On the origin of the stereoselective affinity of Nutlin-3 geometrical isomers for the MDM2 protein

The stereoselective affinity of small-molecule binding to proteins is typically broadly explained in terms of the thermodynamics of the final bound complex. Using Brownian dynamics simulations, we show that the preferential binding of the MDM2 protein to the geometrical isomers of Nutlin-3, an effective anticancer lead that works by inhibiting the interaction between the proteins p53 and MDM2, can be explained by kinetic arguments related to the formation of the MDM2:Nutlin-3 encounter complex. This is a diffusively bound state that forms prior to the final bound complex. We find that the MDM2 protein stereoselectivity for the Nutlin-3a enantiomer stems largely from the destabilization of the encounter complex of its mirror image enantiomer Nutlin-3b, by the K70 residue that is located away from the binding site. On the other hand, the trans-Nutlin-3a diastereoisomer exhibits a shorter residence time in the vicinity of MDM2 compared with Nutlin-3a due to destabilization of its encounter complex by the collective interaction of pairs of charged residues on either side of the binding site: Glu25 and Lys51 on one side, and Lys94 and Arg97 on the other side. This destabilization is largely due to the electrostatic potential of the trans-Nutlin-3a isomer being largely positive over extended continuous regions around its structure, which are otherwise well-identified into positive and negative regions in the case of the Nutlin-3a isomer. Such rich insight into the binding processes underlying biological selectivity complements the static view derived from the traditional thermodynamic analysis of the final bound complex. This approach, based on an explicit consideration of the dynamics of molecular association, suggests new avenues for kinetics-based anticancer drug development and discovery.     

Effect of boundary type and season on predatory arthropods associated with field margins on New Zealand farmland

Pitfall traps were used to monitor predatory arthropod numbers along two types of field boundary, a post and wire fence line and a Cupressus macrocarpa hedge, along the same paddock margin in Canterbury, New Zealand, over 24 months. The seven most abundant predator groups recorded were: Araneae > Phalangiidae > Staphylinidae > Coccinellidae > Chilopoda > Hemerobiidae > Carabidae. Araneae, Phalangiidae, Staphylinidae, Chilopoda and Hemerobiidae were found in larger numbers at the wire fence than at the hedge site, whereas the numbers of Carabidae and Coccinellidae adults exhibited no field margin preference. However, more species of Araneae and Staphylinidae were caught at the hedge site, whereas species richness of carabid beetles was greatest at the wire fence. Principal component analysis clearly separated the samples collected from the two habitats based on the assemblages of Araneae, Staphylinidae and Carabidae, and certain species in each of these taxonomic groups appeared to be particularly associated with one boundary type or the other. All the main taxonomic groups exhibited clear seasonal patterns, with distinct peaks in abundance occurring at certain times of the year. The results of the study reinforce the idea that management of field boundaries can be used to manipulate the type and abundance of particular groups of predatory arthropods, and that seasonal patterns should be taken into account in schemes of integrated pest management so that any adverse effects of biocide application on these beneficial species may be minimised.     

Effects of transdermal nicotine treatment on structure and function of coronary artery bypass grafts.

Smoking is a major risk factor for failure of coronary artery bypass grafts (CABG). Experiments were designed to determine effects of transdermal nicotine, independent of smoking, on structure and function of CABG. Saphenous veins were placed as CABG in untreated dogs (control) or in dogs treated with transdermal nicotine (one 11-mg or two 22-mg patches/day) for 5 wk. Serum nicotine and plasma nitric oxide were measured. Grafts were removed and prepared for organ chamber studies and histology. Serum nicotine averaged 12.1 and 118.7 ng/ml in the 11 mg/day and 44 mg/day groups, respectively. Plasma nitric oxide was higher in dogs treated with 11 mg/day doses compared with controls. In organ chamber studies, endothelium-dependent relaxations to thrombin and A-23187 and endothelium-independent relaxations to nitric oxide were greatest in grafts from dogs treated with 11 mg/day doses. Intimal thickness of the grafts were similar among groups. However, staining for bone sialoprotein was increased in the media of grafts from the 11 mg/day treatment group. These data suggest that transdermal nicotine in doses comparable and double to those used for conventional smoking cessation treatment in humans does not adversely affect early patency of canine CABG up to 4 wk postoperatively. Transdermal nicotine, however, may increase production of and response to nitric oxide in bypass grafts.     

Effects of aloe extracts on human normal and tumor cells in vitro

Fractions of leaf extracts from 2 local types, labeledAloe vera (subsequently identified asAloe barbadensis Mill, andA. saponaria Haw.), were prepared by differential centrifugation and tested by in vitro assays for the presence of lectinlike activities and for effects on the attachment and growth of human normal and tumor cells. Fractions of extracts of fresh leaves and commercially “stabilized”Aloe vera gel had high levels of lectin-like substances measured by immunodiffusion and hemagglutination assays. Substances in fluid fractions from both fresh leaf sources were found to markedly promote attachment and growth of human normal, but not tumor, cells and to enhance healing of wounded cell monolayers. In contrast, fractions of “stabilized”Aloe vera gel were equally cytotoxic for human normal and tumor cells in vitro. Results from cell assays suggested that the observed growth promotion and wound healing effects of aloe substances in vitro may be analogous to what has been observed in vivo during healing of wounds and burns.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila melanogaster oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal-ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. We show that Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. We show that cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal-anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation.     

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).     

Molecular Phylogeny of Coral-Reef Sea Cucumbers (Holothuriidae: Aspidochirotida) Based on 16S Mitochondrial Ribosomal DNA Sequence

Members of the Holothuriidae, found globally at low to middle latitudes, are often a dominant component of Indo–West Pacific coral reefs. We present the first phylogeny of the group, using 8 species from the 5 currently recognized genera and based on approximately 540 nucleotides from a polymerase chain reaction–amplified and conserved 3′ section of 16S mitochondrial ribosomal DNA. Parsimony and likelihood analyses returned identical topologies, permitting several robust inferences to be drawn. Several points corroborated the Linnean classification. Actinopyga and Bohadschia each appear monophyletic and Pearsonothuria is sister to Bohadschia. Other aspects of our phylogeny, however, were not in accord with the taxonomy of Holothuriidae or previous speculations about the group’s evolutionary history. Most notably, the genus Holothuria appears paraphyletic. Actinopyga and Bohadschia, sometimes held to be closely related to one another because of certain morphologic similarities, are only distantly related. The morphologically distinct Labidodemas, even thought to warrant separation at the family level, is nested well within Holothuria. A maximum parsimony reconstruction of ancestral ossicle form on the phylogeny indicated that, in addition to a probable bout of elaboration in ossicle form (the modification of rods or rosettes to holothuriid-type buttons), at least 2 rounds of ossicle simplification also transpired in which buttons reverted to rods or rosettes. Cuvierian tubules, defensive organs unique to numerous members of Holothuriidae, were probably present before the initial radiation of the family, but the reconstruction is ambiguous as to their ancestral function.     

Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources

Purified DNA from the liver of rats, mice, rabbits, and guinea pigs, from guinea pig lymph nodes, from hyperplastic nodules induced in rat liver by feeding with 2-(acetylamino)fluorene, and from Escherichia coli cells was made apurinic by reaction with diphenylamine. After chromatographic separation of pyrimidine tracts (isostichs or isoplyths) according to the number of contiguous pyrimidines, semilog plots of tract frequency vs. the number of contiguous pyrimidines were linear, plots for DNA from several sources differed from one another, and all deviated significantly from randomness. Similar semilog plots for coding sequences among 60 mammalian genomes or 28 rat tissue genomes were intermediate among slopes for isolated DNA. Individual isostichs were hydrolyzed, and their constituent pyrimidine bases were analyzed by high-pressure liquid chromatography. Among isostichs from isolated DNAs, the distribution of Thy and Cyt contents differed markedly from the distribution of 5-methylcytosine (5-Me-Cyt); e.g., although isostich 1 contained 45-49% of 5-Me-Cyt, amounts of Thy or Cyt did not exceed 25%. Semilog plots of normalized values for tract frequency or the content of 5-Me-Cyt vs. isostich number were essentially superimposable; thus, among the first five pyrimidine tracts of a particular tissue or E. coli DNA, the number of tracts per 5-Me-Cyt moiety was essentially constant. The data showed that 5-Me-Cyt and/or dCyd-dGuo dinucleotides have a distribution throughout DNA structure that superimposes the distribution of pyrimidine tract frequency and suggests that regulatory 5-Me-Cyt moieties are principally located at 3' termini of pyrimidine tracts.