Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Role of pituitary adenylate cyclase-activating peptide (PACAP) in the cyclic recruitment of immature follicles in the rat ovary

Following the midcyclic gonadotropin surge, PACAP is transiently expressed for approximately 12 h in the cyclic adult rat ovary. PACAP is observed in granulosa/lutein cells of the large mature follicles destined to ovulate and is believed to be a regulator of acute progesterone production and luteinization in these follicles. PACAP is also observed in solitary theca cells of immature follicles and in interstitial glandular cells intimately surrounding immature follicles. To examine if PACAP could be involved in the process of cyclic recruitment of such immature follicles, we primed immature granulosa cells from prepubertal ovaries with PACAP (1 nM and 100 nM) for 12 h. The treatment significantly stimulated the subsequent 24 h FSH-induced estradiol production (2.2 and 2.4 fold, respectively). The response seemed to be caused by a stimulation of aromatase activity. Estradiol production induced by testosterone was increased 2.4 and 2.6 fold, respectively, whereas functional FSH-receptors (cAMP production following FSH stimulation) or spontaneous apoptosis (immunohistochemical detection of DNA fragments) was unaffected. We conclude that PACAP priming of immature rat granulosa cells for 12 h increases subsequent FSH induced estradiol production and that PACAP could be involved in the cyclic recruitment of immature follicles in the adult rat ovary.     

5-Fluoro-4-thiouridine phosphoramidite: new synthon for introducing photoaffinity label into oligodeoxynucleotides

The synthesis of phosphoramidite of 5-fluoro-4-thio-2'-O-methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2' and 3' hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.     

Histocompatibility-2 system in wild mice

Six semicongenic lines carrying differentt haplotypes on the background of strain C57BL/10Sn (B10.t strains) and a (B10 ×T/t ⁰) F₁ hybrid were tested against one another in the mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML) assays. In every instance, the MLR results paralleled those of the CML typing: strain combinations giving a positive result in one assay gave a positive result in the second; combinations in which no response was observed in the MLR assay also failed to kill target cells specifically in the CML assay. Furthermore, the MLR and CML results were concordant with the results of the serological typing of these strains, as reported previously by us. The combined results suggest sharing ofH-2 hyplotypes between B10.t¹² and B10.t³², between B10.t⁶ and B10.t^w1, and between B10.t^w2 and (B10. ×T/t ⁰) F₁. These data support the conclusion, reached in our previous publication, that members of the samet-complementation group, with few exceptions, shareH-2 haplotypes.     

Floristic diversity patterns in the White Carpathians biosphere reserve,Czech Republic

The flora of the White Carpathians, a mountain range in the south-east of the Czech Republic, is documented by about 485,000 records of vascular plant occurrences collected since the mid-19^th century. A total of 1299 species recorded in 93 grid cells of 2.8 × 3.1 km were used for an analysis of spatial patterns of floristic diversity in the White Carpathians. Multivariate statistical techniques such as ordination and classification were used to reveal the main gradients in floristic composition and species richness, and measured environmental data and Ellenberg indicator values were used to assess underlying environmental factors. There is a striking floristic contrast between the western and eastern part of the study area, which is associated with differences in climate, mean altitude, topographic heterogeneity measured as altitudinal range, and land use. The western part is characterised by thermophilous, continental and calcicolous species of open habitats. In contrast, the more forested eastern part along the state border with Slovakia and the north-eastern part of the area are characterised by acidophilous species with higher moisture requirements. This pattern is consistent with the established phytogeographical division of the Czech Republic into the phytogeographical regions of Thermophyticum and Mesophyticum. The further division of the area into four regions, based on classified grid data, is also similar to the current division into phytogeographical districts, except for the Javorníky district. There are two distinct hot spots of species richness, in the western and the extreme north-eastern part. A poorer flora was found in landscapes with intensive agriculture. Species richness is associated with different environmental factors than species composition, namely with soil types and land-use categories. Alien species are more common in areas with a higher incidence of arable land and built-up areas, and less common in areas dominated by grasslands and forests.     

Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.