Inhibition of basophil histamine release by anti-inflammatory steroids. II. Studies on the mechanism of action

The glucocorticosteroids inhibit the IgE-dependent release of histamine by human basophils with an order of potency that very closely parallels that found in vivo (i.e., triamcinolone acetonide greater than dexamethasone greater than beta-methasone greater than prednisolone greater than hydrocortisone much greater than progesterone approximately tetrahydrocortisone approximately 0). The effect is seen after a 24-hr preincubation with nanomolar to micromolar concentrations of glucocorticoid. In contrast, release of histamine stimulated by the formyl methionine containing peptide f-met-leu-phe, the calcium ionophore A23187, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate was not inhibited by 24-hr incubation with the potent glucocorticoid dexamethasone. Dexamethasone inhibited anti-IgE-induced histamine release without altering its rate, suggesting that the glucocorticoids do not inhibit histamine release by elevating the intracellular level of cAMP. Dexamethasone did not consistently alter either the total or occupied basophil IgE Fc receptor number, and therefore the glucocorticoid effect does not appear to be due to the modulation of cell surface Fc epsilon receptor content. These data indicate that steroid hormones inhibit basophil IgE-dependent activation through a specific glucocorticoid receptor. The mechanism by which they do so appears not to involve an elevation of cAMP or a shedding of cell surface Fc epsilon receptors. Further, because the glucocorticoids did not inhibit release initiated by the PLA2-dependent stimuli f-met-leu-phe, A23187 and TPA, the inactivation of IgE-dependent histamine release by glucocorticoids may not be the result of PLA2 inhibition.     

Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II.

The mechanism of enzymatic elongation by Escherichia coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primer-template molecule during extensive intervals of DNA synthesis. Despite the presence of E. coli DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme.In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.These findings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication.     

Studies on the mechanism of enzymatic hydrolysis of cellulosic substances.

Most cellulosic substances contain appreciable amounts of cellulose and hemicellulose, which on enzymatic hydrolysis mainly yield a mixture of glucose, cellobiose, and xylose. In this paper, studies on the mechanisms of hydrolysis of bagasse (a complex native cellulosic waste left after extraction of juice from cane sugar) by the cellulase enzyme components are described in light of their adsorption characteristics. Simultaneous adsorption of exo- and endoglucanases on hydrolyzable cellulosics is the causative factor of the hydrolysis that follows immediately after. It supports the postulate of synergistic enzyme action proposed by Eriksson. Xylanase pretreatment enhanced the hydrolysis of bagasse owing to the creation of more accessible cellulosic regions that are readily acted upon by exo- and endoglucanases. The synergistic action of the purified exoglucanase, endoglucanase, and xylanse has been found to be most effective for hydrolysis of bagasse but not for pure cellulose. Significant quantities of glucose are produced in beta-glucosidase-free cellulase action on bagasse. Individual and combined action of the purified cellulase components on hydrolysis of native and delignified bagasse are discussed in respect to the release of sugars in the hydrolysate.     

Studies on the mechanism of secretion of rat adrenal steroids in vitro

The possibility that in the rat adrenal cortex synthesis and secretion of steroid hormones may be distinct. processes has been studied using in vitro methods.The uptake and subsequent release of radioactive corticosterone and 18-OH-DOC added to incubation media showed little difference between the two steroids. Both were taken up rapidly by the tissue, and after exchange of medium, 50% was released within 15 min. The properties of muscle and adrenal tissue were qualitatively similar in these respects, and were consistent with steroid movement by diffusion.Different results were obtained when steroids formed from the tissues' endogenous precursor supply were studied. Following incubation for 2h tissue and medium steroid cohtent were measured, and tissue/medium ratios of steroid were used as an index of secretion. With varying volumes of incubation medium (1.4–10 ml), secretion of corticosterone was related to volume, although synthesis was constant. With 18-hydroxydeoxycorticosterone (18-OH-DOC) on the other hand neither synthesis nor secretion was affected by medium volume.The effects of ouabain (10⁻⁴ M) on synthesis and secretion were also studied, using a 5 ml volume of medium. Under these conditions, tissue levels of corticosterone were very low, while 18-OH-DOC was extractable from tissues in amounts up to 30% of the total synthesised. In capsules, steroid synthesis was decreased by ouabain, or by a lower potassium medium (3.6 mM) or both together. Secretion of corticosterone was not affected by these treatements, whereas secretion of 18-OH-DOC and aldosterone was greatly inhibited: maximally 50% of the synthesised 18-oxygenated steroid was retained within the tissue. These treatments also affected inner zone function, ouabain inhibited steroid synthesis, but the low potassium medium did not. Secretion of corticosterone was unaffected, whereas secretion of 18-OH-DOC was again inhibited both by low potassium medium and by ouabain. Maximally as in the capsules, 50% of the synthesised 18-OH-DOC was retained within the tissue.The results suggest that the secretion of 18-OH-DOC formed from endogenous precursors by rat adrenal tissue is controlled by a mechanism involving a ouabain sensitive process in the cell membrane. Corticosterone is released by a different mechanism, quite possibly passive diffusion.