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.     

Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.     

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.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer''s Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease

Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µ M ) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M₁ receptor, the selective M₁ agonist PD 142505-0028 (10 µ M ) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M₁ antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.     

Characterisation of the Glycine Modulatory Site of the N-Methyl-d-Aspartate Receptor-Ionophore Complex in Human Brain

[3H]Glycine binding and glycine modulation of [3H]MK-801 binding have been used to study the glycine allosteric site associated with the N-methyl-D-aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK-801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK-801 binding by glycine are consistent with [3H]glycine binding occurring to an N-methyl-D-aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimer's disease and reduced glycine enhancement of [3H]MK-801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease-related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.     

Electron probe X-ray microanalysis of calcium and phosphorus distribution in developing hamster tooth germs in vitro and in vivo

The aim of the present study was to investigate the spatial distribution of Ca and P in dentin and enamel of developing first (M1) and second (M2) maxillary hamster molars (age: 3-5 days) in comparison with cultured molars. For culturing the germs were dissected from 3-day-old hamsters and incubated for 1 and 2 days, respectively, in a modified BGJb medium. Electron probe X-ray measurements were carried out on 3 regions extending in a vertical axis from cusp tip over cusp middle to cusp base next to the cervical loop region. Neither the in vivo nor the in vitro group was statistically different in the Ca and P concentration in the regions of dentin. In both groups the measurements in enamel showed a gradient with an increase in Ca and P from enamel surface towards dentin-enamel junction and a gradient with an increase from cusp base towards cusp tip. Direct comparison of the in vivo group with the in vitro group did not demonstrate a statistical difference between the mineral content of the 4-day-old germs and the 1-day culture germs, respectively the 5-day-old germs and the 2-day culture germs. The results indicate a high correspondence between the mineralization process of in vitro and in vivo tooth germ development.