Purification of a protein binding quinacrine and histrionicotoxin from membrane fragments rich in cholinergic receptors in Torpedo marmorata]

A protein is purified by differential centrifugation from membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ after dissolution by a mixture of non denaturing detergents. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and Coomassie blue staining the purified protein yields a single band of apparent molecular weight 43,000. Spectroscopic experiments carried out in the absence of Ca++ and detergents reveal that the 43 K protein interacts with the fluorescent local anesthetic quinacrine and with the frog toxin histrionicotoxin (apparent KD : 7 X 10(-7) M) but not with carbamylcholine and the alpha toxin from N. nigricollis.     

Importance of protein-protein interactions for the structural integrity of membrane framents from Torpedo marmorata electric organ]

Electron spin resonance and electron microscopy were used to show that alkaline extraction of a 43,000 dalton protein from torpedo membrane induces structural destabilization of the membrane and rotational diffusion of the cholinergic receptor protein.     

Existence of cholinergic receptors in the very young chick embryo]

The injection of certain cholinergic agents in the yolk sac of young Chick embryos (40 to 48 hrs. of incubation) gives rise to a twisting of the cervical notochord and spinal cord and contraction of the cervical somites. These morphogenetic changes result in spinal column malformations in older embryos. The effects of the cholinergic agents are inhibited by simultaneous treatments with a cholinergic agonist and an antagonist or a cholinergic receptor ligand. The results lead to the assumption that the early embryos already possess cholinergic receptors, probably located in the notochord.     

RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda]

Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides). RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E. coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease. They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174. These RNA fragments are also required for in vitro replication of lambda phage DNA.     

Serotonergic and cholinergic interaction in the regulation of pituitary-adrenal function in rats

It has been proposed that the central serotonergic inputs which modulate pituitary-adrenal secretion are mediated by cholinergic neurons. We have tested this hypothesis in intact rats. Male Sprague-Dawley rats were injected with cholinergic and serotonergic agents which enhanced transmitter function and with receptor blocking agents. Agents were injected, singly and in combination, into both unstressed and stressed animals. Since the response to cholinergic agents might be due to changes to vasopressin release, Brattleboro (vasopressin deficient) rats were also injected with cholinergic agents. The level of plasma corticosterone at 1-h post-injection was determined. Results indicate that the serotonin receptor blockade decreased the stimulatory, cholinergic effect of physostigmine. Cholinergic receptor blockers did not significantly reduce the corticosterone rise induced by 5-hydroxytryptophan. These results do not support the hypothesis of cholinergic mediation of serotonergic input. Nicotinic and muscarinic receptors appeared to exert opposing influences on the system. The nicotinic receptor antagonist was able to block the stimulatory effect of physostigmine. The muscarinic receptor antagonist significantly elevated plasma corticosterone levels. No differences were found in the effect of physostigmine on Brattleboro rats as compared to controls. These data are interpreted as suggesting that 1) the acetylcholine-induced stimulation of pituitary-adrenal function is mediated, in part, by serotonergic neurons; and 2) stimulation of nicotinic receptors is facilitatory whereas stimulation of muscarinic receptors is inhibitory to pituitary-adrenal function.     

Model of the interaction between trypsin and alpha 2-macroglobulin of porcine serum]

A comparative study of the dissociation into subunits of Porcine alpha2 M, either native or bound to trypsin (Tn), has been carried out in order to determine the modifications of the alpha2 M structure due to the formation of the Tn-alpha2 M complex. Analytical ultra-centrifugation at pH 3.5 shows that the dissociation is smaller when alpha 2 M is bound to trypsin. Electrophoresis in 4% polyacrylamide gels, in presence of 0.1% SDS, of alpha2 M and Tn-alpha2 M incubated in 1% SDS leads to the same conclusion; the enzyme must stabilize the quaternay structure of alpha2 M. In presence of SDS + beta-mercaptoethanol, only a molecular weight (M.W.) 200,000 band is revealed in electrophoresis pattern of native alpha2 M. In the case of reduced Tn-alpha2 M, some other bands of M.W. 100,000, 50,000, 30,000 appear. When trypsin is inactivated by TLCK 100,000 M.W. band is present, accompanied by the 200,000 M.W. band whose intensity is function of the alpha2 M concentration. The 100,000 M.W. band appears therefore characteristic of the formation of the complex which must imply a proteolytic cleavage in the middle of the 100,000 polypeptidic chain of alpha2 M. A model of the complex is proposed in which the enzyme forms a proteic bridge between the two halves of the alpha2 M molecule.     

Study of a correlation between glycolipids and tumorigenesis in 6 cell lines derived from human brain tumors]

By silica gel chromatography we have studied the various glycolipids of six established lines derived from human brain tumors. Among the glycolipids, the GM3 ganglioside appeared as one of the major components in the three lines rejected by athymic nude Mice. On the contrary, GM3 was absent, or present in trace, in the three tumorigenic lines.     

Correlation between inhibition of stimulatory membrane repolarization and positive inotropic response caused by ouabain in rat heart]

During a stimulus train, the diastolic membrane potential of rat atria exhibits a depolarization phase followed by a slower repolarization phase which has been attributed to the activation of an electrogenic sodium pump (ATPase Na+, K+). This pump seems to be all the more active as stimulation frequency is higher. The parallel evolution of the sodium pump inhibition and a positive inotropic effect in response to ouabain perfusion, suggests that the enzymatic inhibition is directly involved in the development of the cardiotonic effect of digitalis.     

Concentration and method of cadmium fixation by a marine Vibrio]

In many organisms, intracellular fixation of cadmium involves specific proteins, the metallothioneins. A similar phenomenon was demonstrated in a marine bacterium belonging to the genus Vibrio. The accumulation of the metal is a function of its concentration in the medium. The uptake is also increased by cysteine, which decreases the toxicity of the metal.     

Tubular and glomerular nephropathy induced in rabbits by injection of a heterologous antitubular basement membrane serum]

A nephropathy affecting both tubules and glomeruli has been induced in Rabbits by the injection of an heterologous immune serum directed against Rabbit tubular basement membrane. It differs from experimental antiglomerular basement membrane nephropathy by the fixation of antibodies and C3 on tubular basement membrane and by the occurrence of glucosuria.     

The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore

Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.     

Purification of a membrane protein modifying the sensitivity of Na+/K+ ATPase to ouabain from a supernatant of plasma membrane treated with EDTA]

A membrane protein of M. W. 32,000 D and pI 5 has been purified from EDTA-treated plasma membrane supernatants by Page without detergents. These membranes are purified from the MF2S cell line issued from the murine plasmacytoma MOPC 173. The purified protein was able to induce a 300 fold increase in the Na+/K+ ATPase resistance to ouaba?n thus mimicking the original resistance of the enzyme to the drug.     

A rapid and simple method for the isolation of pure eosinophilic leukocytes from horse blood

Summary An improved and short method is described for the isolation of intact eosinophilic leukocytes from horse blood with high yield (1–1.5 g/20 1). Viability and purity of the preparations were verified by light and electron microscopy and by the trypan blue exclusion test. Isolated eosinophils were 98–100% pure, intact and viable, and they could be shown to phagocytise immune-complexes.The authors are indebted to the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung for financial support.