Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

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.     

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.     

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.