Mutually exclusive expression of a helix-loop-helix gene and N-myc in human neuroblastomas and in normal development.

We have isolated a novel human gene encoding a helix-loop-helix (HLH) protein by molecularly cloning chromosome 1p36-specific CpG islands. The gene termed heir-1 was localized to the neuroblastoma consensus deletion at 1p36.2-p36.12. Its predicted protein is 95.8% identical to the mouse HLH462 protein and has clear homology to the mouse Id and Drosophila emc proteins. Heir-1 does not encode a basic DNA binding domain as found in basic HLH proteins. The gene is expressed specifically at high abundance in adult lung, kidney and adrenal medulla, but not in adult brain. Despite prominent heir-1 expression in adrenal medulla, which is a prime target for neuroblastomas, 10 out of 12 neuroblastoma-derived cell lines revealed very low levels of heir-1 mRNA. Low heir-1 expression was generally found in tumor cell lines with N-myc overexpression, whereas the two cell lines displaying high heir-1 levels did not overexpress N-myc. Mutually exclusive expression of both genes was also found by in situ hybridization in developing mouse tissues, particularly in the forebrain neuroectoderm. We conclude that heir-1 expression is reduced specifically in the majority of neuroblastomas and suggest an inverse correlation between heir-1 and N-myc expression in neuroblastoma tumors and in embryonic development.     

Absence of the prion protein homologue Doppel causes male sterility

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm-egg interaction.     

Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP

Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.     

Isolation of a new foamy retrovirus from orangutans.

We have isolated a new foamy virus from blood samples taken from two apparently healthy orangutans (Pongo pygmaeus). The older orangutan has since died with encephalopathy after a brief acute illness, while the younger one, his grandson, remains well. These animals and 12 other orangutans had specific antibodies to foamy virus as measured by immunofluorescence. The new foamy virus and the antisera showed strong and specific neutralization, with only weak cross-reaction with other simian foamy virus strains. Southern blotting with gag and env probes of human foamy virus and PCR amplification showed that the new foamy virus, designated SFV-11, is related to, yet distinct from, previously characterized strains from humans, chimpanzees, and monkeys.     

Scrapie Pathogenesis in Subclinically Infected B-Cell-Deficient Mice

Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.     

Ecomorphology of morpho‐functional relationships in the family of sparidae: A quantitative statistic approach

In many fish species, morphological similarity can be considered as a proxy for similarities in habitat use. The Sparidae family includes species that are recognized for common morphological features such as structure and positioning of the fins and specialized dentition. The aim of this study was to quantitatively describe the relationship of body shape morphology with habitat use, trophic level, and systematics in the majority of known Sparidae species (N = 92). This ecomorphological comparison was performed with a geometric morphometric approach considering as variables the Trophic Index (TROPH), the habitat (i.e., classified as demersal, benthopelagic and reef associated) and the phylogenetic relationship of species at the subfamily level. The analysis by the TROPH variable showed a positive relation with shape because the morphological features of all the species are strongly correlated with their trophic behavior (e.g., herbivore species have a smaller mouth gap that make them able to feed upon sessile resources). The morphological analysis according to the Habitat variable was used to classify species according to a feeding‐habitat niche in terms of portion of the water column and seabed space where species mostly perform their behavioral activities. We described three kinds of morphological designs in relation to a benthopelagic, demersal and reef‐associated habit. The six subfamily groups were morphologically well distinguishable and the cladogram relative to Mahalanobis' morphological distances was compared with those proposed by other authors. We also quantified the phylogenetic relationship among the different subfamilies based on the analysis of shape in relation to trophic ecology, confirming the observations of the authors. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.     

Prion Pathogenesis Is Faithfully Reproduced in Cerebellar Organotypic Slice Cultures

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.