Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of the suppression of the spinal pain syndrome by serotonin derivatives

The ability of serotonin derivatives to stimulate cAMP accumulation in isolated nerve terminals and lumbar enlargement of the spinal cord of normal rats was compared. The effect of the compounds on the intensity of spinal pain syndrome was also assessed. It has been established that substitutes injected into NH2-group of serotonin in 5-OH position attenuate the ability to stimulate cAMP accumulation in synaptosomes, with the effect more pronounced with substitutes of larger volume. A certain correlation between the ability of serotonin derivatives to stimulate adenylate cyclase in vivo and in vitro, on the one hand, and their analgetic effect, on the other hand, is suggested.     

Protease activity of the nuclear matrix of rat hepatocytes

It was demonstrated that the nuclear matrix of rat liver possesses the protease activity. The specific activity of nuclear matrix proteases exceeds that of intact nuclei 7-fold. The optimum activity of nuclear matrix proteases is observed at pH 8-9. The protease activity of the nuclear matrix is inhibited by p-chloromercuribenzoate, N-ethylmaleimide, EDTA, phenylmethylsulfonyl fluoride. This suggests that thiol, serine and metalloproteases are associated with the nuclear matrix.     

Therapy of severe stenocardia with ultraviolet blood irradiation (UVB) and various action mechanisms of this therapy

We observed 70 male patients with a seriously proceeding Chronic myocardial ischemia. They were hospitalised because of frequent, permanent and serious attacks of stenocardia at rest and in stress situations. More than 2/3 of these patients had suffered from a myocardial infarct. In the course of two weeks an intensive therapy with all modern preparations for vasodilatation was made. This therapy proved to be unsuccessful. Nearly all patients were administered more than 10 tables of nitroglycerin per day and, in addition, they were injected analgetics as a compensation of attack. The ultraviolet own blood irradiation (UVB) had a positive therapeutic effect in all patients. There was a good success in 46 patients, in all patients satisfactory results could be registered. The effect of therapy was evident by the decrease of administration of nitroglycerin required, by an increase in the degree of stress capacity, and by an easier treatment of stenocardia attacks. The observation time for patients amounted to 2-8 months. The success of therapy remained in 38 patients. After this time the success of therapy could partially be regained by a repeated number of irradiation series. Then, it remained positive in 9 of 22 patients who had been followed-up for 10 months. The half decay period of eliminating 131I from an intradermal depot could be normalised under the influence of ultraviolet own blood irradiation. This ultraviolet own blood irradiation had no significant influence on the fibrinogen level, fibrinolytic activity, and erythrocyte aggregation (examined in 11 patients). A 2 1/2-fold diminution of monomer fibrin complexes in the blood could be observed. The titre of antistreptolysin-O was increased in all patients who had got over the infarct. It had completely normalised a week after finishing the ultraviolet own blood irradiation. Spectroscopic examinations of the blood and plasma made after ultraviolet own blood irradiation revealed that this irradiation will not only affect the properties of Hb, but will also cause a photochemical transformation accompanied by a destruction of some plasma proteins, of the membrane of formed blood elements, and a photosynthesis of biochemically active compounds. The mechanism of action of ultraviolet own blood irradiation is complicated and requires further exact investigations. Even today, however, this method can be recommended as a complex therapy in patients with severe myocardial ischemia.     

Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex.

Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.