Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.

Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation both in vitro and in vivo by a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to bind to a portion of the supF mutation reporter gene. Using in vitro gel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, the in vitro binding of the c7X-TFOs was not affected, but the efficiency of in vivo triple helix formation was reduced. These results indicate the utility of the c7X substitution for in vivo gene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.     

Contribution of fungi to chronic dermatitis and lymphangitis in patients with filariasis]

Adenolymphangitis is a common occurrence in filarial lymphedema. Damage to the lymphatics by F. bancrofti is followed by obliteration of lymph vessel and lymph stasis. Obstruction of lymphatics prevents the microbs skin penetration. Presented studies were performed to evaluate the role of fungi colonization of the skin among patients with filarial lymphedema. The fungal colonization of skin in patients with filarial lymphedema may be an important reason for chronic inflammatory disorders.     

Novel 5-HT7R antagonists,arylsulfonamide derivatives of (aryloxy)propyl piperidines: Add-on effect to the antidepressant activity of SSRI and DRI,and pro-cognitive profile

A novel series of arylsulfonamide derivatives of (aryloxy)propyl piperidines was designed to obtain potent 5-HT₇R antagonists. Among the compounds evaluated herein, 3-chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)2-hydroxypropyl]piperidin-4-yl}benzenesulfonamide (25) exhibited antagonistic properties at 5-HT₇R and showed selectivity over selected serotoninergic and dopaminergic receptors, as well as over serotonin, noradrenaline and dopamine transporters. Compound 25 demonstrated significant antidepressant-like activity in the forced swim test (0.625–2.5 mg/kg, i.p.) and in the tail suspension test (1.25 mg/kg, i.p.), augmented the antidepressant effect of inactive doses of escitalopram (selective serotonin reuptake inhibitor) and bupropion (dopamine reuptake inhibitor) in the FST in mice, and similarly to SB-269970, exerted pro-cognitive properties in the novel object recognition task in cognitively unimpaired conditions in rats (0.3 mg/kg, i.p.). Such an extended pharmacological profile, especially the augmentation effect of the identified 5-HT₇R antagonist on SSRI activity, seems promising regarding the complexity of affective disorders and potentially improved outcomes, including mnemonic performance.     

Algorithm of OMA for large-scale orthology inference

Background  

OMA is a project that aims to identify orthologs within publicly available, complete genomes. With 657 genomes analyzed to date, OMA is one of the largest projects of its kind.     

The influence of dioxygen on luminol chemiluminescence

Assays of peroxy compounds are commonly performed after chromatographic separation of analysed mixtures. In high‐performance liquid chromatography (HPLC), solvent reservoirs are sparged by helium or inline vacuum‐degassed in order to control the compressibility of the solvents for efficient pumping. In this study, we investigated the influence of degassing the reaction solution on the light output of the hemin‐catalyzed luminol oxidation by various oxidants. We found that, when t‐butyl hydroperoxide, hydrogen peroxide, n‐butyl hydroperoxide, iodosobenzene and iodobenzene diacetate were used as oxidants, the luminol chemiluminescence was lowered by 50–70% compared with an equilibrated and degassed solution. The opposite effect was observed when dibenzoyl peroxide and 3‐chloroperoxybenzoic acid were used as oxidants, as the chemiluminescence increased by approximately 20–30%. The reduced chemiluminescence was explained based on the known role of dioxygen in luminol chemiluminescence. The enhancement of chemiluminescence was rationalized by suggesting an alternative mechanism of luminol oxidation valid for peroxyacids and diacyl peroxides in which the reaction of a peroxyacid anion with the diazaquinone led to light emission with a higher quantum yield than the usual path, which is suppressed by the removal of dioxygen from the reaction solution. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural and functional analysis of the GABARAP interaction motif (GIM)

Through the canonical LC3 interaction motif (LIR), [W/F/Y]‐X₁‐X₂‐[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a I nteraction 相似文献   

Impact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.     

Molecular controls of antigen receptor clustering and autoimmunity

Cellular organization of the cytoskeleton, assembly of intracellular signaling complexes and movement of membrane receptors into supramolecular activation complexes (SMACs) are crucial prerequisites for lymphocyte activation and function. Full T-cell activation requires costimulatory signals in addition to antigen-mediated signals. Costimulatory signals facilitate T-cell activation by inducing SMAC formation, resulting in sustained signal transduction, cell-cycle progression and cytokine production. The guanine nucleotide exchange factor Vav1 and the Wiscott-Aldrich syndrome protein (WASP) regulate the actin cytoskeleton in T cells and also regulate SMAC formation. In mice lacking the E3 ubiquitin ligase Cbl-b, the Vav-WASP signaling pathway is active in the absence of costimulation resulting in deregulated cytoskeletal reorganization, enhanced priming and expansion of autoreactive T cells, and the development of autoimmunity. This review discusses the role of Cbl-b, Vav and WASP in the regulation of SMAC formation and the implications for the maintenance of tolerance and the development of autoimmunity.