Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding.

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other.

Evaluation of hydrophobicity and adherence of Neisseria meningitidis strains and a study of their correlation by analysis of alterations induced by antibiotics.

Surface hydrophobicity and adherence to human buccal epithelial cells were evaluated in four Neisseria meningitidis strains. Hydrophobicity was measured by partition into p-xylene in the presence or absence of ammonium sulphate, whereas radioactivity-labelled bacteria were used to estimate the ability to adhere to buccal cells by liquid scintillation techniques. Eight antibiotics were employed to induce modifications in the two parameters in order to estimate their possible relationship.

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor.

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor.

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping.

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments.

Studies on the implication of surface hydrophobicity in the adherence of Neisseria meningitidis to buccal epithelial cells.

The study of surface hydrophobicity and adherence to human buccal epithelial cells of four Neisseria meningitidis strains and the correlation analysis of the data obtained for both parameters has demonstrated that hydrophobicity of meningococci is not a causative factor in adherence in normal conditions. Modification of the bacterial surfaces (by freezing, heating, ultraviolet irradiation, fixation with glutaraldehyde or sodium metaperiodate cleavage of external sugars) induced significant changes in one or both parameters, but these changes were not correlative in both properties. In contrast to other bacteria, the hydrophobicity levels shown by our meningococci are not relevant to their adherent processes.

Cytotoxic granules from killer cells: specificity of granules and insertion of channels of defined size into target membranes.

The channel-forming polyperforins P1 and P2 are thought to be formed from the contents of dense core vesicles of cytolytic effector cells. To test this hypothesis, granules from various cytotoxic effector cells were assayed for cytolytic activity on nucleated or unnucleated targets. The results show that in general, granules from cytolytic effector cells are cytolytic, whereas granules from noncytotoxic cells are not.

Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor.

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor.

Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits.

Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the alpha subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, alpha subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide alpha 159-169 was less by a factor of 10(4), suggesting that the mAbs bind primarily to the sequence alpha 152-159. Cholinergic ligands did not inhibit mAb binding.

Adherence and hydrophobicity in Neisseria meningitidis and their relationship with surface charge.

The influence of surface charge on hydrophobicity and adherence of four strains of Neisseria meningitidis was studied. After neutralization of negative (treatment with EDC-methylamine), positive (treatment with formaldehyde) or both charges, adherence to buccal cells and hydrophobicity were determined and analysed. It was found that surface charges are relevant for adherent processes.

Immunochemical tests of acetylcholine receptor subunit models.

Acetylcholine receptors of fish electric organs and mammalian skeletal muscle comprise four structurally homologous glycoprotein subunits in the mole ratio alpha 2 beta gamma delta (refs 1-4). All four subunits have leader sequences and are exposed on both sides of the membrane. From amino acid sequencing, three groups have predicted that each subunit has four hydrophobic alpha-helical transmembranous domains.

Functional properties of the acetylcholine receptor incorporated in model lipid membranes. Differential effects of chain length and head group of phospholipids on receptor affinity states and receptor-mediated ion translocation.

Torpedo acetylcholine receptor was reconstituted into liposomes of pure synthetic lipids in order to study the influence of the lipid environment on affinity state transitions and the ion translocation function of the receptor. A critical concentration of 30 to 40% of cholesteryl hemisuccinate was necessary in liposomes made of cholesteryl hemisuccinate and dimyristoyl phosphatidylcholine to mimic the kinetics of agonist-induced state transitions observed in native membranes. With increasing chain length of the saturated lecithins, a marked increase in carbamylcholine dissociation constants was observed.

Expression of surface hydrophobicity encoded by R-plasmids in Escherichia coli laboratory strains.

The inclusion of drug-resistance plasmids (R-plasmids) in Escherichia coli strains has been shown to determine the formation of specific surface structures which could modify bacterial surface characteristics relevant for pathogenic processes. Thirtyone R-plasmids (from different incompatibility groups) have been transferred to three E. coli laboratory strains, and surface hydrophobicity modifications have been measured by three methods: "salting-out", adsorption to hexadecane and adsorption to xylene.

Toxicity of an algal mucopolysaccharide for Escherichia coli and Neisseria meningitidis strains.

Several bacterial strains of clinical significance have been tested to assess the toxic effect of a lectin-like algal mucopolysaccharide from Fucus vesiculosus on their growth. The toxic effect of the mucopolysaccharide has been found to be exerted only on Escherichia coli and Neisseria meningitidis strains. The degree of toxicity, measured by the effect on the growth of the bacteria, is variable depending on the strains of E.

Conversion of acetylcholine receptor dimers to monomers upon depletion of non-receptor peripheral proteins.

The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorata. Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction.

Influence of K99 on the phagocytosis and killing of K99-positive Escherichia coli by mouse peritoneal cells in vitro.

The expression of K99-plasmid encoded products was examined to assess their influence on phagocytosis and killing by mouse peritoneal cells. Strains of K99-positive Escherichia coli were cultured under varying conditions which either allowed or inhibited the expression of K99-plasmid products. The survival of phagocytosis by the bacteria was found to depend more on the expression of the K99 antigen than the expression of its associated adhesin.

Study of several factors affecting the agglutinating activity of K99-positive Escherichia coli strains.

The effect of several factors on Escherichia coli K99-plasmid associated agglutination has been studied. The results obtained indicate that Escherichia coli 637 (K99+) mediated red blood cell agglutination is unspecific although the agglutination titres for several erythrocyte species are significantly different. The agglutination is highly stable (at least with sheep red blood cells) to changes in temperature (from 4 degrees C to 37 degrees C), to changes in pH (from 5 to 9) and to the presence or absence of several metallic cations.

Sulphydryl groups and iodo-[3H]acetic acid labeling in proteolipids from Torpedo electroplax.

Several fractions of proteolipids from Torpedo electroplax were separated by DEAE-cellulose chromatography in organic solvents, and the sulphydryl groups were determined by a spectrophotometric method. On the same fractions the covalent labeling with iodo-[3H]acetic acid to sulphydryl groups was studied. In total proteolipids there were 30.

Purification and partial characterization of a Fucus Vesiculosus agglutinin.

Fucus vesiculosus agglutinin has been purified to homogeneity by conventional chromatographic procedures and characterized as a mucopolysaccharide with 90% carbohydrate content. Estimated molecular weight is about 2 X 10(6) daltons. It has no sub-unit structure and its isoelectric point is 3.

Selective interaction of a Fucus vesiculosus lectin-like mucopolysaccharide with several Candida species.

A complex carbohydrate specific lectin-like mucopolysaccharide extracted from the brown alga Fucus vesiculosus was found to agglutinate Candida guilliermondii cells but not those of other species of the same genus (except for a weak agglutination with C. krusei). The selective binding of this mucopolysaccharide correspondingly affected the growth of the yeasts.

Purification and partial characterization of a K99-antigen associated adhesin in Escherichia coli (637 strain).

The K99-antigen associated adhesin in Escherichia coli (637 Strain) has been purified to homogeneity by using conventional chromatographic procedures. Sodium deoxycholate was used in the precipitation steps to avoid hydrophobic interactions between the fimbriae and other membrane-associated components. Homogeneity of the purified adhesin was assessed by electrophoresis, isoelectrofocusing, analytical gel filtration and immunoprecipitation against K99 specific antiserum, being homogeneous in all cases.

The delipidation of brain proteolipid protein by ultrafiltration.

It has been very difficult to prepare the apoprotein moiety of brain white matter proteolipid so that it is completely devoid of complex lipids, without suffering aggregation and protein denaturation. The reason is that complex lipids are tightly bound to the proteolipid apoprotein. Using a new ultrafiltration method, we obtained, in a gradual way and in a relatively short time, more than 99% delipidation in water-saturated n-butanol, with and without 0.

Method for lyophilizing brain proteolipid preparations that increases subsequent solubilization by detergents.

A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins.