique. Biochemical evidence indicated that the microtubules obtained were exclusively composed of tubulin, as demonstrated by slab gel polyacrylamide electrophoresis and by immunoblot staining with highly specific tubulin antibodies.

Purification and characterization of a trypsin-like protein from rat pancreas.

The purification of the latent form of a rat pancreas trypsin-like protein was performed by ion-exchange and hydrophobic chromatographies. After partial activation, the affinity on immobilized soybean trypsin inhibitor allowed the isolation of an active and an inactive form. They had 30,000 and 32,000 molecular weight, respectively, as checked by polyacrylamide slab gel electrophoresis. Active enzyme (named TLP) was not glycosylated and had an isoelectric point of 4.4. The rate of hydrolysis of different substrates and the effects of various proteinase inhibitors indicated clearly that TLP differs from proteinases previously described and belongs to the trypsin family.

Glyceraldehyde-3-phosphate dehydrogenase is a microtubule binding protein in a human colon tumor cell line.

A protein which binds to tubulin polymer was isolated from a human colonic tumor cell line. This protein has a molecular mass of 35 kDa, as determined by polyacrylamide slab gel electrophoresis. The protein was purified by affinity chromatography on taxol-stabilized microtubules, and it did not cross-react with anti-MAP2 or anti-tau antibodies. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase by its enzyme activity and immunoblotting experiments. The purified protein caused a pronounced enhancement in the turbidity increase produced by in vitro tubulin polymerization, and electron microscopic observations revealed the presence of bundles of microtubules.

Characterization of a 67 kDa microtubule-binding protein in the pancreas from different species.

Microtubule-interacting proteins have been studied from a pancreas supernatant. These proteins were first identified by affinity chromatography on taxol-stabilized microtubules. Among these interacting polypeptides, we show, for the first time, the presence of a protein which has a molecular mass of 67 kDa, as determined by polyacrylamide slab gel electrophoresis. The heat stability and the ability of this 67 kDa polypeptide to copolymerize with phosphocellulose-purified tubulin suggest that this protein may be a microtubule-associated protein.

Adhesion complexes implicated in intestinal epithelial cell-matrix interactions.

This article review summarizes data on cell-substratum adhesion complexes involved in the regulation of cellular functions in the intestine. We first focus on the molecular composition of the two main adhesion structures-the beta1 integrin-adhesion complex and the hemidesmosome-found in vivo and in two human intestinal cell lines. We also report the key findings on the cellular behavior and response to the extracellular matrix that involve integrins, the main transmembrane anchors of these complexes. How the dynamics of cell/extracellular matrix interactions contribute to cell migration, proliferation, differentiation, and tumorigenicity is discussed in the light of the data provided by the human intestinal cells.

Improved purification of rat intestinal lactase.

A rapid and improved method to obtain purified lactase from rat intestine is described. The purification procedure involved only two chromatographic steps. The degree of purification was far above (500 fold) the values reached with classical methods. Rabbit antisera raised to the purified lactase were characterized using conventional immunological techniques. The specificity of the lactase antibodies was confirmed by the lack of interference on maltase, aminopeptidase and alkaline phosphatase activities measured after papain extraction of the membrane proteins.

Pancreatic anionic trypsin: evidence for the existence of a 30 kDa form.

1. An anionic form of trypsin has been isolated from pancreas of various species (rat, pig, dog and cow). 2. The purification procedure included affinity chromatography on STI-Sepharose 4B and ion-exchange chromatography on DEAE-Sephadex A-50. 3. The preparation was homogenous as checked by SDS-polyacrylamide slab gel electrophoresis, resulting in an estimated molecular weight of 30 kilodaltons (kDa) for this anionic form. 4. Antibodies against the anionic form from rat pancreas cross-reacted towards the anionic enzyme from porcine pancreas but not with the dog or bovine enzyme, nor with all the studied cationic forms. 5. Limited proteolysis of tubulin, a cytoskeletal protein, with an anionic or cationic form of trypsin showed striking differences in the size of produced peptides.

Stimulation of intestinal chromatin template activity by dietary carbohydrates in adult rats.

Oral administration of a 70% solution of sucrose to starved adult rats resulted 1 h after feeding in a 3.5-fold stimulation of intestinal chromatin template activity assayed in vitro using E. coli RNA polymerase. A similar stimulatory effect was observed with fructose, whereas glucose exhibited a weaker effect, indicating that the nature of the ingested carbohydrate may have a direct effect on the extent of intestinal chromatin template activation.

Stimulus secretion coupling: role of cyclic GMP and calcium in the regulation of secretion from rat exocrine pancreas.

In rat pancreatic fragments, stimulation of amylase and labeled protein release by carbachol, caerulein, and ionophore A 23187 results within minutes in a short rise in cyclic GMP levels. Cyclic AMP levels do not change significantly. The secretory response elicited by each secretagogue is not modified when combined in pairs. Under intracellular calcium depleting conditions, both the cyclic GMP and the secretory responses to secretagogues are inhibited in parallel, suggesting a good correlation between both processes. Furthermore, 8-Bromocyclic GMP induces pancreatic secretion, but to a lesser extent, and fails to alter the increase in secretion caused by the various secretagogues. However, other agents such as imidazole, ascorbic acid, phenylhydrazine, and sodium azide also increase cyclic GMP levels but fail to stimulate pancreatic secretion. On the other hand, dibutyryl cyclic AMP also stimulates amylase and labeled protein discharge and potentiates the increase caused by cabachol, caerulein, and ionophore A 23187. These results do not permit conclusions regarding a cause and effect relationship between cyclic GMP and secretion. A role for calcium seems to be the most likely.

Microtubule affinity chromatography: a new technique for isolating microtubule-binding proteins from rat pancreas.

We have developed an affinity chromatography method for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts of rat pancreas. Among the ten proteins which copurify with pancreas tubulin on a colchicine derivatives-affinity chromatography, three polypeptides of respectively 58, 55 and 48 kDa strongly bind to the microtubule affinity column. To begin to characterize these proteins, we have generated polyclonal antibodies against tau polypeptides from brains of immature chicken or rat. As judged by immunoblots, the three polypeptides seem to be immunologically related to the tau proteins previously localized in brain.

Modulation by thyroxine of the amount of lactase protein in the jejunum of adult rats.

Adult rats starved for 48 h received a daily injection of thyroxine over a 3-day period before they were killed. When compared to nourished animals, starvation provoked a 4- to 5-fold increase in immunoreactive lactase protein, which paralleled a similar stimulation of lactase activity in the brush border membranes of the proximal jejunum. Exogenous thyroxine completely inhibited the starvation-induced increase in immunoreactive lactase protein in both the intracellular and the brush border membranes.

Rat pancreas kinesin: identification and potential binding to microtubules.

We have demonstrated the presence of kinesin in the secretory pancreatic tissue using SDS-PAGE, immunoblot and immunoelectron microscopy techniques. Polyclonal antibodies were raised against the rat brain kinesin heavy chain and affinity-purified. Immunoblot studies showed that these antibodies were bound to a 116 kDa protein found in rat pancreas crude extracts and in partially purified kinesin fractions. Kinesin identification was also performed by a cosedimentation procedure based on its strong binding to microtubules in the presence of sodium fluoride. The microtubule-kinesin complex was observed by immunoelectron microscopy gold staining. The reversible association of kinesin with microtubules was generated by MgATP.

Establishment of serum based essentiall free of proteolytic activity for the culture of mouse pancreatic islets.

The present study demonstrates that a) serum based culture medium degrades 125I inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the hear treated serum.

Rat pancreas actin: purification and characterization.

Isolation of rat pancreas actin was performed with three different technics: polymerization-depolymerization method, affinity chromatography on DNase I-Sepharose 4B or ion exchange chromatography on DEAE-cellulose. Inhibition of DNase I activity, localization by SDS polyacrylamide slab gel electrophoresis and presence of microfilaments allowed its identification. Affinity process led us to obtain actin which kept inhibitory activity (30,000 U per mg) on DNase I when using vacuum dialysis. Actin eluted from DEAE-cellulose associated reversibly in 50-70 A microfilaments in the presence of phalloidin, was pure at 95% and had a satisfactory inhibitor activity (77,000 U per mg).

Regulation of the type II hemidesmosomal plaque assembly in intestinal epithelial cells.

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.

On the secretagogue effect of dibutyryl cyclic AMP in the rat exocrine pancreas.

DbcAMP greater than or equal to 0.1 mM induces the discharge of exportable enzymes from rat pancreas fragments incubated in vitro. This effect is qualitatively similar to the action of physiological secretagogues acting via hormone receptors: 1) it is accompanied by the appearance of exocytotic images at the acinar cell apex; 2) it is energy dependent but energy supply is low while that required for the carbamylcholine or caerulein response is high and can only be afforded by oxidative phosphorylation; 3) it is calcium dependent, but no alteration of inward or outward calcium movement can be observed; 4) it is altered by agents known to disrupt the microfilamentous microtubular system [41]. However, the secretory response to DbcAMP is quantitatively less than that obtained with hormonal stimuli. A damaging effect of DbcAMP on pancreatic acinar cells is ruled out on histological and biochemical grounds: there is no significant leakage of LDH; protein synthesis, 2-deoxy-D-glucose and L-leucine uptake are unaltered. The secretagogue effect of DbcAMP is reversible, dose-related and specific. It is not mediated by neurotransmitter release or by interaction with their receptors. The evidence presented points to a direct interaction of DbcAMP on the pancreatic acinar cell and suggests the last step of the secretory cycle as the most probable site of action of the nucleotide derivative.

Biochemical and immunochemical identification of a microtubule-binding protein from bovine pancreas.

We have identified a 67 kDa heat-stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat-stable microtubule-associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau.