Regulation of lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin.

The bindings of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin-I + C binds to actin-tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin-I binds to actin and the binding is improved in the presence of tropomyosin even though troponin-I does not bind to tropomyosin alone. Troponin-C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.     

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed—despite being orally bioavailable and not a P-glycoprotein substrate—much lower brain/plasma exposure ratios than PD325901.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

Challenges to global surveillance and response to infectious disease outbreaks of international importance

This article presents a notional scheme of global surveillance and response to infectious disease outbreaks and reviews 14 international surveillance and response programs. In combination, the scheme and the programs illustrate how, in an ideal world and in the real world, infectious disease outbreaks of public health significance could be detected and contained. Notable practices and achievements of the programs are cited; these may be useful when instituting new programs or redesigning existing ones. Insufficiencies are identified in four critical areas: health infrastructure; scientific methods and concepts of operation; essential human, technical, and financial resources; and international policies. These insufficiencies challenge global surveillance of and response to infectious disease outbreaks of international importance. This article is intended to help policymakers appreciate the complexity of the problem and assess the impact and cost-effectiveness of proposed solutions. An assessment of the potential contribution of appropriate diagnostic tests to surveillance and response is included.     

Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography

We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.