Investigations of a prion infectivity assay to evaluate methods of decontamination

Prions are unique infectious agents which have been shown to be transmitted iatrogenically through contaminated surfaces. Surface contamination is a concern on reusable medical devices and various industrial surfaces, but there is currently no standard, accepted model to evaluate surface prion decontamination. In this report, a set of both in vitro and in vivo methods were investigated based on the contamination of surface through artificial exposure to infected brain. An in vitro surface contamination protocol was developed with subsequent biochemical detection of the prion protein (PrPres). In parallel, the in vivo investigations included the contamination of different types of surface materials (stainless steel or plastic wires) with different prion strains (scrapie strain adapted to hamsters 263K or bovine spongiform encephalopathy strain adapted to mouse 6PB1). The in vivo models with various prion strains and brain homogenate dilutions reproducibly transmitted the disease and a relationship was established between the infectivity titre, the transmission rate and the incubation period. Moreover, the in vivo models were studied for their ability to demonstrate the efficacy of heat and chemical-based decontamination methods, with similar results. The in vivo scrapie method described is proposed as a standard to evaluate existing and developing prion decontamination technologies.     

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.     

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.     

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.     

Dimeric DOTA-alpha-melanocyte-stimulating hormone analogs: synthesis and in vivo characteristics of radiopeptides with high in vitro activity

Dimeric analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for alpha-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled alpha-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [(111)In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([(111)In]DOTA)-amide, and 0.36 for [(111)In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [(111)In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled alpha-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.     

Bringing antivenoms to Sub-Saharan Africa

To reduce unacceptably high death rates from snakebite envenomation, sub-Saharan Africa must adopt not only a new generation of multivalent biotech antivenoms, but also an infrastructure to deliver them.     

Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum

A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized.     

A structural alignment kernel for protein structures

MOTIVATION: This work aims to develop computational methods to annotate protein structures in an automated fashion. We employ a support vector machine (SVM) classifier to map from a given class of structures to their corresponding structural (SCOP) or functional (Gene Ontology) annotation. In particular, we build upon recent work describing various kernels for protein structures, where a kernel is a similarity function that the classifier uses to compare pairs of structures. RESULTS: We describe a kernel that is derived in a straightforward fashion from an existing structural alignment program, MAMMOTH. We find in our benchmark experiments that this kernel significantly out-performs a variety of other kernels, including several previously described kernels. Furthermore, in both benchmarks, classifying structures using MAMMOTH alone does not work as well as using an SVM with the MAMMOTH kernel. AVAILABILITY: http://noble.gs.washington.edu/proj/3dkernel     

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.