Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.     

Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination

Wolbachia is a group of maternally inherited endosymbiotic bacteria that infect and induce cytoplasmic incompatibility (CI) in a wide range of arthropods. In contrast to other species, the mosquito Culex pipiens displays an extremely high number of CI types suggesting differential infection by multiple Wolbachia strains. Attempts so far failed to detect Wolbachia polymorphism that might explain this high level of CI diversity found in C. pipiens populations. Here, we establish that Wolbachia infection is near to or at fixation in worldwide populations of the C. pipiens complex. Wolbachia polymorphism was addressed by sequence analysis of the Tr1 gene, a unique transposable element of the IS5 family, which allowed the identification of five C. pipiens Wolbachia strains, differing either by nucleotide substitution, presence or absence pattern, or insertion site. Sequence analysis also showed that recombination, transposition and superinfection occurred at very low frequencies. Analysis of the geographical distributions of each Wolbachia strain among C. pipiens populations indicated a strong worldwide differentiation independent from mosquito subspecies type, except in the UK. The availability of this polymorphic marker now opens the way to investigate evolution of Wolbachia populations and CI dynamics, in particular in regions where multiple crossing types coexist among C. pipiens populations.     

Oxidation-induced ferritin turnover in microglial cells: role of proteasome

Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules.     

New short route to unsaturated fluoroketonucleosides: case of 5-fluoro-1-(6-O-acetyl-3,4-di-deoxy-3-fluoro-beta-D-glycero-hex-3-eno-pyranos-2-ulosyl) uracil

The proposed short synthesis involves two key steps: Oxidation of the isopropylidene derivative of the 3-fluoronucleoside possessing a free hydroxyl group in 2-position and acetylation of deprotected 3-fluoro-2-ketonucleoside which, after a beta-elimination reaction, gives the desired unsaturated ketonucleoside 5.     

Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.     

Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.     

Tissue magnesium loss during prolonged hypokinesia in magnesium supplemented and unsupplemented rats

This study aims at showing that during hypokinesia (HK) tissue magnesium (Mg2+) content decreases more with higher Mg2+ intake than with lower Mg2+ intake and that Mg2+ loss increases more with higher than lower tissue Mg2+ depletion due to inability of the body to use Mg2+ during HK. Studies were conducted on male Wistar rats during a pre-HK period and a HK period. Rats were equally divided into four groups: unsupplemented vivarium control rats (UVCR), unsupplemented hypokinetic rats (UHKR), supplemented vivarium control rats (SVCR) and supplemented hypokinetic rats (SHKR). SVCR and SHKR consumed 42 mEq Mg2+ per day. The gastrocnemius muscle and right femur bone Mg2+ content decreased significantly, while plasma Mg2+ level and urine and fecal Mg2+ loss increased significantly in SHKR and UHKR compared with their pre-HK values and their respective vivarium controls (SVCR and UVCR). However, muscle and bone Mg2+ content decreased more significantly and plasma Mg2+ level, and urine and fecal Mg2+ loss increased more significantly in SHKR than in UHKR. The greater tissue Mg2+ loss with higher Mg2+ intake and the lower tissue Mg2+ loss with lower Mg2+ intake shows that the risk of higher tissue Mg2+ depletion is directly related to the magnitude of Mg2+ intake. The higher Mg2+ loss with higher tissue Mg2+ depletion and the lower Mg2+ loss with lower Mg2+ tissue depletion shows that the risk of greater Mg2+ loss is directly related to the magnitude of tissue Mg2+ depletion. It was concluded that tissue Mg2+ depletion increases more when the Mg2+ intake is higher and that Mg2+ loss increases more with higher than lower tissue Mg2+ depletion indicating that during prolonged HK the tissue Mg2+ depletion is not due to the Mg2+ shortage in food but to the inability of the body to use Mg2+.     

Patterns of species richness on very small islands: the plants of the Aegean archipelago

Aim To investigate the species–area relationship (SAR) of plants on very small islands, to examine the effect of other factors on species richness, and to check for a possible Small Island Effect (SIE). Location The study used data on the floral composition of 86 very small islands (all < 0.050 km²) of the Aegean archipelago (Greece). Methods We used standard techniques for linear and nonlinear regression in order to check several models of the SAR, and stepwise multiple regression to check for the effects of factors other than area on species richness (‘habitat diversity’, elevation, and distance from nearest large island), as well as the performance of the Choros model. We also checked for the SAR of certain taxonomic and ecological plant groups that are of special importance in eastern Mediterranean islands, such as halophytes, therophytes, Leguminosae and Gramineae. We used one‐way anova to check for differences in richness between grazed and non‐grazed islands, and we explored possible effects of nesting seabirds on the islands’ flora. Results Area explained a small percentage of total species richness variance in all cases. The linearized power model of the SAR provided the best fit for the total species list and several subgroups of species, while the semi‐log model provided better fits for grazed islands, grasses and therophytes. None of the nonlinear models explained more variance. The slope of the SAR was very high, mainly due to the contribution of non‐grazed islands. No significant SIE could be detected. The Choros model explained more variance than all SARs, although a large amount of variance of species richness still remained unexplained. Elevation was found to be the only important factor, other than area, to influence species richness. Habitat diversity did not seem important, although there were serious methodological problems in properly defining it, especially for plants. Grazing was an important factor influencing the flora of small islands. Grazed islands were richer than non‐grazed, but the response of their species richness to area was particularly low, indicating decreased floral heterogeneity among islands. We did not detect any important effects of the presence of nesting seabird colonies. Main conclusions Species richness on small islands may behave idiosyncratically, but this does not always lead to a typical SIE. Plants of Aegean islets conform to the classical Arrhenius model of the SAR, a result mainly due to the contribution of non‐grazed islands. At the same time, the factors examined explain a small portion of total variance in species richness, indicating the possible contribution of other, non‐standard factors, or even of stochastic effects. The proper definition of habitat diversity as pertaining to the taxon examined in each case is a recurrent problem in such studies. Nevertheless, the combined effect of area and a proxy for environmental heterogeneity is once again superior to area alone in explaining species richness.