Differential nuclear localization of complexes may underlie in vivo intrabody efficacy in Huntington's disease.

Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.This is the final published version. It's also available from OUP at http://peds.oxfordjournals.org/content/27/10/359.full.html

Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ∼80–90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation

Evidence That Mutation Is Universally Biased towards AT in Bacteria

Mutation is the engine that drives evolution and adaptation forward in that it generates the variation on which natural selection acts. Mutation is a random process that nevertheless occurs according to certain biases. Elucidating mutational biases and the way they vary across species and within genomes is crucial to understanding evolution and adaptation. Here we demonstrate that clonal pathogens that evolve under severely relaxed selection are uniquely suitable for studying mutational biases in bacteria. We estimate mutational patterns using sequence datasets from five such clonal pathogens belonging to four diverse bacterial clades that span most of the range of genomic nucleotide content. We demonstrate that across different types of sites and in all four clades mutation is consistently biased towards AT. This is true even in clades that have high genomic GC content. In all studied cases the mutational bias towards AT is primarily due to the high rate of C/G to T/A transitions. These results suggest that bacterial mutational biases are far less variable than previously thought. They further demonstrate that variation in nucleotide content cannot stem entirely from variation in mutational biases and that natural selection and/or a natural selection-like process such as biased gene conversion strongly affect nucleotide content

Scopolamine Administration Modulates Muscarinic, Nicotinic and NMDA Receptor Systems

Studies on the effect of scopolamine on memory are abundant but so far only regulation of the muscarinic receptor (M1) has been reported. We hypothesized that levels of other cholinergic brain receptors as the nicotinic receptors and the N-methyl-D-aspartate (NMDA) receptor, known to be involved in memory formation, would be modified by scopolamine administration

Baby Business: a randomised controlled trial of a universal parenting program that aims to prevent early infant sleep and cry problems and associated parental depression

<p>Abstract</p> <p>Background</p> <p>Infant crying and sleep problems (e.g. frequent night waking, difficulties settling to sleep) each affect up to 30% of infants and often co-exist. They are costly to manage and associated with adverse outcomes including postnatal depression symptoms, early weaning from breast milk, and later child behaviour problems. Preventing such problems could improve these adverse outcomes and reduce costs to families and the health care system. Anticipatory guidance-i.e. providing parents with information about normal infant sleep and cry patterns, ways to encourage self-settling in infants, and ways to develop feeding and settling routines <it>before </it>the onset of problems-could prevent such problems. This paper outlines the protocol for our study which aims to test an anticipatory guidance approach.</p> <p>Methods/Design</p> <p>750 families from four Local Government Areas in Melbourne, Australia have been randomised to receive the <it>Baby Business </it>program (intervention group) or usual care (control group) offered by health services. The <it>Baby Business </it>program provides parents with information about infant sleep and crying via a DVD and booklet (mailed soon after birth), telephone consultation (at infant age 6-8 weeks) and parent group session (at infant age 12 weeks). All English speaking parents of healthy newborn infants born at > 32 weeks gestation and referred by their maternal and child health nurse at their first post partum home visit (day 7-10 postpartum), are eligible. The primary outcome is parent report of infant night time sleep as a problem at four months of age and secondary outcomes include parent report of infant daytime sleep or crying as a problem, mean duration of infant sleep and crying/24 hours, parental depression symptoms, parent sleep quality and quantity and health service use. Data will be collected at two weeks (baseline), four months and six months of age. An economic evaluation using a cost-consequences approach will, from a societal perspective, compare costs and health outcomes between the intervention and control groups.</p> <p>Discussion</p> <p>To our knowledge this is the first randomised controlled trial of a program which aims to prevent both infant sleeping and crying problems and associated postnatal depression symptoms. If effective, it could offer an important public health prevention approach to these common, distressing problems.</p> <p>Trial registration number</p> <p>ISRCTN: <a href="http://www.controlled-trials.com/ISRCTN63834603">ISRCTN63834603</a></p

Cellular and humoral sensitivity to gluten fractions in patients with treated nontropical sprue

The presence of circulating antibodies and lymphocyte response to gliadin and fraction III were measured in three groups of 12 patients each. Group I consisted of patients with nontropical sprue maintained on a gluten-free diet; Group II contained patients with other gastrointestinal diseases manifesting malabsorption and Group III was composed of normal controls. Rabbits immunized to both antigens provided positive controls for each method of antibody determination. Results agree with those previously reported in that negligible antibody titers were present to either antigen in normals, patients with other forms of malabsorption or patients with nontropical sprue maintained, for some time, on a gluten-free diet. Lymphocyte stimulation failed to occur with either gluten fraction although the hyporesponsiveness to phytohemagglutinin, previously reported by others, was not observed. Further studies are needed in patients with nontropical sprue following controlled antigenic challenge. Antibody levels in jejunal fluid should also be studied. Until such studies are carried out, evaluation of immunologic factors in the pathogenesis of nontropical sprue will be incomplete.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44378/1/10620_2005_Article_BF02232292.pd

Identification and functional characterization of a novel monotreme-specific antibacterial protein expressed during lactation

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.Swathi Bisana, Satish Kumar, Peggy Rismiller, Stewart C. Nicol, Christophe Lefèvre, Kevin R. Nicholas, Julie A. Shar