A continuous, fluorescent, high-throughput assay for human dimethylarginine dimethylaminohydrolase-1

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possess adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4446 compounds, and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date and enables screening of compound libraries using [S] = K (M) conditions while displaying Z' factors from 0.6 to 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.     

Escherichia coli responds with a rapid and large change in growth rate upon a shift from glucose-limited to glucose-excess conditions

Glucose pulse experiments at seconds time scale resolution were performed in aerobic glucose-limited Escherichia coli chemostat cultures. The dynamic responses of oxygen-uptake and growth rate at seconds time scale were determined using a new method based on the dynamic liquid-phase mass balance for oxygen and the pseudo-steady-state ATP balance. Significant fold changes in metabolites (10-1/10) and fluxes (4-1/4) were observed during the short (200 s) period of glucose excess. During glucose excess there was no secretion of by-products and the increased glucose uptake rate led within 40s to a 3.7 fold increase in growth rate. Also within 40-60s a new pseudo-steady-state was reached for both metabolite levels and fluxes. Flux changes of reactions were strongly correlated to the concentrations of involved compounds. Surprisingly the 3.7 fold increase in growth rate and hence protein synthesis rate was not matched by a significant increase in amino acid concentrations. This poses interesting questions for the kinetic factors, which drive protein synthesis by ribosomes.     

The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.     

Building a beverage for recovery from endurance activity: a review

Recovery beverages are commonly used by endurance and team-sport athletes during the time between exercise sessions. Practical recommendations on the optimal nutrient composition of these drinks and timing of their consumption are therefore needed. This article summarizes research to date on the use of recovery beverages after aerobic activities and provides the following recommendations for practitioners on the optimal formula and timing of use for endurance and team-sport athletes. Current evidence suggests that, to maximize glycogen resynthesis, athletes should consume about 1.2 g carbohydrate per kilogram body weight as glucose and sucrose immediately after exercise and each hour thereafter for 4-6 hours postexercise. Alternatively, they may consume 0.8 g·kg(-1)·h(-1) in combination with 0.4 g·kg(-1)·h(-1) amino acids or protein. Liquids provide valuable fluids for rehydration, and an ideal recovery beverage should not only contain carbohydrate and protein but also contain electrolytes, including about 0.3-0.7 g sodium·per liter fluid to help restore sodium lost through sweat. Commercial beverages with this type of nutrient composition are effective, and recent work indicates that chocolate milk may be as effective as or superior to these in promoting recovery. Research regarding the effects of specific types of amino acids and antioxidants on recovery is mixed; thus, further investigation is needed before specific recommendations about these nutrients can be made. Future studies that include women and athletes representing a variety of sports, ages, and training levels and that use consistent methodology will lead to a better understanding of the effects of postexercise intake on recovery.     

Hypochondroplasia due to FGFR3 gene mutation (N540K) and mosaic form of Down syndrome in the same patient

The simultaneous presence of Down syndrome and achondroplasia has rarely been reported in the literature, and our search revealed only six patients with such an association. We are reporting the first case of a patient with Down syndrome and hypochondroplasia. In this patient, Down syndrome was clinically recognised and confirmed by the cytogenetic finding of mosaic karyotype (47,XX,+21/46,XX) shortly after birth. She was subsequently diagnosed with hypochondroplasia at the age of 6 years when disproportional short stature, stocky habitus and macrocephaly were observed. These phenotypic findings were later confirmed by the presence of fibroblast growth factor receptor 3 (FGFR3) gene mutation N540K. The overlapping common clinical features of Down syndrome and hypochondroplasia resulted in delayed diagnosis of hypochondroplasia in our patient and the associated deleterious effect on her linear growth. Her final height is 126.5 cm, which is −3.76 standard deviations (SD) lower than the median height in patients with Down syndrome, and is under the lower borderline of the adult height range for women with hypochondroplasia.     

Phage display can select over-hydrophobic sequences that may impair prediction of natural domain-peptide interactions

MOTIVATION: The phage display peptide selection approach is widely used for defining binding specificities of globular domains. PDZ domains recognize partner proteins via C-terminal motifs and are often used as a model for interaction predictions. Here, we investigated to which extent phage display data that were recently published for 54 human PDZ domains can be applied to the prediction of human PDZ-peptide interactions. RESULTS: Promising predictions were obtained for one-third of the 54 PDZ domains. For the other two-thirds, we detected in the phage display peptides an important bias for hydrophobic amino acids that seemed to impair correct predictions. Therefore, phage display-selected peptides may be over-hydrophobic and of high affinity, while natural interaction motifs are rather hydrophilic and mostly combine low affinity with high specificity. We suggest that potential amino acid composition bias should systematically be investigated when applying phage display data to the prediction of specific natural domain-linear motif interactions.     

Antifouling Bastadin Congeners Target Mussel Phenoloxidase and Complex Copper(II) Ions

Synthetically prepared congeners of sponge-derived bastadin derivatives such as 5,5′-dibromohemibastadin-1 (DBHB) that suppress the settling of barnacle larvae were identified in this study as strong inhibitors of blue mussel phenoloxidase that is involved in the firm attachment of mussels to a given substrate. The IC₅₀ value of DBHB as the most active enzyme inhibitor encountered in this study amounts to 0.84 μM. Inhibition of phenoloxidase by DBHB is likely due to complexation of copper(II) ions from the catalytic centre of the enzyme by the α-oxo-oxime moiety of the compound as shown here for the first time by structure activity studies and by X-ray structure determination of a copper(II) complex of DBHB.     

Adaptive phenotypic plasticity in the Midas cichlid fish pharyngeal jaw and its relevance in adaptive radiation

Background  

Phenotypic evolution and its role in the diversification of organisms is a central topic in evolutionary biology. A neglected factor during the modern evolutionary synthesis, adaptive phenotypic plasticity, more recently attracted the attention of many evolutionary biologists and is now recognized as an important ingredient in both population persistence and diversification. The traits and directions in which an ancestral source population displays phenotypic plasticity might partly determine the trajectories in morphospace, which are accessible for an adaptive radiation, starting from the colonization of a novel environment. In the case of repeated colonizations of similar environments from the same source population this "flexible stem" hypothesis predicts similar phenotypes to arise in repeated subsequent radiations. The Midas Cichlid (Amphilophus spp.) in Nicaragua has radiated in parallel in several crater-lakes seeded by populations originating from the Nicaraguan Great Lakes. Here, we tested phenotypic plasticity in the pharyngeal jaw of Midas Cichlids. The pharyngeal jaw apparatus of cichlids, a second set of jaws functionally decoupled from the oral ones, is known to mediate ecological specialization and often differs strongly between sister-species.