A neuraminidase from Streptococcus sanguis that can release O-acetylated sialic acids

The naturally occurring sialic acids can have different types of N- and O-substitutions, resulting in more than 20 known isomers and compounds. Most methods for the detailed study of these various sialic acids require that the molecules be first released from their alpha-glycosidic linkage. When mild acid hydrolysis is used for this purpose, significant destruction of O-substituent groups occur. On the other hand, the presence of O-substituent groups renders the sialic acid molecule partially or completely resistant to the action of the currently known neuraminidase. To circumvent this problem, we searched for a neuraminidase whose activity is not affected by O-substitution. We reasoned that because Streptococcus sanguis from the human oral cavity is continually exposed to O-substituted sialic acids, its extracellular neuraminidase might not be blocked by O-substitution. We therefore purified this enzyme 3100-fold (56% yield) using ammonium sulfate precipitation, N-(p-aminophenyl)oxamic acid-agarose affinity chromatography, and chromatography on quaternary aminoethyl (QAE)-Sephadex, sulfopropyl (SP)-Sephadex, and Sephacryl S-200. The purified preparation is free of other significant glycosidase activities and proteolytic activities. It is capable of quantitatively releasing all the O-acetylated sialic acids that we studied with the single exception of the 4-O-acetylated sialic acid of equine submaxillary mucin. The activity of the enzyme is also not restricted by the type pf sialic acid linkage or the nature of the underlying oligosaccharide. However, it has maximal activity on gangliosides only in the presence of detergents. The general properties of this enzyme are described and its substrate specificities are contrasted with those of the commonly used neuraminidase from Vibrio cholerae.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

Monoclonal antibodies modify acetylcholine-induced ionic channel properties in cultured chick myoballs

Summary Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick myoballs. Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

The effect of osmotic `shock'' on the swelling pattern and respiratory control of rat-liver mitochondria

1. Rat-liver mitochondria suspended in 0.25m-sucrose were exposed for a few seconds to strongly hypo-osmotic conditions, and then the osmolarity of the medium was raised again to 0.25 with the aid of tris chloride (osmotic ;shock'). 2. Mitochondria after hypo-osmotic pretreatment lost their capacity for slow energy-dependent swelling in iso-osmotic tris buffer and showed no respiratory control. 3. Swelling could be induced in the ;shocked' mitochondria by ATP but not by addition of respiratory substrates. 4. It was shown that cytochrome c is lost from ;shocked' mitochondria when they come into contact with the tris buffer present in the assay medium, and that the changes observed in the pattern of swelling, as well as in respiratory control, are directly connected with this loss of cytochrome c. 5. The results of the investigation are discussed with regard to the role of cytochrome c in swelling and respiratory control.     

Differential Effect of Polyamines on T4 Morphogenesis

The 5-fluorouracil (5 FU) technique for the phenotypic reversion of amber mutants was used to demonstrate that under certain circumstances, in the presence of putrescine or spermidine, early mutants have an enhanced response to 5 FU, whereas late mutants have a delayed response. Bacteria infected by T4D wild-type bacteriophage did not produce phage in the presence of high putrescine concentrations. Pulse treatments with putrescine showed that the production of lysozyme depends on a putrescine-sensitive process that begins immediately after infection at 26 C and ends at 36 min or even later. The addition of putrescine at any time during the critical period between 0 and 36 min led to a corresponding delay in lysozyme synthesis after the inhibitor was removed. Intracellular phage maturation was delayed by the addition of 100 mumoles of putrescine per ml. Early enzymes were not affected by the diamine, but the level of phage deoxyribonucleic acid was considerably decreased by the inhibitor. The putrescine-sensitive process that affects the timing of maturation is suggested to be the natural process controlling the T4 "clock."     

Relation between optical configuration and immunogenicity of synthetic polypeptides

1. Three random linear copolymers composed of two or three of the amino acids d-tyrosine, d-glutamic acid, d-alanine and d-lysine, and a branched multichain copolymer with a poly-d-lysine backbone and polymeric side chains of d-tyrosine and d-glutamic acid, were found to be non-antigenic in rabbits, by precipitin and passive cutaneous anaphylaxis, and in guinea pigs, by delayed hypersensitivity tests. The corresponding four copolymers of l-amino acids were shown to be antigenic by all the three criteria. 2. No immunological cross-reactions were observed between the polypeptides composed of d-amino acids and the corresponding l-amino acid copolymers. 3. Similarly, an azobenzenearsonic acid conjugate of poly-d-tyrosine was shown to be non-antigenic in guinea pigs, in contrast with an analogous conjugate of poly-l-tyrosine. Animals sensitized with the conjugate of poly-l-tyrosine did not exhibit delayed skin reactions, when cross-tested with the d-conjugate. 4. A linear polymer composed of d-tyrosine, l-glutamic acid and l-alanine was found to be immunogenic and to cross-react with the corresponding polymer composed exclusively of d-amino acids.     

GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion.

Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.