THE MECHANISM OF COMPLEMENT ACTION

It has been shown: 1. That complement exposed to ultra-violet light is not thereby sensitized to the action of heat (which indicates that it is not protein). 2. That inactivation of complement by ultra-violet light is accompanied by a decrease in its surface tension. 3. That photoinactivation of complement is not a result of any changes in hydrogen ion concentration since these are less than 0.05 pH. 4. That hydrogen ion concentrations high enough to transform serum proteins from the cation to the anion condition (i.e. past the isoelectric point) permanently inactivate complement. These facts together with those given in previous papers lead to the following hypotheses. 1. That there is present in serum a hemolytic substance which is formed from a precursor (which may resemble lecithin) and is constantly being formed and simultaneously being broken down into inactive products. 2. That both precursor and lysin contain the same photosensitive molecular group. 3. That the lytic substance is dependent for its activity upon the state of the serum proteins.     

THE MECHANISM OF α-ADRENERGIC AGONIST ACTION IN LIVER

1. Although the mechanism of action of a-adrenergic agonists in liver tissue is somewhat complex, a number of experimental approaches can be usefully employed to identify the molecular details of the events that occur. 2. Receptors specific for α₁-adrenergic agonists located on the plasma membranes of rat liver cells have been partially characterized using pharmacological agents, affinity labels and monoclonal antibodies. Much of this work has employed isolated plasma membrane fractions and does not take account of tissue-related factors which may now be studied in the intact perfused rat liver, following the development of an appropriate assay system. 3. Because a redistribution of cellular Ca²⁺ is central to the mechanism of action of a-adrenergic agonists in liver, it is important to first gain an understanding of basic cellular Ca²⁺ regulation. Knowledge about the compartmentation of cellular calcium and about Ca²⁺-translocation systems located in the mitochondria, plasma membrane and endoplasmic reticulum is now quite extensive. However, the role of mitochondria in the regulation of intracellular Ca²⁺ is still unclear; it now appears that the mitochondrial calcium content is much less than considered previously. This may have important implications for such a regulatory role. 4. The sequence of Ca²⁺ movements that may occur when a-adrenergic agonists interact with liver have been identified and are as follows: (a) Ca²⁺ is mobilized from an intracellular pool(s) (mitochondria plus endoplasmic reticulum and/or plasma membranes). (b) This elevates the cytoplasmic free Ca²⁺ concentration and leads to an efflux of the ion from the cell. (c) At this time, Ca²⁺-sensitive metabolic events in the cytoplasm are activated and an increase in Ca²⁺-cycling occurs across the plasma membrane. (d) Immediately after the hormone is withdrawn, there is a net influx of Ca²⁺ into the cell, and the intracellular Ca²⁺ pools and transmembrane fluxes are restored to the pre-induced states. In this model, Ca²⁺ movements across the plasma membrane play a key role in regulating the cytoplasmic Ca²⁺ concentration. 5. In the perfused rat liver it has been possible to define in quite precise terms the amounts and rates of Ca²⁺ mobilized in each of these stages. 6. Although several proposals for ‘second messengers’ to link the hormone-receptor interaction with initial Ca²⁺ mobilization have been made, at this time only polyphosphoinositide turnover appears to be a suitable candidate.     

STUDIES ON THE MECHANISM OF ACTION OF PEDERINE

Pederine, a drug extracted from the coleopter Paederus fuscipes, inhibits the growth of in vitro cultured cell lines at concentrations of the order of 1.5 nanogram/ml. Cytological examination shows a generalized cytotoxic effect. Analysis of macromolecular syntheses by the use of radioactive precursors shows that pederine causes an almost immediate block of protein and DNA synthesis, without affecting RNA synthesis. The effects on the synthesis of the two types of macromolecules remain nearly simultaneous even at the lowest active concentrations of pederine. Studies with cell-free systems show that the drug inhibits protein synthesis, whereas it is ineffective on the DNA-polymerizing activity. It seems, therefore, that the drug acts primarily on the amino acid-polymerizing system, and that the effect on DNA is secondary. This idea is strengthened by the observation that puromycin, a specific inhibitor of protein synthesis, also affects promptly DNA synthesis of in vitro cultured cells. Other authors have shown the same phenomenon with a number of inhibitors of protein synthesis; the properties of pederine support, therefore, the view that continuous protein synthesis is necessary for the maintenance of DNA replication in higher organisms.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.     

NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium-3 (HC-3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [³²P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [^l4C]HC-3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC-3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.     

MECHANISM OF SYNCOPE AND ACTION OF DRUGS IN COMPLETE HEART BLOCK

The syncopal attacks of complete heart block may be due either to ventricular standstill or to ventricular acceleration including fibrillation. As treatment may be harmful unless the underlying mechanism in each case is determined, it is important to apply the available methods for differentiation.Epinephrine and certain related compounds (sympathomimetic amines) are the only effective substances in the therapy of ventricular arrest.Isopropyl nor-epinephrine is a most potent drug in the prevention and treatment of ventricular arrest and has the advantage that it does not dispose to fibrillation.Quinidine is unreliable and probably hazardous in the control of ventricular fibrillation in heart block as it appears to precipitate this arrhythmia.Preliminary observations indicate that ectopic ventricular rhythms are also induced by procaine amide in complete heart block.Isuprel® may be of value in the therapy of ventricular acceleration, by preventing the ventricular arrest which frequently follows the initial acceleration.