Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.     

The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase.

Mutation pAR5 replaces residues 145'-153' at the C terminus of the regulatory (r) chains of Escherichia coli ATCase by a new sequence of six residues. The mutated enzyme has been shown to lack substrate cooperativity and inhibition by CTP. Solution X-ray scattering curves demonstrate that, in the absence of ligands, its structure is intermediate between the T form and the R form. In the presence of N-phosphonacetyl-L-aspartate, the mutant is similar to the wild type. An examination of the crystal structure of unligated ATCase reveals that the mutated site is at an interface between r and catalytic (c) chains, which exists only in the T allosteric form. A computer simulation by energy minimization suggests that the pAR5 mutation destabilizes this interface and induces minor changes in the tertiary structure of r chains. The resulting lower stability of the T form explains the loss of substrate cooperativity. The lack of allosteric inhibition may be related to a new electrostatic interaction made in mutant r chains between the C-terminal carboxylate and a lysine residue of the allosteric domain.     

Haemoglobin: the surface buried between the alpha 1 beta 1 and alpha 2 beta 2 dimers in the deoxy and oxy structures

Using the newly available refined co-ordinates of deoxy and oxyhaemoglobin, we have re-examined and compared the interfaces between the dimers alpha 1 beta 1 and alpha 2 beta 2. The most extensive monomer-monomer contacts are between alpha 1 and beta 2, and, symmetrically, alpha 2 and beta 1. In oxyhaemoglobin these interfaces bury 700 A2 less protein surface than in deoxyhaemoglobin. The alpha 1 alpha 2 interface involves similar salt bridges in both forms, but in oxyhaemoglobin buries 240 A2 more surface than in deoxyhaemoglobin. There is a loosely packed beta 1 beta 2 interface burying 320 A2 of surface in oxyhaemoglobin; there is no beta 1 beta 2 interface in deoxyhaemoglobin. The greater stability of the deoxy form, in the absence of ligands, can be attributed to a combination of hydrophobic, van der Waals' and electrostatic interactions.     

Prevalence and Phylogenetic Analysis of Hepatitis B in Captive and Wild-Living Pileated Gibbons (Hylobates pileatus) in Cambodia

International Journal of Primatology - The risk of transmitting novel pathogens from released animals to wild conspecifics is an important consideration for reintroduction initiatives. Apart from...     

Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase

The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.     

Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-alpha -dependent mechanism

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 microCi of (131)I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 +/- 0.01 in the C group to 0.19 +/- 0.03 in OA group (P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 +/- 0.05) compared with C (1.38 +/- 0.03, P = 0.01) and Se groups (1.42 +/- 0.03, P = 0.04). Administration of anti-tumor necrosis factor-alpha antibodies within the instillate significantly decreased this ratio (1.32 +/- 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-alpha-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.