Cytochrome oxidase histochemistry in the rat hippocampus

Summary The diaminobenzidine (DAB) method was adapted for the microphotometric determination of cytochrome c oxidase (cyt ox) in the rat hippocampus. The qualitative and quantitative investigations at the light microscopic level showed that acetone and cytochrome c pretreatment of cryostat sections resulted in a significant increase of demonstrable cyt ox activities. The final incubation medium consisted of 7.5 mM DAB, 2% polyvinylalcohol (PVA) and 6% dimethyl sulfoxide in 0.1 M Hepes buffer; final pH 7.5. PVA was used to keep DAB and artificially oxidized DAB in solution. In the kinetic and endpoint measurements a linear response of the reaction with highest slope was observed only in the initial 5–6 min of reaction. Thereafter the slope decreased. Ultracytochemical demonstrations, which were performed as a topochemical control, showed reaction product only in mitochondria (cristae and intermembranous space). In contrast to vibrotome sections all mitochondria reacted positively in cryostat sections of aldehyde-fixed hippocampi. The enhancement of reaction after acetone pretreatment of cryostat sections (light microscopic level) and after a freezing step in ultracytochemistry is discussed in connection with diffusion problems of DAB through mitochondrial membranes.Dedicated to Professor Dr. G. Lang on the occasion of his 65th birthdaySupported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

Blütenökologische Untersuchungen Mit Hummeln

Ohne ZusammenfassungMit 9 Textabbildungen.     

Identifying rare and common disease associated variants in genomic data using Parkinson’s disease as a model

Background

Genome-wide association studies have been successful in identifying common genetic variants for human diseases. However, much of the heritable variation associated with diseases such as Parkinson’s disease remains unknown suggesting that many more risk loci are yet to be identified. Rare variants have become important in disease association studies for explaining missing heritability. Methods for detecting this type of association require prior knowledge on candidate genes and combining variants within the region. These methods may suffer from power loss in situations with many neutral variants or causal variants with opposite effects.

Results

We propose a method capable of scanning genetic variants to identify the region most likely harbouring disease gene with rare and/or common causal variants. Our method assigns a score at each individual variant based on our scoring system. It uses aggregate scores to identify the region with disease association. We evaluate performance by simulation based on 1000 Genomes sequencing data and compare with three commonly used methods. We use a Parkinson’s disease case–control dataset as a model to demonstrate the application of our method.Our method has better power than CMC and WSS and similar power to SKAT-O with well-controlled type I error under simulation based on 1000 Genomes sequencing data. In real data analysis, we confirm the association of α-synuclein gene (SNCA) with Parkinson’s disease (p = 0.005). We further identify association with hyaluronan synthase 2 (HAS2, p = 0.028) and kringle containing transmembrane protein 1 (KREMEN1, p = 0.006). KREMEN1 is associated with Wnt signalling pathway which has been shown to play an important role for neurodegeneration in Parkinson’s disease.

Conclusions

Our method is time efficient and less sensitive to inclusion of neutral variants and direction effect of causal variants. It can narrow down a genomic region or a chromosome to a disease associated region. Using Parkinson’s disease as a model, our method not only confirms association for a known gene but also identifies two genes previously found by other studies. In spite of many existing methods, we conclude that our method serves as an efficient alternative for exploring genomic data containing both rare and common variants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0088-9) contains supplementary material, which is available to authorized users.     

Evidence for linkage of migraine in Rolandic epilepsy to known 1q23 FHM2 and novel 17q22 genetic loci

Migraine headaches are a common comorbidity in Rolandic epilepsy (RE) and familial aggregation of migraine in RE families suggests a genetic basis not mediated by seizures. We performed a genome‐wide linkage analysis of the migraine phenotype in 38 families with RE to localize potential genetic contribution, with a follow‐up in an additional 21 families at linked loci. We used two‐point and multipoint LOD (logarithm of the odds) score methods for linkage, maximized over genetic models. We found evidence of linkage to migraine at chromosome 17q12‐22 [multipoint HLOD (heterogeneity LOD) 4.40, recessive, 99% penetrance], replicated in the second dataset (HLOD 2.61), and suggestive evidence at 1q23.1‐23.2, centering over the FHM2 locus (two‐point LOD 3.00 and MP HLOD 2.52). Sanger sequencing in 14 migraine‐affected individuals found no coding mutations in the FHM2 gene ATP1A2. There was no evidence of pleiotropy for migraine and either reading or speech disorder, or the electroencephalographic endophenotype of RE when the affected definition was redefined as those with migraine or the comorbid phenotype, and pedigrees were reanalyzed for linkage. In summary, we report a novel migraine susceptibility locus at 17q12‐22, and a second locus that may contribute to migraine in the general population at 1q23.1‐23.2. Comorbid migraine in RE appears genetically influenced, but we did not obtain evidence that the identified susceptibility loci are consistent with pleiotropic effects on other comorbidities in RE. Loci identified here should be fine‐mapped in individuals from RE families with migraine, and prioritized for analysis in other types of epilepsy‐associated migraine.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation of Drosophila Vasa In Vivo through Paralogous Cullin-RING E3 Ligase Specificity Receptors

In Drosophila species, molecular asymmetries guiding embryonic development are established maternally. Vasa, a DEAD-box RNA helicase, accumulates in the posterior pole plasm, where it is required for embryonic germ cell specification. Maintenance of Vasa at the posterior pole requires the deubiquitinating enzyme Fat facets, which protects Vasa from degradation. Here, we found that Gustavus (Gus) and Fsn, two ubiquitin Cullin-RING E3 ligase specificity receptors, bind to the same motif on Vasa through their paralogous B30.2/SPRY domains. Both Gus and Fsn accumulate in the pole plasm in a Vasa-dependent manner. Posterior Vasa accumulation is precocious in Fsn mutant oocytes; Fsn overexpression reduces ovarian Vasa levels, and embryos from Fsn-overexpressing females form fewer primordial germ cells (PGCs); thus, Fsn destabilizes Vasa. In contrast, endogenous Gus may promote Vasa activity in the pole plasm, as gus females produce embryos with fewer PGCs, and posterior accumulation of Vas is delayed in gus mutant oocytes that also lack one copy of cullin-5. We propose that Fsn- and Gus-containing E3 ligase complexes contribute to establishing a fine-tuned steady state of Vasa ubiquitination that influences the kinetics of posterior Vasa deployment.Establishment and maintenance of polarity is essential for multicellular development. Asymmetric distribution of proteins within cells is often realized by localizing specific mRNAs to distinct positions and by tightly regulating their translation (1). During Drosophila oogenesis, polarized deployment of key mRNAs is crucial for the maternal determination of the embryonic body axes (17). However, although asymmetric mRNA localization within cells is widespread (1, 22), some proteins localize directly. An example is Vasa (Vas), which accumulates in a highly polarized fashion in Drosophila oocytes from a uniformly distributed mRNA that is not believed to be under translational control (8, 20, 21). Vas accumulates in the pole plasm of the oocyte, where it is necessary for embryonic posterior patterning and primordial germ cell (PGC) formation (26). Accumulation of high levels of Vas in the pole plasm requires the deubiquitinating enzyme (DUB) Fat facets (Faf) (25). In faf mutants, levels of posterior Vas are reduced, and polyubiquitinated forms of Vas accumulate. This indicates that Vas stability in the pole plasm is regulated by ubiquitin-dependent pathways.Ubiquitination culminates in the E3 ligase-catalyzed formation of a covalent bond between the C terminus of ubiquitin and a lysine residue of the ubiquitinated protein (12). Target proteins can be ubiquitinated simultaneously and/or sequentially on different lysine residues, and the presence of seven internal lysine residues in ubiquitin itself allows for the formation of topologically distinct polyubiquitin chains (9, 12, 27). Different forms of ubiquitination usually produce different effects on the target protein, and modulation of the steady-state dynamics of ubiquitin conjugation can strongly influence a target''s activity and/or stability. The regulatory logic governing the steady state of target ubiquitination can consist of nonlinear pathways that involve feedback mechanisms, responses to cellular stimuli such as phosphorylation, and complex cross-regulation between individual components of the ubiquitin conjugation machinery and corresponding DUBs.Cullin-RING ubiquitin E3 ligases (CRLs) comprise the largest class of ubiquitin E3 ligases (30). CRLs contain a substrate specificity receptor that binds the ubiquitinated target and a RING protein that is involved in recruiting an E2-conjugating enzyme, which catalyzes transfer of ubiquitin to the associated substrate through the E3 ligase. RING proteins and particular substrate specificity receptors are brought together by scaffold proteins called Cullins, often through small adaptor proteins that link the Cullin with the receptor. Cullin-1 (Cul-1) CRLs recruit their substrate through F-box proteins, with a Skp family adaptor protein forming a bridge between the Cullin and the F-box. In contrast, CRLs containing Cullin-5 (Cul-5) recognize their substrates through receptor proteins that contain a SOCS-box, which are linked to the Cullin by the Elongin B/Elongin C (EloBC) adaptor complex.In this study we identified the F-box protein Fsn and the SOCS-box protein Gus as in vivo regulators of Vas. Fsn and its Caenorhabditis elegans orthologue are required for normal synaptic development and associate with RING proteins encoded by highwire and rpm-1, respectively (24, 46). Gus was previously shown to interact with Vas and was implicated in its posterior localization (37). Gus features a B30.2/SPRY domain through which it directly interacts with a five-amino-acid motif on Vas (DINNN), and it can be cocrystallized with the EloBC complex (44, 45). Fsn has a B30.2/SPRY domain that is very similar to that of Gus (40% identity). We show experimentally that Fsn also binds the DINNN motif and that Gus associates more stably with Vas than does Fsn. Using genetic methods, we investigated the contributions of Gus and Fsn to regulating Vas activity and deployment.