Non-peptidic substrate-mimetic inhibitors of Akt as potential anti-cancer agents

Akt has emerged as a critical target for the development of anti-cancer therapies. It has been found to be amplified, overexpressed, or constitutively activated in numerous human malignancies with oncogenesis derived from the simultaneous promotion of cell survival and suppression of apoptosis. A valuable alternative to the more common ATP-mimetic based chemotherapies is a substrate-mimetic approach, which has the potential advantage of inherent specificity of the substrate-binding pocket. In this paper we present the development of high affinity non-peptidic, substrate-mimetic inhibitors based on the minimum GSK3β substrate sequence. Optimization of initial peptidic leads resulted in the development of several classes of small molecule inhibitors, which have comparable potency to the initial peptidomimetics, while eliminating the remaining amino acid residues. We have identified the first non-peptidic substrate-mimetic lead inhibitors of Akt 29a–b, which have affinities of 17 and 12 μM, respectively. This strategy has potential to provide a useful set of molecular probes to assist in the validation of Akt as a potential target for anti-cancer drug design.     

Localized Populations of CD8low/? MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8⁺. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer⁺CD8^low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer⁺CD20⁻ cells inside B cell follicles and that tetramer⁺ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer⁺CD8^low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.     

Differential Regulation of Glycogenolysis by Mutant Protein Phosphatase-1 Glycogen-targeting Subunits

PTG and G_L are hepatic protein phosphatase-1 (PP1) glycogen-targeting subunits, which direct PP1 activity against glycogen synthase (GS) and/or phosphorylase (GP). The C-terminal 16 amino residues of G_L comprise a high affinity binding site for GP that regulates bound PP1 activity against GS. In this study, a truncated G_L construct lacking the GP-binding site (G_Ltr) and a chimeric PTG molecule containing the C-terminal site (PTG-G_L) were generated. As expected, GP binding to glutathione S-transferase (GST)-G_Ltr was reduced, whereas GP binding to GST-PTG-G_L was increased 2- to 3-fold versus GST-PTG. In contrast, PP1 binding to all proteins was equivalent. Primary mouse hepatocytes were infected with adenoviral constructs for each subunit, and their effects on glycogen metabolism were investigated. G_Ltr expression was more effective at promoting GP inactivation, GS activation, and glycogen accumulation than G_L. Removal of the regulatory GP-binding site from G_Ltr completely blocked the inactivation of GS seen in G_L-expressing cells following a drop in extracellular glucose. As a result, G_Ltr expression prevented glycogen mobilization under 5 mm glucose conditions. In contrast, equivalent overexpression of PTG or PTG-G_L caused a similar increase in glycogen-targeted PP1 levels and GS dephosphorylation. Surprisingly, GP dephosphorylation was significantly reduced in PTG-G_L-overexpressing cells. As a result, PTG-G_L expression permitted glycogenolysis under 5 mm glucose conditions that was prevented in PTG-expressing cells. Thus, expression of constructs that contained the high affinity GP-binding site (G_L and PTG-G_L) displayed reduced glycogen accumulation and enhanced glycogenolysis compared with their respective controls, albeit via different mechanisms.Hepatic glycogen metabolism plays a central role in the maintenance of circulating plasma glucose levels under various physiological conditions. The rate-controlling enzymes in glycogen metabolism, glycogen synthase (GS)² and glycogen phosphorylase (GP), are subject to multiple levels of regulation, including allosteric binding of activators and inhibitors, protein phosphorylation, and changes in subcellular localization. GS is phosphorylated on up to 9 residues by a variety of kinases, although site 2 appears to be the most important regulator of hepatic GS (1). In contrast, GP is phosphorylated on a single N-terminal serine residue by phosphorylase kinase, which increases GP activity and its sensitivity to allosteric activators. Both GS and GP are in turn also regulated by protein phosphatases, most notably PP1. Although PP1 is a cytosolic protein, a family of five molecules has been reported that targets the enzyme to glycogen particles (2–7), whereas another two glycogen-targeting subunits have been putatively identified based on sequence homology (8). Published work has indicated that each targeting subunit confers differential regulation of PP1 activity by extracellular hormonal signals and/or intracellular changes in metabolites (9–11).Four PP1-glycogen-targeting proteins are expressed in rodent liver, although G_L and PTG/R5 have been most extensively studied (9, 12–15). G_L is present at higher levels in rat liver than PTG (12), but the expression of both proteins is subject to coordinate regulation by fasting/refeeding and insulin (12, 13). Previous studies indicated that the PTG-PP1 complex is primarily responsible for GP dephosphorylation and regulation of glycogenolysis (13, 16), whereas the G_L-PP1 complex preferentially mediates the activation of GS upon elevation of extracellular glucose (9, 13). However, the molecular mechanisms underlying these differential properties of PTG and G_L have not been completely defined.Both PTG and G_L directly bind to specific PP1 substrates involved in glycogen metabolism, albeit for different physiological reasons. The extreme C-terminal 16 amino acids of G_L comprises a unique, high affinity binding site for phosphorylated GP (GPa (17)), which has been further delineated to two critical tyrosine residues (18, 37). Interaction of PP1 with G_L reduces phosphatase activity against GPa (3). In turn, GPa binding to the G_L-PP1 complex potently inhibits phosphatase activity against GS in vitro (3, 19) and regulates glycogen-targeted PP1 activity in liver cells and extracts (20–22). PTG contains a single substrate-binding site that interacts with GS and GP (5, 23). In contrast to the regulatory role of the GPa binding to G_L, interaction of substrates with PTG increases PP1 activity against these proteins (24). Indeed, disruption of the substrate-binding site by point mutagenesis abrogated the ability of mutant PTG expression to increase cellular glycogen levels (23), indicating an important role for substrate binding to the PTG-PP1 complex.Previous work has comprehensively compared the metabolic impact of PTG versus G_L overexpression in hepatocytes and thus was not the goal of this study (9, 10). Instead, two novel PP1 targeting constructs were generated in which the high affinity GPa-binding site was removed from G_L or added to the C terminus of PTG. The effects of expressing wild-type and mutant constructs on GS and GP activities and on the regulation of glycogen metabolism by extracellular glucose were investigated using primary mouse hepatocytes.     

Differential modulation of 3T3-L1 adipogenesis mediated by 11beta-hydroxysteroid dehydrogenase-1 levels

The localized activation of circulating glucocorticoids in vivo by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays a critical role in the development of the metabolic syndrome. However, the precise contribution of 11beta-HSD1 in the initiation of adipogenesis by inactive glucocorticoids is not fully understood. 3T3-L1 fibroblasts can be terminally differentiated to mature adipocytes in a glucocorticoid-dependent manner. Both inactive rodent dehydrocorticosterone and human cortisone were able to substitute for the synthetic glucocorticoid dexamethasone in 3T3-L1 adipogenesis, suggesting a potential role for 11beta-HSD1 in these effects. Differentiation of 3T3-L1 cells caused a strong increase in 11beta-HSD1 protein levels, which occurred late in the differentiation protocol. Reduction of 11beta-HSD1 activity in 3T3-L1 fibroblasts, achieved by pharmacological inhibition or adenovirally mediated delivery of short hairpin RNA constructs, specifically blocked the ability of inactive glucocorticoids to drive 3T3-L1 differentiation. However, even modest increases in exogenous 11beta-HSD1 expression in 3T3-L1 fibroblasts, to levels comparable with endogenous 11beta-HSD1 in differentiated 3T3-L1 adipocytes, were sufficient to block adipogenesis. Luciferase reporter assays indicated that overexpressed 11beta-HSD1 was catalyzing the inactivating dehydrogenase reaction, because the ability of both active and inactive glucocorticoids to activate the glucocorticoid receptor were largely suppressed. These results suggest that the temporal regulation of 11beta-HSD1 expression is tightly controlled in 3T3-L1 cells, so as to mediate the initiation of differentiation by inactive glucocorticoids and also to prevent the inhibitory activity of prematurely expressed 11beta-HSD1 during adipogenesis.     

Identifying and prioritizing greater sage-grouse nesting and brood-rearing habitat for conservation in human-modified landscapes

Background

Balancing animal conservation and human use of the landscape is an ongoing scientific and practical challenge throughout the world. We investigated reproductive success in female greater sage-grouse (Centrocercus urophasianus) relative to seasonal patterns of resource selection, with the larger goal of developing a spatially-explicit framework for managing human activity and sage-grouse conservation at the landscape level.

Methodology/Principal Findings

We integrated field-observation, Global Positioning Systems telemetry, and statistical modeling to quantify the spatial pattern of occurrence and risk during nesting and brood-rearing. We linked occurrence and risk models to provide spatially-explicit indices of habitat-performance relationships. As part of the analysis, we offer novel biological information on resource selection during egg-laying, incubation, and night. The spatial pattern of occurrence during all reproductive phases was driven largely by selection or avoidance of terrain features and vegetation, with little variation explained by anthropogenic features. Specifically, sage-grouse consistently avoided rough terrain, selected for moderate shrub cover at the patch level (within 90 m²), and selected for mesic habitat in mid and late brood-rearing phases. In contrast, risk of nest and brood failure was structured by proximity to anthropogenic features including natural gas wells and human-created mesic areas, as well as vegetation features such as shrub cover.

Conclusions/Significance

Risk in this and perhaps other human-modified landscapes is a top-down (i.e., human-mediated) process that would most effectively be minimized by developing a better understanding of specific mechanisms (e.g., predator subsidization) driving observed patterns, and using habitat-performance indices such as those developed herein for spatially-explicit guidance of conservation intervention. Working under the hypothesis that industrial activity structures risk by enhancing predator abundance or effectiveness, we offer specific recommendations for maintaining high-performance habitat and reducing low-performance habitat, particularly relative to the nesting phase, by managing key high-risk anthropogenic features such as industrial infrastructure and water developments.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

Summary The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, wherep=DPro,k=DLys, andh=DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu²⁺. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu²⁺ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu²⁺ but not in its absence. The observed width of the fibrils (7±2 nm) was consistent with the width (6.8nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.     

A model of stress and strain in the interosseous ligament of the forearm based on fiber network theory

Fiber network theory was developed to describe cloth, a thin material with strength in the fiber directions. The interosseous ligament (IOL) of the forearm is a broad, thin ligament with highly aligned fibers. The objectives of this study were to develop a model of the stress and strain distributions in the IOL, based on fiber network theory, to compare the strains from the model with the experimentally measured strains, and to evaluate the force distribution across the ligament fibers from the model. The geometries of the radius, ulna, and IOL were reconstructed from CT scans. Position and orientation of IOL insertion sites and force in the IOL were measured during a forearm compression experiment in pronation, neutral rotation, and supination. An optical image-based technique was used to directly measure strain in two regions of the IOL in neutral rotation. For the network model, the IOL was represented as a parametric ruled three-dimensional surface, with rulings along local fiber directions. Fiber strains were calculated from the deformation field, and fiber stresses were calculated from the strains using average IOL tensile properties from a previous study. The in situ strain in the IOL was assumed uniform and was calculated so that the net force predicted by the network model in neutral rotation matched the experimental result. The net force in the IOL was comparable to experimental results in supination and pronation. The model predicted higher stress and strain in fibers near the elbow in neutral rotation, and higher stresses in fibers near the wrist in supination. Strains in neutral forearm rotation followed the same trends as those measured experimentally. In this study, a model of stress and strain in the IOL utilizing fiber network theory was successfully implemented. The model illustrates variations in the stress and strain distribution in the IOL. This model can be used to show surgeons how different fibers are taut in different forearm rotation positions-this information is important for understanding the biomechanical role of the IOL and for planning an IOL reconstruction.     

PRODUCTION AND CELLULAR LOCALIZATION OF NEUTRAL LONG‐CHAIN LIPIDS IN THE HAPTOPHYTE ALGAE ISOCHRYSIS GALBANA AND EMILIANIA HUXLEYI1

Isochrysis galbana Parke, Emiliania huxleyi (Lohm.) Hay and Mohler, and some related prymnesiophyte algae produce as neutral lipids a set of polyunsaturated long‐chain (C_37–39) alkenones, alkenoates, and alkenes (PULCA). These biomarkers are widely used for paleothermometry, but the biosynthesis and cellular location of these unique lipids remain largely unknown. By staining with the fluorescent lipophilic dye Nile Red, we found that I. galbana and E. huxleyi, like many other algae, package their neutral lipid into cytoplasmic vesicles or lipid bodies. We found that these lipid bodies increase in abundance under nutrient limitation and disappear under prolonged darkness and show that this pattern correlates well with the concentration of PULCA as measured by TLC. In addition, we show that lipid vesicles purified by sucrose density gradient centrifugation consist predominantly of PULCA. We also found significant pools of neutral lipid associated with chloroplasts, and PULCA component profiles in lipid vesicles and chloroplasts are similar. Examination of cell ultrastructure shows conspicuous cytoplasmic and chloroplast lipid bodies, and we suggest that PULCA may be synthesized in chloroplasts and then exported to cytoplasmic lipid bodies for storage and eventual metabolism. Our results connect and extend prior observations of lipid bodies and membrane‐unbound PULCA in I. galbana and E. huxleyi, as well as the behavior of PULCA during nutrient and light stress.