A Drug-Based Cellular Assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: A practical guide

Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrP(C)) in this process, remain enigmatic. A window into the normal function of PrP(C), and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines. We have used this drug-sensitizing effect to develop a simple Drug-Based Cell Assay (DBCA) that reproduces several features of mutant PrP toxicity observed in vivo, including the rescuing activity of wild-type PrP. In this paper, we present a detailed guide for executing the DBCA in several, different experimental settings, including a new slot blot-based format. This assay provides a unique tool for studying PrP cytotoxic and cytoprotective activities in cell culture.     

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA^Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.     

The "far-west" of Anopheles gambiae molecular forms

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed.     

Fractal dimension of the middle meningeal vessels: variation and evolution in Homo erectus, Neanderthals, and modern humans

The middle meningeal vascular network leaves its traces on the endocranial surface because of the tight relationship between neurocranial development and brain growth. Analysing the endocast of fossil specimens, it is therefore possible to describe the morphology of these structures, leading inferences on the cerebral physiology and metabolism in extinct human groups. In this paper, general features of the meningeal vascular traces are described for specimens included in the Homo erectus, Homo neanderthalensis, and Homo sapiens hypodigms. The complexity of the arterial network is quantified by its fractal dimension, calculated through the box-counting method. Modern humans show significant differences from the other two taxa because of the anterior vascular dominance and the larger fractal dimension. Neither the fractal dimension nor the anterior development are merely associated with cranial size increase. Considering the differences between Neanderthals and modern humans, these results may be interpreted in terms of phylogeny, cerebral functions, or cranial structural network.     

Lens-forming competence in the epidermis of Xenopus laevis during development

In larval X. laevis the capacity to regenerate a lens under the influence of inductive factors present in the vitreous chamber is restricted to the outer cornea and pericorneal epidermis (Lentogenic Area, LA). However, in early embryos, the whole ectoderm is capable of responding to inductive factors of the larval eye forming lens cells. In a previous paper, Cannata et al. (2003) demonstrated that the persistence of lens-forming competence in the LA is the result of early signals causing lens-forming bias in the presumptive LA and of late signals from the eye causing cornea development. This paper analyzes 1) the decrease of the lens-forming capacity in ectodermal regions both near LA (head epidermis) and far from LA (flank epidermis) during development, 2) the capacity of the head epidermis and flank epidermis to respond to lens-competence promoting factors released by an eye transplanted below these epidermal regions, and 3) the eye components responsible for the promoting effect of the transplanted eye. Results were obtained by implanting fragments of ectoderm or epidermis into the vitreous chamber of host tadpoles and by evaluating the percentage of implants positive to a monoclonal antibody anti-lens. These results demonstrated that the lens-forming competence in the flank region is lost at the embryonic stage 30/31 and is weakly restored by eye transplantation; however, lens-forming competence in the head region is lost at the larval stage 48 and is strongly restored by eye transplantation. The authors hypothesize that during development the head ectoderm outside the LA is attained by low levels of the same signals that attain the LA and that these signals are responsible for the maintenance of lens-forming competence in the cornea and pericorneal epidermis of the larva. In this hypothesis, low levels of these signals slacken the decrease of the lens-forming competence in the head ectoderm and make the head epidermis much more responsive than the flank epidermis to the effect of promoting factors released by a transplanted eye. Results obtained after transplantation of eyes deprived of some components indicate that the lens and the retina are the main source of these promoting factors. The immunohistochemical detection of the FGFR-2 (bek variant) protein in the epidermis of stage 53 larvae submitted to eye transplantation at stage 46 showed that the eye transplantation increased the level of FGFR-2 protein in the head epidermis but not in the flank epidermis, indicating that the lens-forming competence in X. laevis epidermis could be related to the presence of an activated FGF receptor system in the responding tissue.     

Synthesis of a D-rhamnose branched tetrasaccharide, repeating unit of the O-chain from Pseudomonas syringae pv. Syringae (cerasi) 435

The first synthesis of a d-rhamnose branched tetrasaccharide, corresponding to the repeating unit of the O-chain from Pseudomonas syringae pv. cerasi 435, as methyl glycoside is reported. The approach used is based on the synthesis of an opportune building-block, that is the methyl 3-O-allyl-4-O-benzoyl-alpha-D-rhamnopyranoside, which was then converted into both a glycosyl acceptor and two different protected glycosyl trichloroacetimidate donors. Successive couplings of these three compounds afforded the target oligosaccharide. The reported synthesis is also useful to perform the oligomerization of the repeating unit.     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

From symmetrical to asymmetrical nitrido phosphino-thiol complexes: a new class of neutral mixed-ligand (99m)Tc compounds as potential brain imaging agents

A general procedure is presented for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core that could be used to design either essential or target specific imaging agents. This procedure is based on the chemical properties of a new monosubstituted [Tc(N)(R2PS)Cl(PPh3)] species composed of a TcN multiple bond and an ancillary phosphine thiol ligand (R2PSH). This intermediate readily reacts with bidentate mononegative ligands (S--Y) containing soft pi-donor coordinating atoms to give neutral pentacoordinate asymmetrical complexes of the type [Tc(N)(R2PS)(S--Y)]. The ability of several bidentate ligands containing different combination of heteroatoms (S, N, O) to form complexes with the [Tc(N)(R2PS)]+ building block was investigated. It was found that mononegative dithiocarbamate (DTC) or cysteine carboxyl derivate ligands promptly react with the monosubstituted species to form the final mixed compound in high yield. Preliminary biodistribution data in rats of some representative [Tc(N)(R2PS)(DTC)] compounds revealed an interesting initial brain uptake (in the range 0.20 +/- 0.01% ID/g and 0.91 +/- 0.06% ID/g), indicating their ability to cross in and out of the intact BBB. In these complexes the dithiocarbamate, or more generally the bidentate ligand (S--Y), can be designed to carry a functional group or a bioactive molecule, which could be involved in a trapping mechanism to increase brain retention for longer time intervals. These results could be conveniently utilized to devise a new procedure for the production of a novel class of brain perfusion and/or brain receptor imaging agents.