Effect of the cytoskeleton dynamic condition on the neuronal plasticity]

Infringement of the Lymnaea stagnalis cytoskeleton condition affected preservation and repeated development of plastic responses. Stabilising of the microtubules led to a dependence of the development and preservation dynamics of the plastic responses. Stabilising of the microfilaments transformed short-term plastic responses into long-term ones. The findings suggest a key role of reorganisation of the cytoskeleton in neuronal plasticity.     

Cell polarity regulates the release of secretory component, the epithelial receptor for polymeric immunoglobulins, from the surface of HT-29 colon carcinoma cells.

The HT-29 human colon carcinoma cell line differentiates in glucose-free medium to an enterocytic phenotype. We previously isolated a series of HT-29 subclones selected for high levels of expression of secretory component (SC), the epithelial receptor for polymeric immunoglobulins. To develop a model system for studying effects of cell polarity on SC expression and release from the cell surface, the HT-29.74 subclone was induced to differentiate in glucose-free medium. Expression of SC was induced by glucose deprivation in both the parental HT-29 cell line and, to an even greater extent, in the HT-29.74 subclone. Prolonged glucose deprivation of HT-29.74 cells resulted in morphological changes consistent with enterocytic differentiation. Metabolic radiolabeling of SC in differentiated HT-29.74 cells indicated that proteolytic cleavage of membrane-bound to free SC occurred both on the cell surface and intracellularly, possibly in a vacuolar apical compartment or intrapeithelial lumen. To study effects of cell polarity on SC release, differentiated HT-29.74 cells were depolarized by culturing in low calcium medium. Within 2 hours after transfer of the cells into low calcium medium, a burst of SC release was observed concomitant with cell depolarization. Subsequently, release of SC declined significantly and remained low as long as cells were maintained in a depolarized state. The extent of cell depolarization could be controlled by varying the extracellular calcium concentration or by substituting the divalent cation Sr++, which partially prevents depolarization, for Ca++. In either case, the magnitude of the initial burst and subsequent decline in release of SC was proportional to the extent of cell depolarization. We conclude that cell polarity plays an important role in controlling the release of SC in intestinal epithelial cells, most likely by regulating the distribution of membrane-bound SC and SC protease, which are on the basolateral and apical cell surfaces, respectively, in differentiated cells.     

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor,peripheral ν-polypeptide (Mr 43,000)

NaIO₄ oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (α, β, γ and δ) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ν-peptide (M_r 43,000), pH and temperature. Very low NaIO₄ concentrations (50 μM) suffice to prevent the penetration of the ν-peptide into NaDodSO₄ polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (α, M_r 40,000) into a doublet. In contrast, NaIO₄ treatment of the detergent-solubilized receptor aggregated the α-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO₄-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (～2 mM NaIO₄).Neuraminidase digestion affected exclusively the γ- and δ-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. β-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO₄ nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-α-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and β-glucosidase treatments. The latter enzymatic digestion reduced the α-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.     

Interaction of enzymatically modified low-density lipoproteins with fibronectin

Interaction of human low-density lipoproteins (LDL) with homologous fibronectin fixed on collagen-Sepharose was studied. LDL were digested with pepsin, the degree of hydrolysis amounting to 10%. Upon passing modified LDL through a fibronectin-collagen-Sepharose column the desorption of fibronectin occurred. Addition of the increasing amount of fibronectin to the pepsin-treated LDL solution in the presence of Ca2+ ions led to the formation of LDL-fibronectin insoluble complexes. Interaction of native LDL with fibronectin was not observed. The data suggest that enzymatic modification of LDL increasing interaction of modified LDL with fibronectin, a component of extracellular matrix, could promote the accumulation of such LDL in arterial walls.