Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase.

While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis.     

Updates in Gastrointestinal Oncology – insights from the 2008 44th annual meeting of the American Society of Clinical Oncology

Tyrosine Kinase Inhibitors (TKI) have significantly changed the landscape of current cancer therapy. Understanding of mechanisms of aberrant TK signaling and strategies to inhibit TKs in cancer, further promote the development of novel agents. ABT-869, a novel ATP-competitive receptor tyrosine kinase inhibitor is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families. ABT-869 showed potent antiproliferative and apoptotic properties in vitro and in animal cancer xenograft models using tumor cell lines that were "addicted" to signaling of kinases targeted by ABT-869. When given together with chemotherapy or mTOR inhibitors, ABT-869 showed at least additive therapeutic effects. The phase I trial for ABT-869 was recently completed and it demonstrated respectable efficacy in solid tumors including lung and hepatocellular carcinoma with manageable side effects. Tumor cavitation and reduction of contrast enhancement after ABT-869 treatment supported the antiangiogenic activity. The correlative laboratory studies conducted with the trial also highlight potential biomarkers for future patient selection and treatment outcome. Parallel to the clinical development, in vitro studies on ABT-869 resistance phenotype identified novel resistance mechanism that may be applicable to other TKIs. The future therapeutic roles of ABT-869 are currently been tested in phase II trials.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

Preliminary results on the photoluminescence and optically stimulated luminescence in Cu‐doped and Ag‐doped ZnB2X4 (B = Li,Na, K: X = Cl,Br) compounds

Photoluminescence, and optically stimulated luminescence in ZnB₂X₄ (B; Li,Na,K: X; Cl,Br) compounds doped with Cu⁺ or Ag⁺ were studied. Double humped emission bands attributable to the activators were observed in all the samples. The observed photoluminescence of Cu⁺ and Ag⁺ could be identified with 3d⁹4s¹?3d¹⁰ and 4d⁹5s¹?5d¹⁰ transitions respectively. The longer wavelength band (400–500 nm range) could be attributed to the Cu⁺ or Ag⁺ ion replacing alkali ion at the octahedral alkali site whereas short wavelength band (340–400 nm range) is attributed to a Cu or Ag ion at tetrahedral zinc site. The short wavelength band was found to be intense compared with long wavelength and gave an indication that most of the Cu or Ag ions prefered a tetrahedral Zn site compared with the octahedral alkali site. All the samples exhibit optically stimulated luminescence (OSL). The sensitivity was found to be lattice dependent. The lowest sensitivity of about 1% compared with Al₂O₃:C was observed in lithium lattices whereas highest the sensitivity of about 290% was observed in the case of Cu‐doped ZnNa₂Br₄.     

Ferrocene-based inhibitors of hepatitis C virus replication that target NS5A with low picomolar in vitro antiviral activity

An unprecedented series of organometallic HCV (hepatitis C virus) NS5A (nonstructural 5A protein) replication complex inhibitors that incorporates a 1,1′-ferrocenediyl scaffold was explored. This scaffold introduces the elements of linear flexibility and non-planar topology that are unconventional for this class of inhibitors. Data from 2-D NMR spectroscopic analyses of these complexes in solution support an anti (unstacked) arrangement of the pharmacophoric groups. Several complexes demonstrate single-digit picomolar in vitro activity in an HCV genotype-1b replicon system. One complex to arise from this investigation (10a) exhibits exceptional picomolar activity against HCV genotype 1a and 1b replicons, low hepatocellular cytotoxicity, and good pharmacokinetic properties in rat.     

Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.     

Chemodenitrification in the cryoecosystem of Lake Vida,Victoria Valley,Antarctica

Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, ?13.4 °C, lack of O₂, and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N₂O) is present at a concentration among the highest reported for an aquatic environment. Only a minor ¹⁷O anomaly was observed in N₂O, indicating that this gas was predominantly formed in the lake. In contrast, the ¹⁷O anomaly in nitrate () in Lake Vida brine indicates that approximately half or more of the present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of ¹⁵N‐enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of to ammonium (), anaerobic ammonium oxidation, or denitrification of N₂O under the conditions tested. In the presence of ¹⁵N‐enriched nitrite (), both N₂ and N₂O exhibited substantial ¹⁵N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of ¹⁵N– alone and in the presence of HgCl₂ and ZnCl₂ to aged brine at ?13 °C resulted in linear increases in the δ¹⁵N of N₂O with time. As HgCl₂ and ZnCl₂ are effective biocides, we interpret N₂O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of ¹⁵N‐enriched and ZnCl₂ and determined rates of N₂O and N₂ production of 4.11–41.18 and 0.55–1.75 nmol L^?1 day^?1, respectively. If these rates are representative of natural production, the current concentration of N₂O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the high levels of N₂O present in Lake Vida.