A simple cell based assay to measure Parkin activity

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.     

Genetic diversity patterns in Curcuma reflect differences in genome size

The relationships between genome size and the systematic and evolutionary patterns in vascular plants are equivocal, although a close relationship between genome size and evolutionary patterns has been previously reported. However, several studies have also revealed the dynamic nature of genome size evolution and its considerable ‘ups’ and ‘downs’. Thus, in this study, the phylogenetic relationships among three previously revealed genome size groups and among species of the highly polyploid genus Curcuma were evaluated using AFLP. Our results suggest two main lineages within Indian Curcuma reflecting evolution of genome size. The first one includes hexaploids and higher polyploids of the previously recognized genome size group I, and the second one includes mainly hexaploids of genome size groups II and III. Within genome size group I, relationships among species seem to be influenced by reticulate evolution and higher polyploids are likely to be of allopolyploid origin. Reproductive systems in Indian Curcuma vary considerably among ploidy levels and these differences considerably affect morphological and genetic variation. In general, clonally reproducing species are expected to exhibit low genotypic diversity, but, at the same time, species of allopolyploid origin are expected to maintain higher levels of heterozygosity compared with their progenitors. We investigated intra‐populational genetic variability in Curcuma spp. to evaluate whether mode of reproduction or ploidy represent the main factor influencing the degree of genetic diversity. We found that hexaploid species exhibited significantly higher genetic diversity than higher polyploids (9x, 15x). Our results suggest that this genetic diversity pattern is largely influenced by the mode of reproduction, as higher polyploids reproduce exclusively vegetatively, whereas hexaploids reproduce mainly sexually. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 388–401.     

Morphology, Anatomy and Ontogeny of Female Cones in Acmopyle pancheri(Brongn. & Gris) Pilg. (Podocarpaceae)

The developmental morphology and anatomy of the female conesof Acmopyle pancheri(Brongn. & Gris) Pilg. (Podocarpaceae)are described and illustrated, based on observations, histology,scanning electron microscopy (SEM) and nuclear magnetic resonance(NMR) imaging. Ovulate development is typically podocarpaceous.Ovules are unitegmic, and horizontal or inclined upwards throughoutontogeny; the pollination drop is inverted because of the declinatemicropyle. Ontogeny of the epimatium-ovule complex is acropetal,the epimatium developing first. A terminal, distal sterile bractcreates a pollen-scavenging area. During development, the wholecone re-orientates through some 270°, and the seed realignsapprox. 60° with respect to the receptacle axis. The ‘receptacle’or podocarpium supporting the seed is formed by gradual fusionof initially free bracts. The structures adnate to these bractsrepresent homologues of ovuliferous scales; they bear vestigialepimatia which may develop into supernumerary ovules or non-functionalepimatia. Thus, female cones ofA. pancheri are vestigially multi-ovulate.NMR imaging effectively and non-invasively revealed the three-dimensionalarrangement of vascular bundles and resin canals in the cones.Copyright 2001 Annals of Botany Company Acmopyle pancheri(Brongn. & Gris) Pilg., anatomy, developmental morphology, gymnosperms, histology, nuclear magnetic resonance (NMR) imaging, ontogeny, ovules, Podocarpaceae, scanning electron microscopy (SEM), seed cones     

A Markov chain Monte Carlo approach for joint inference of population structure and inbreeding rates from multilocus genotype data

Nonrandom mating induces correlations in allelic states within and among loci that can be exploited to understand the genetic structure of natural populations (Wright 1965). For many species, it is of considerable interest to quantify the contribution of two forms of nonrandom mating to patterns of standing genetic variation: inbreeding (mating among relatives) and population substructure (limited dispersal of gametes). Here, we extend the popular Bayesian clustering approach STRUCTURE (Pritchard et al. 2000) for simultaneous inference of inbreeding or selfing rates and population-of-origin classification using multilocus genetic markers. This is accomplished by eliminating the assumption of Hardy-Weinberg equilibrium within clusters and, instead, calculating expected genotype frequencies on the basis of inbreeding or selfing rates. We demonstrate the need for such an extension by showing that selfing leads to spurious signals of population substructure using the standard STRUCTURE algorithm with a bias toward spurious signals of admixture. We gauge the performance of our method using extensive coalescent simulations and demonstrate that our approach can correct for this bias. We also apply our approach to understanding the population structure of the wild relative of domesticated rice, Oryza rufipogon, an important partially selfing grass species. Using a sample of n = 16 individuals sequenced at 111 random loci, we find strong evidence for existence of two subpopulations, which correlates well with geographic location of sampling, and estimate selfing rates for both groups that are consistent with estimates from experimental data (s approximately 0.48-0.70).     

Selective incorporation of polyanionic molecules into hamster prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro.Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.     

High titers of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrPSc in vivo

Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrP(Sc)) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrP(Sc). This work questions the correlation between PrP(Sc) level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrP(Sc) as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.     

Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.