Investigation of the conformational dynamics of the apo A2A adenosine receptor

The activation/deactivation processes for G-protein coupled receptors (GPCRs) have been computationally studied for several different classes, including rhodopsin, the β2 adrenergic receptor, and the M2 muscarinic receptor. Despite determined cocrystal structures of the adenosine A_2A receptor (A_2AAR) in complex with antagonists, agonists and an antibody, the deactivation process of this GPCR is not completely understood. In this study, we investigate the convergence of two apo simulations, one starting with an agonist-bound conformation (PDB: 3QAK)¹⁴ and the other starting with an antagonist-bound conformation (PDB: 3EML)¹¹. Despite the two simulations not completely converging, we were able to identify distinct intermediate steps of the deactivation process characterized by the movement of Y288^7.53 in the NPxxY motif. We find that Y288^7.53 contributes to the process by forming hydrogen bonds to residues in transmembrane helices 2 and 7 and losing these interactions upon full deactivation. Y197^5.58 also plays a role in the process by forming a hydrogen bond only once the side chain moves from the lipid interface to the middle of the helical bundle.     

A study of the action of phagolessin A58 on the T phages

Phagolessin A58, an antibiotic substance active against a number of bacterial viruses, was studied for activity against the seven T phages. Only three of the seven phages—T1, T3, and T7—proved to be sensitive to the antibiotic. The antibiotic caused a direct, apparently irreversible inactivation of free phage particles. A study of the properties of the inactivated phage particles showed that the particles retained the ability to kill host cells and to exert mutual exclusion against an unrelated phage after infectivity was lost. There was a progressive loss in these two properties when higher concentrations of antibiotic were used to inactivate the phage. Results with inactivated T3 and T7 revealed that these two properties—the ability to kill host cells and to exclude an unrelated phage—were lost at a different rate. They were, therefore, presumed to be different properties of these particular phage particles. The inactivation of phage by phagolessin A58 was inhibited by desoxyribose nucleic acid and to a lesser extent by ribose nucleic add. Cytosine, thymine, adenine, guanine, and cysteine failed to inhibit the reaction.     

A quantitative treatment of the kinetics of the folding transition of ribonuclease A.

New experimental data and a quantitative theoretical treatment are given for the kinetics of the thermal folding transition of ribonuclease A at pH 3.0. A three-species mechanism is used as a starting point for the analysis: U1 (slow) in equilibrium U2(fast) in equilibrium N, where U1 and U2 are two forms of the unfolded enzyme with markedly different rates of refolding and N is the native enzyme. This mechanism is based on certain facts established in previous studies of refolding. The kinetics of unfolding and refolding show two phases a fast phase and a slow phase, over a range of temperatures extending above the transition midpoint, Tm. The three-species mechanism can be used in this range. At higher temperatures a new much faster kinetic phase is also observed corresponding to the transient formation of a new intermediate (I). Although the general solution for a four-species mechanism is complex it is not difficult to extend the three-species analysis for the special case found here, in which the fast reaction (I in equilibrium N) is well separated from the other two reactions. At temperatures below the transition zone the slow phase of refolding becomes kinetically complex. No attempt has been made to extend the analysis to include this effect. The basic test of the three-state analysis is the prediction as a function of temperature of alpha2, the relative amplitude of the fast phase, both for unfolding and refolding. At temperatures above Tm for which the three-state analysis must be extended to include the new intermediate I, a crresponding quanitity alpha2(cor) is predicted and compared with measured values. Data used in the three-state prediction are values of tau2 and tau1, the time constants of the fast and slow kinetic phases, plus a single value of alpha2 measured when tau2 and tau1 are well separated. The observed and predicted values of alpha2 agree within experimental error. The analysis predicts correctly that, for these experiments, alpha2 should have the same value in unfolding as in refolding in the final conditions. The analysis also predicts satisfactorily the equilibrium transition curve from kinetic data alone. Four striking properties of the kinetics are explained or correlated by the analysis: (a) the drop in alpha2 to a minimum near Tm as well as the delayed rise in alpha2 above Tm;(b) the vanishing of alpha1 above the transition zone; (c) the sharp drop in tau1 inside the transition zone followed by a partial leveling off outside this zone; and (d) the passage of tau2 through a maximum near Tm. Through a comparison of observed and predicted values of alpha2, the analysis also rules out the alternative three-species mechanism U1 (slow) in equilibrium N (fast) in equilibrium U2. Finally, the temperature dependence of the amplitude for the fast reaction (I in equilibrium N) is discussed; the behavior of I is like that of U2 and I may be an unfolded species populated at equilibrium...     

A Multimedia Assessment of the Environmental Fate of Bisphenol A

A comprehensive multimedia assessment of the environmental fate of bisphenol A (BPA) is presented. Components of the assessment include an evaluation of relevant partitioning and reactive properties, estimation of discharge quantities in the U.S. and the European Union (E.U.) resulting in conservative and realistic emission scenarios, and a review of monitoring data. Evaluative assessments of chemical fate using the Equilibrium Criterion (EQC) model are described from which it is concluded that the low volatility of BPA will result in negligible presence in the atmosphere. It is relatively rapidly degraded in the environment with half-lives in water and soil of about 4.5 days and less than 1 day in air, and with an overall half-life of 4.5 to 4.7 days, depending on the medium of release. The degradation rate in water is such that it may be transported some hundreds of kilometres in rivers, but long-range transport potential in air is negligible. Its low bioconcentration factor is consistent with rapid metabolism in fish (half-life less than 1 day). The estimated concentrations were generally consistent with the monitoring data, with the exception of sediment-water concentration ratios. Several hypotheses for the apparent nonequilibrium sediment-water partitioning are presented.     

Identity of the antibiotic ramihyphin A and cyclosporin A

The antifungal antibiotic ramihyphin A, isolated fromFusarium solani strain S-435 in 1974, was shown to be identical with cyclosporin A.     

A probe of the active site acidity of carboxypeptidase A

The substrate analogue 2-(1-carboxy-2-phenylethyl)-4-phenylazophenol is a potent competitive inhibitor of carboxypeptidase A. Upon ligation to the active site, the azophenol moiety undergoes a shift of pKa from a value of 8.76 to a value of 4.9; this provides an index of the Lewis acidity of the active site zinc ion. Examination of the pH dependence of Ki for the inhibitor shows maximum effectiveness in neutral solution (limiting Ki = 7.6 X 10(-7) M), with an increase in Ki in acid (pK1 = 6.16) and in alkaline solution (pK2 = 9.71, pK3 = 8.76). It is concluded that a proton-accepting enzymic functional group with the lower pKa (6.2) controls inhibitor binding, that ionization of this group is also manifested in the hydrolysis of peptide substrates (kcat/Km), and that the identity of this group is the water molecule that binds to the active site metal ion in the uncomplexed enzyme (H2OZn2+L3). Reverse protonation state inhibition is demonstrated, and conventional concepts regarding the mechanism of peptide hydrolysis by the enzyme are brought into question.     

A New Process for the Production of Coenzyme A

A new process has been described for the preparation of coenzyme A of high purity from the cultured broth of Brevibacterium ammoniagenes IFO 12071. The product was obtained in a high yield by the use of Duolite S–30, charcoal, and Dowex 1×2, and identified chemically and enzymatically. This method is simple, rapid, and compact, requires no special equipment, and has been shown to be adaptable for preparing large amounts of highly pure coenzyme A.     

A functional map of the fruit-specific promoter of the tomato 2A11 gene

The 5 region of the fruit-specific tomato gene, 2A11, contains both positive and negative regulatory elements. We divided the 5 promoter region of the 2A11 gene into small fragments, ranging in size from 211 to 634 bp and used these short DNA fragments in in vitro protein-binding studies. These studies revealed the presence of at least four fruit-specific and one leaf- and fruit-active protein-binding domains. These promoter fragments, as well as other overlapping fragments, were tested for their ability to enhance expression from a truncated heterologous promoter in transgenic plants. This analysis showed the presence of four fruit-specific and three general or leaf-active positive regulatory elements. Comparison of the results obtained with these two approaches allowed us to draw a functional map of the 2A11 promoter.     

A close-up of the ESCRTs

The ESCRT protein complexes assemble on the endosome into a protein network that sorts ubiquitinated transmembrane proteins into lumenal vesicles of the compartment. New structural information highlights the complexity of the ESCRT network.     

A review of the Cucurbitaceae

Progress in studies of Cucurbitaceae since 1964 is reviewed, particular attention being paid to the roles of seed-coat anatomy, palynology and phytochemistry in elucidating the taxonomy of the family; to the use of wild species and land-races as sources of horticulturally desirable traits for the improvement of crops; to the potentialities of wild species for domestication as new crops for tropical areas, and to the biology of reproduction, sex control and sex expression. The classification of the lady is outlined, illustrated by discussion of the more important members of each tribe, and a revised classification of Cucumis is presented.