Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury

In this paper the stimuli for and pattern of Schwann cell proliferation are defined under various experimental conditions. We used a tissue culture system in which fetal rat dorsal root ganglia, treated to eliminate contaminating fibroblasts (Wood, P., 1976, Brain Res. 115:361--375), appear to recapitulate many aspects of the developing peripheral nervous system. We observed that: (a) proliferation of Schwann cells on neurites is initially rapid, but, as each neurite becomes fully ensheathed, division slows considerably and is confined to the periphery of the outgrowth; (b) during the period of rapid proliferation, excision of the ganglion causes a rapid decay in the number of dividing cells; (c) excision of the ganglion from more established cultures in which there was little ongoing proliferation resulted in a small increase in labeling at the site of excision for all Schwann cells and a substantial increase in labeling for myelin-related cells with a peak labeling period at 4 d; (d) direct mechanical injury during Wallerian degeneration is mitogenic for Schwann cells; (e) a variety of potential mitogens failed to stimulate Schwann cell proliferation, and (f) replated cells have a slightly higher level of proliferation and show a small and variable response to the addition of cAMP.     

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)     

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.     

Density gradient analysis of newly replicated DNA from synchronized mouse lymphoma cells

The replication of DNA in synchronous cultures of mouse lymphoma cells was investigated by use of CsCl density gradient centrifugation. We found that the buoyant density of newly replicated DNA depended upon the particular stage of S phase in which synthesis occurred. In early S phase, newly replicated DNA exhibited buoyant densities which were slightly higher, on the average, than that of pre-existing DNA. As S phase progressed, newly replicated DNA shifted to lower buoyant densities, until, near the end of S phase, densities less than pre-existing DNA were observed. These observations are discussed in terms of their possible relevance to base compositional differences between nucleotide sequences made in early as opposed to middle or late S phase.