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.

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.

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.

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.

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).

Lactate kinetics after short strenuous exercise in man.

Arterial blood lactate was measured at 10 s time intervals after a 3 min strenuous exercise for six athletes pedaling a bicycle ergometer in the sitting position. Recovery curves were fitted to the equation: Y(t) = A1(1-e-gamma1t)+A2(1-e-gamma2t)(0). The evolution of arterial lactate concentrations during recovery can accurately be represented by this equation. The values of the coefficients A and gamma found were used for a numerical application to an open two-compartment model: the "working muscle space" (1) and the "lactate space" (2). Intramuscular concentrations, the transfer coefficients from compartment 1 to compartment 2 and from compartment 2 to compartment 1 and the fractional turnover and basal turnover rate were calculated. Computed intramuscular lactate concentrations at the end of exercise compare favorably with those found earlier by muscular biopsic samplings. The turnover data are higher than those previously reported. This discrepancy may possibly be attributed to the method of mathematical analysis.