Characterization of chicken TNFR superfamily decoy receptors,DcR3 and osteoprotegerin

Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively expressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously identified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species.     

Gaussian docking functions

A shape-based Gaussian docking function is constructed which uses Gaussian functions to represent the shapes of individual atoms. A set of 20 trypsin ligand-protein complexes are drawn from the Protein Data Bank (PDB), the ligands are separated from the proteins, and then are docked back into the active sites using numerical optimization of this function. It is found that by employing this docking function, quasi-Newton optimization is capable of moving ligands great distances [on average 7 A root mean square distance (RMSD)] to locate the correctly docked structure. It is also found that a ligand drawn from one PDB file can be docked into a trypsin structure drawn from any of the trypsin PDB files. This implies that this scoring function is not limited to more accurate x-ray structures, as is the case for many of the conventional docking methods, but could be extended to homology models.     

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.     

Neuregulins: functions,forms, and signaling strategies

The neuregulins (NRGs) are cell-cell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB family. The neuregulin family of genes has four members: NRG1, NRG2, NRG3, and NRG4. Relatively little is known about the biological functions of the NRG2, 3, and 4 proteins, and they are considered in this review only briefly. The NRG1 proteins play essential roles in the nervous system, heart, and breast. There is also evidence for involvement of NRG signaling in the development and function of several other organ systems, and in human disease, including the pathogenesis of schizophrenia and breast cancer. There are many NRG1 isoforms, raising the question "Why so many neuregulins?" Study of mice with targeted mutations ("knockout mice") has demonstrated that isoforms differing in their N-terminal region or in their epidermal growth factor (EGF)-like domain differ in their in vivo functions. These differences in function might arise because of differences in expression pattern or might reflect differences in intrinsic biological characteristics. While differences in expression pattern certainly contribute to the observed differences in in vivo functions, there are also marked differences in intrinsic characteristics that may tailor isoforms for specific signaling requirements, a theme that will be emphasized in this review.     

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

Rhenium(III) and rhenium(V) complexes stabilized by the potentially tetradentate ligand tris(2-diphenylphosphinoethyl)amine

The reaction of the rhenium(V) nitrido complex [Re(N)Cl₂(PPh₃)₂] with the tripodal ligand N(CH₂CH₂PPh₂)₃ (NP₃) in THF gave [Re(N)Cl₂(η²-P,P-NP₃)] (1) in which NP₃ acts as a tridentate ligand using the nitrogen and two phosphorus donors for coordination. Refluxing 1 in a polar solvent such as ethanol produced [(η⁴-NP₃)Re(N)Cl]Cl (2) in which NP₃ acts as a tetradentate ligand. Treatment of complex [Re(O)Cl₃(AsPh₃)₂] containing the [ReO]³⁺ core with NP₃ in THF yielded [ReCl₃{η³-N,P,P-(N{CH₂CH₂Ph₂}₂{CH₂CH₂P(O)Ph₂})}] (3). Complexes 1 and 3 have been characterized by single-crystal X-ray analyses.     

Reactions of quadruply bonded dimolybdenum(II) and dirhenium(III) complexes with 2,6-bis(dicyclohexylphosphinomethyl)pyridine and the related behavior of the mixed P,S,P ligand bis(diphenylphosphinomethyl)sulfide

The first complexes that contain the 2,6-bis(dicyclohexylphosphinomethyl)pyridine ligand (PNP) have been isolated and characterized. The reactions of K₄Mo₂Cl₈, (n-Bu₄N)₂Re₂Cl₈ and PdBr₂(1,5-COD) afford Mo₂Cl₄(PNP)(HPCy₂) (1), ReCl₃(PNP) (2) and PdBr₂(PNP) (4), respectively, while from the reaction of PNP with cis-Re₂(μ-O₂CCH₃)₂Cl₄(H₂O)₂ the heteromacrocylic dication [Cy₂P{CH₂pyCH₂}₂PCy₂]²⁺ has been isolated as its mixed [Cl]⁻/[ReO₄]⁻ salt (3). The reaction of cis-Re₂(μ-O₂CCH₃)₂Cl₄(H₂O)₂ with bis(diphenylphosphinomethyl)sulfide (PSP) gives the mononuclear Re(V) complex ReO(OEt)Cl₂(PSP) (5) in which the S atom is not coordinated. The structures of 1-5 have been established by X-ray crystallography, that of 5 being the first for a complex of this ligand.     

The expanding interleukin-1 family and its receptors: do alternative IL-1 receptor/signaling pathways exist in the brain?

Interleukin-1 (IL-1) has been implicated in neuroimmune responses and has pleiotropic actions in the brain. Compelling evidence has shown that IL-1 is a major mediator of inflammation and the progression of cell death in response to brain injury and cerebral ischemia. Its expression is strongly increased in these pathological conditions, and central administration of exogenous IL-1 significantly exacerbates ischemic brain damage. In contrast, inhibiting IL-1 actions (by intracerebroventricular [icv] injection of IL-1ra, neutralizing antibody to IL-1 or caspase-1 inhibitor) significantly reduces ischemic brain damage. IL-1 acts by binding to the IL-1 type-1 receptor (IL-1RI), which is to date, the only known functional receptor for IL-1. However, our recent investigations suggest that IL-1 can act independently of IL-1RI, raising the possibility that additional, as yet undiscovered, receptor(s) for IL-1 exist in the brain. The recent characterization of putative, new IL-1 ligands and new IL-1 receptor-related molecules leads to the hypothesis that there might be alternative IL-1 signaling pathway(s) in the central nervous system (CNS).     

Comparative enantiocontrol with allyl phenyldiazoacetates in asymmetric catalytic intramolecular cyclopropanation

Dirhodium(II) azetidinone-carboxylates are effective asymmetric catalysts for diazo decomposition of allyl diazoacetates and their subsequent intramolecular cyclopropanations. The effect of alkene substituents on enantiocontrol has been examined and modest selectivities have been achieved. Steric influences from substituents on the diazo carbon are seen to diminish enantioselectivities.