Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

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Highlights

• •MS-based clinical assay that accurately determines phospho Rab10 occupancy.
• •Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.
• •Determination of pRab levels in neutrophils of Parkinson disease patients.
• •Relevance of pRab levels as marker of PD.

     

Shape and location of eukaryotic initiation factor eIF-2 on the 40S ribosomal subunit of rat liver. Immunoelectron-microscopic and hydrodynamic investigations

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.     

Banning Fisheries Discards Abruptly Has a Negative Impact on the Population Dynamics of Charismatic Marine Megafauna

Food subsidies have the potential to modify ecosystems and affect the provision of goods and services. Predictable Anthropogenic Food Subsidies (PAFS) modify ecosystems by altering ecological processes and food webs. The global concern over the effects of PAFS in ecosystems has led to development of environmental policies aimed at curbing the production or ultimately banning of PAFS. However, the effects of reducing or banning PAFS are not known. We explore the consequences of PAFS removal in a marine ecosystem under two scenarios: 1) gradual reduction, or 2) an abrupt ban, using a mass balance model to test these hypotheses–The reduction or loss of PAFS will: i) modify trophic levels and food webs through effects on foraging by opportunistic species, ii) increase the resilience of opportunistic species to food shortages, and iii) modify predator–prey interactions through shifts in prey consumption. We found that PAFS lower the trophic levels of opportunistic scavengers and increase their food pathways. Scavengers are able to switch prey when PAFS are reduced gradually but they decline when PAFS are abruptly banned. PAFS reduction to a certain minimal level causes a drop in the ecosystem’s stability. We recommend gradual reduction of PAFS to a minimal level that would maintain the ecosystem’s stability and allow species exploiting PAFS to habituate to the food subsidy reduction.     

The role of the cytosolic fraction and of initiation factor eIF-2 for changes of the rate of protein synthesis during liver regeneration

The protein synthesis-stimulating activity of the cytosolic fraction from regenerating rat liver was tested in a cell-free system using washed polysomes from normal rat liver. This activity undergoes significant changes during liver regeneration after partial hepatectomy (p.h.). An initial decrease until 16 h after p.h. is followed by a significant increase until 24 h after p.h. Beyond 32 h after p.h. the activity begins to decline again. Evidence is presented that these changes of the cytosolic activity may not be due to alterations in the distribution of protein synthesis-stimulating factors between the microsomal and the cytosolic fraction. The Met-tRNAf-binding activity of the cytosolic fraction changes during liver regeneration analogously to the protein synthesis-stimulating activity measured in the polysomal assay. This indicates that initiation factor eIF-2 is involved in the observed changes of the cytosolic activity. This conclusion could be confirmed by addition of purified eIF-2 to the polysomal assay system. Addition of eIF-2 to cytosolic fractions of low endogenous protein synthesis-stimulating activity (16 h after p.h.) enhances amino-acid incorporation to a significantly higher extent than addition to highly active cytosolic fractions (24 h and 32 h after p.h.). From these results it is concluded that changes in eIF-2 plays an essential role in the described alterations of the cytosolic activities during liver regeneration.     

Inhibition of nicotine-induced secretion from bovine chromaffin cells by the amidated C-terminal sequence of the opioid peptide amidorphin

The nicotine-induced release of catecholamines and opioid peptides from bovine chromaffin cells is inhibited by the amidated opioid peptide amidorphin. The active site of this inhibitory activity is located at the peptide's C-Terminus, which is, in contrast to the N-terminal sequence TYR-GLY-GLY-PHE, not responsible for the opioidergic activity of opioid peptides. The noradrenaline-secretion induced by histamine, a non-cholinergic secretagogue, has not been inhibited by amidorphin.