Discovery of a potent CDK2 inhibitor with a novel binding mode, using virtual screening and initial, structure-guided lead scoping

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.     

Internal Binding of Halogenated Phenols in Dehaloperoxidase-Hemoglobin Inhibits Peroxidase Function

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.     

Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters

Anthropogenic activities on coastal watersheds increase nutrient concentrations of groundwater. As groundwater travels downslope it transports these nutrients toward the adjoining coastal water. The resulting nutrient loading rates can be significant because nutrient concentrations in coastal groundwaters may be several orders of magnitude greater than those of receiving coastal waters. Groundwater-borne nutrients are most subject to active biogeochemical transformations as they course through the upper 1 m or so of bottom sediments. There conditions favor anaerobic processes such as denitrification, as well as other mechanisms that either sequester or release nutrients. The relative importance of advective vs. regenerative pathways of nutrient supply may result in widely different rates of release of nutrients from sediments. The relative activity of denitrifiers also may alter the ratio of N to P released to overlying waters, and hence affect which nutrient limits growth of producers. The consequences of nutrient (particularly nitrate) loading include somewhat elevated nutrient concentrations in the watercolumn, increased growth of macroalgae and phytoplankton, reduction of seagrass beds, and reductions of the associated fauna. The decline in animals occurs because of habitat changes and because of the increased frequency of anoxic events prompted by the characteristically high respiration rates found in enriched waters.     

Phylogenetic analyses suggest that Psammomitra (Ciliophora,Urostylida) should represent an urostylid family,based on small subunit rRNA and alpha‐tubulin gene sequence information

The morphologically unique ciliate Psammomitra has long been considered as a systematically uncertain stichotrich. This is mainly because of its highly specialized morphology and a lack of either detailed information concerning its ontogenesis, or molecular data. Based on the small subunit rRNA (SSrRNA) gene and alpha‐tubulin gene sequences, we re‐evaluated the phylogenetic position of Psammomitra retractilis using multiple algorithms. Phylogenetic trees inferred from the SSrRNA gene sequences representing a total of 53 spirotrichs demonstrated the closest relationship of Psammomitra was with Holosticha‐like taxa, with strong support, which clearly suggested that Psammomitra should be placed into the order Urostylida although it branched at a rather deep level, and is likely to be closely related to Holostichidae. With consideration to molecular evidence and morphological characters, Psammomitra should be a clearly outlined taxon at about the rank of family, i.e. Psammomitridae stat. nov. , within the order Urostylida. The improved diagnosis for this family is as follows: Urostylida possessing extremely contractile, elongated body which consists of three parts: head, trunk, and slender tail; midventral complex composed of midventral pairs only and restricted to about anterior 1/3 of ventral surface; frontal, frontoterminal, and transverse cirri present; one left and one right marginal rows which commence near proximal end of adoral zone and extend to near rear body end.     

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

Background  

Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.     

Directional gene flow patterns in disjunct populations of the black ratsnake (Pantheropis obsoletus) and the Blanding’s turtle (Emydoidea blandingii)

The estimation and maintenance of connectivity among local populations is an important conservation goal for many species at risk. We used Bayesian statistics and coalescent theory to estimate short- and long-term directional gene flow among subpopulations for two reptiles that occur in Canada as peripheral populations that are geographically disjunct from the core of their respective species’ ranges: the black ratsnake and the Blanding’s turtle. Estimates of directional gene flow were used to examine population connectivity and potential genetic source-sink dynamics. For both species, our estimates of directional short- and long-term gene flow were consistently lower than estimates inferred previously from F _ST measures. Short- and long-term gene flow estimates were discordant in both species, suggesting that population dynamics have varied temporally in both species. These estimates of directional gene flow were used to identify specific subpopulations in both species that may be of high conservation value because they are net exporters of individuals to other subpopulations. Overall, our results show that the use of more sophisticated methods to evaluate population genetic data can provide valuable information for the conservation of species at risk, including bidirectional estimates of subpopulation connectivity that rely on fewer assumptions than more traditional analyses. Such information can be used by conservation practitioners to better understand the geographic scope required to maintain a functional metapopulation, determine which habitat corridors within a working landscape may be most important to maintain connectivity among subpopulations, and to prioritize subpopulations with respect to their potential to act as genetic sources within the metapopulation.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-depen-dent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver :body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate ω- and ω-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-de-alkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.