A physical properties based approach for the exploration of a 4-hydroxybenzothiazolone series of β2-adrenoceptor agonists as inhaled long-acting bronchodilators

The chiral synthesis of a 4-hydroxybenzothiazolone based series of β₂-adrenoceptor agonists is described. Using this methodology a library of N-substituted analogues were prepared for the rapid identification of leads with the potential to be fast onset and long-acting inhaled bronchodilators with improved therapeutic margins. The design of the library to achieve the targeted profile was based upon lipophilicity and metabolism based hypotheses. This approach identified β-phenethyl, α-substituted cyclopentyl and monoterpene N-substituents to be of particular interest for further evaluation, as exemplified by structures 19, 29 and 33, respectively.     

Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP-inhibited turnover of nitrogenase

Two novel electron paramagnetic resonance (EPR) signals arising from the [1Mo-7Fe-9S-homocitrate] (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP-limited conditions. The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals. The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite-reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99. These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.     

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.     

Thiourea inhibitors of herpesviruses. Part 3: Inhibitors of varicella zoster virus

The preparation of alpha-methylbenzyl thioureas and their biological activity against varicella zoster virus is described. Several analogs demonstrated IC50s<0.1 microM and their SAR are discussed. These compounds represent a novel class of potent and selective nonnucleoside inhibitors of varicella zoster virus.     

The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C: In vivo and in vitro studies

There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3,5-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.     

Correction of defective IL 3 responses of T lymphocytes from autoimmune mice

MRL-+ and MRL-lpr congenic mice differ by the presence and expression of the homozygous recessive lymphoproliferation (lpr) gene. One manifestation of this gene is a massive T cell proliferation that results in a generalized lymphadenopathy in older animals. Interleukin 3 (IL 3), a recently described lymphokine, has been shown to influence lymphocyte differentiation. It was possible that abberrant IL 3 production was the mechanism responsible for the lpr controlled lymphadenopathy. Consequently, in this paper we tested the MRL congenic mice for their ability to produce IL 3. We report that the T lymphocytes from MRL-+ and MRL-lpr could neither respond to pokeweed mitogen in the induction of proliferation nor produce IL 3. Moreover, IL 3 was not produced constitutively nor could be induced at any time period up to 5 days in vitro. This hyporesponsiveness was shown to be controlled at the accessory cell level. Addition of T cell-depleted peritoneal exudate cells from normal major histocompatibility complex (MHC) compatible mice was able to restore the ability to secrete IL 3 in response to pokeweed mitogen in MRL-+ and young MRL-lpr mice. The defect in the accessory cells could be overridden by two means: the incorporation of phorbol myristate acetate in the induction system and preincubation of the cells in tissue culture.     

Independent Origin and Global Distribution of Distinct Plasmodium vivax Duffy Binding Protein Gene Duplications

BackgroundPlasmodium vivax causes the majority of malaria episodes outside Africa, but remains a relatively understudied pathogen. The pathology of P. vivax infection depends critically on the parasite’s ability to recognize and invade human erythrocytes. This invasion process involves an interaction between P. vivax Duffy Binding Protein (PvDBP) in merozoites and the Duffy antigen receptor for chemokines (DARC) on the erythrocyte surface. Whole-genome sequencing of clinical isolates recently established that some P. vivax genomes contain two copies of the PvDBP gene. The frequency of this duplication is particularly high in Madagascar, where there is also evidence for P. vivax infection in DARC-negative individuals. The functional significance and global prevalence of this duplication, and whether there are other copy number variations at the PvDBP locus, is unknown.Conclusions/SignificancePvDBP duplications are much more widespread and complex than previously thought, and at least two distinct duplications are circulating globally. The same duplication boundaries were identified in parasites from three continents, and were found at high prevalence in human populations where DARC-negativity is essentially absent. It is therefore unlikely that PvDBP duplication is associated with infection of DARC-negative individuals, but functional tests will be required to confirm this hypothesis.     

Our use,misuse, and abandonment of a concept: Whither habitat?

The foundational concept of habitat lies at the very root of the entire science of ecology, but inaccurate use of the term compromises scientific rigor and communication among scientists and nonscientists. In 1997, Hall, Krausman & Morrison showed that ‘habitat’ was used correctly in only 55% of articles. We ask whether use of the term has been more accurate since their plea for standardization and whether use varies across the broader range of journals and taxa in the contemporary literature (1998–2012). We searched contemporary literature for ‘habitat’ and habitat‐related terms, ranking usage as either correct or incorrect, following a simplified version of Hall et al.'s definitions. We used generalized linear models to compare use of the term in contemporary literature with the papers reviewed by Hall et al. and to test the effects of taxa, journal impact in the contemporary articles and effects due to authors that cited Hall et al. Use of the term ‘habitat’ has not improved; it was still only used correctly about 55% of the time in the contemporary data. Proportionately more correct uses occurred in articles that focused on animals compared to ones that included plants, and papers that cited Hall et al. did use the term correctly more often. However, journal impact had no effect. Some habitat terms are more likely to be misused than others, notably ‘habitat type’, usually used to refer to vegetation type, and ‘suitable habitat’ or ‘unsuitable habitat’, which are either redundant or nonsensical by definition. Inaccurate and inconsistent use of the term can lead to (1) misinterpretation of scientific findings; (2) inefficient use of conservation resources; (3) ineffective identification and prioritization of protected areas; (4) limited comparability among studies; and (5) miscommunication of science‐based findings. Correct usage would improve communication with scientists and nonscientists, thereby benefiting conservation efforts, and ecology as a science.     

Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro

Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.     

The effect of a shift from macrophyte to phytoplankton dominance on phosphorus forms and burial in the sediments of a shallow hard-water lake

In shallow lakes, increasing phosphorus (P) loading has often been accompanied by a shift from a clear-water, macrophyte-dominated state to a turbid state featuring phytoplankton dominance. The effect of a regime shift on P burial and P fractions in lake sediments, however, is poorly understood. We used sediment cores from a eutrophic hard-water lake (Lake Gollinsee, Germany) that had undergone a regime shift (in approximately 1917) to investigate the effect on the accumulation rate of P and on changes in P forms. The cores were dated using Hg contents and radioisotopes (²¹⁰Pb, ¹³⁷Cs, and ²⁴¹Am). A combination of total organic carbon to total nitrogen ratios (TOC:TN), δ¹³TOC values, X-ray fluorescence calcium (Ca) counts, and sediment colour clearly distinguished sediment layers that were deposited during periods of macrophyte or phytoplankton dominance. The accumulation rate of total P (TP) in the sediments was 1.8 times higher after the regime shift and was associated with changes in the distribution of P fractions. The proportions of loosely-(NH₄Cl-extracted TP) and Ca-(HCl-extracted TP) bound P decreased significantly, whilst the proportions of biogenic P (NaOH-extracted NRP) and aluminium-bound P (NaOH-extracted SRP) increased significantly. A higher dry mass deposition rate, reduced burial of stable Ca-P complexes, and increased contents and proportions of the mobile iron-bound (BD-extracted TP) and biogenic P fractions in the near-surface sediment layers are assumed to have enhanced the internal cycling of P and hence to have helped to maintain a state of phytoplankton dominance.