Naturally prion resistant mammals: A utopia?

Each known abnormal prion protein (PrP^Sc) is considered to have a specific range and therefore the ability to infect some species and not others. Consequently, some species have been assumed to be prion disease resistant as no successful natural or experimental challenge infections have been reported. This assumption suggested that, independent of the virulence of the PrP^Sc strain, normal prion protein (PrP^C) from these ‘resistant’ species could not be induced to misfold. Numerous in vitro and in vivo studies trying to corroborate the unique properties of PrP^Sc have been undertaken. The results presented in the article “Rabbits are not resistant to prion infection” demonstrated that normal rabbit PrP^C, which was considered to be resistant to prion disease, can be misfolded to PrP^Sc and subsequently used to infect and transmit a standard prion disease to leporids. Using the concept of species resistance to prion disease, we will discuss the mistake of attributing species specific prion disease resistance based purely on the absence of natural cases and incomplete in vivo challenges. The BSE epidemic was partially due to an underestimation of species barriers. To repeat this error would be unacceptable, especially if present knowledge and techniques can show a theoretical risk. Now that the myth of prion disease resistance has been refuted it is time to re-evaluate, using the new powerful tools available in modern prion laboratories, whether any other species could be at risk.     

Towards a framework for the study of the neural correlates of aesthetic preference

Aiming to provide a tentative framework for the study of the neural correlates of aesthetic preference, we review three recent neuroimaging studies carried out with the purpose of locating brain activity associated with decisions about the beauty of visual stimuli (Cela-Conde et al., 2004; Kawabata and Zeki, 2004; Vartanian and Goel, 2004). We find that the results of the three studies are not in line with previous neuropsychological data. Moreover, there are no coincidences among their results. However, when they are mapped on to Chatterjee's (2003) neuropsychological model of aesthetic preference it becomes clear that neuroimaging data are not contradictory, but complementary, and their interpretation is enriched. The results of these studies suggest that affective processes have an important role in aesthetic preference, and that they are integrated with cognitive processes to reach a decision regarding the beauty of visual stimuli. Future studies must aim to clarify whether certain methodological procedures are better suited to study any of the particular cognitive operations involved in aesthetic preference, and ascertain the extent to which the proposed framework is compatible with the aesthetic appreciation of musical stimuli.     

Fungal root endophytes from natural vegetation in Mediterranean environments with special reference to Fusarium spp

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1alpha gene region (TEF-1alpha) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1alpha in the genus Fusarium.     

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro⁵⁰, Pro⁹¹, and Pro¹⁸⁶ in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro⁹¹, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro⁹¹ and Pro¹⁸⁶ showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.     

Immunohistochemical Distribution of Cytochrome P4501A in Larvae and Fingerlings of the Siberian Sturgeon, Acipenser baeri

In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A), from hatching until 30 days posthatching in developing Siberian sturgeon, Acipenser baeri. For this purpose, a polyclonal (BN-1) antiserum developed against a conservative sequence of piscine CYP1A and a monoclonal (C10-7) antiserum directed against cod CYP1A were used on paraffin-embedded samples. From hatching onwards, distinct CYP1A immunoreactivity was distinctly observed in the following tissues and cells: envelope of oil droplets, matrix and syncytium of the yolk-sac, sinusoids, biliary epithelial cells and hepatocytes. In the digestive tract, buccopharyngeal, oesophageal, gastric and intestinal epithelia, as well as the cytoplasm and brush border of enterocytes were CYP1A-positive. Interestingly, gastric glands and melanin-plug present within lumen of the digestive system were strongly immunoreactive. Kidney (epithelia of renal tubules), gills (pillar and endothelial cells), skin (epithelial cells), muscle fibres of heart and eye (retina) were positive. In brain, we observed a strong CYP1A staining in the developing telencephalon and especially in olfactory system, as well as in those nerve fibres running ventrally toward the posterior brain. A strong CYP1A staining was observed in vascular endothelia of all organs/tissues, especially in the liver. In general, the intensity of CYP1A immunostaining increased during larval development, suggesting besides its known metabolic function (endogenous and/or exogenous), a possible participation of this heme-protein in control of cell division, regulation of growth and differentiation.     

In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

Summary To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.     

Orexin A/Hypocretin Modulates Leptin Receptor-Mediated Signaling by Allosteric Modulations Mediated by the Ghrelin GHS-R1A Receptor in Hypothalamic Neurons

The hypothalamus is a key integrator of nutrient-seeking signals in the form of hormones and metabolites originated in both the central nervous system and the periphery. The main autocrine and paracrine target of orexinergic-related hormones such as leptin, orexin/hypocretin, and ghrelin are neuropeptide Y neurons located in the arcuate nucleus of the hypothalamus. The aim of this study was to investigate the expression and the molecular and functional relationships between leptin, orexin/hypocretin and ghrelin receptors. Biophysical studies in a heterologous system showed physical interactions between them, with potential formation of heterotrimeric complexes. Functional assays showed robust allosteric interactions particularly different when the three receptors are expressed together. Further biochemical and pharmacological assays provided evidence of heterotrimer functional expression in primary cultures of hypothalamic neurons. These findings constitute evidence of close relationships in the action of the three hormones already starting at the receptor level in hypothalamic cells.     

α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5? were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532–587?nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.     

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow‐sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade‐offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.