The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria

The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization,degradation, and toxicity

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry–based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2_911–919, a nine amino acid segment within a flexible interdomain region (LRRK2_853–981), which we designate the “regulatory loop” (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2’s interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase–dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.     

Advances in NANI and NAPI accounting for the Baltic drainage basin: spatial and temporal trends and relationships to watershed TN and TP fluxes

In order to assess the progress toward eutrophication management goals, it is important to understand trends in land-based nutrient use. Here we present net anthropogenic nitrogen and phosphorus inputs (NANI and NAPI, respectively) for 2000 and 2010 for the Baltic Sea watershed. Overall, across the entire Baltic, between the 5-year periods centered on 2000 and 2010, NANI and NAPI decreased modestly by ?6 and ?4%, respectively, but with substantial regional variation, including major increases in the Gulf of Riga drainage basin (+19 and +58%, respectively) and decreases in the Danish Straits drainage basin (?25 and ?40% respectively). The changes were due primarily to changes in mineral fertilizer use. Mineral fertilizers dominated inputs, at 57% of both NANI and NAPI in 2000, increasing to 68 and 70%, respectively, by 2010. Net food and feed imports declined over that period, corresponding to increased crop production; either fewer imports of food and feedstocks were required to feed humans and livestock, or more of these commodities were exported. A strong linear relationship exists between regional net nutrient inputs and riverine nutrient fluxes for both periods. About 17% of NANI and 4.7% of NAPI were exported to the sea in 2000; these relationships did not significantly differ from those for 2010. Changes in NANI from 2000 to 2010 across basins were directly proportional rather than linearly related to changes in total N (TN) fluxes to the sea (i.e., no change in NANI suggests no change in TN flux). Similarly, for all basins except those draining to the Baltic Proper, changes in NAPI were proportional to changes in total P (TP) fluxes. The Danish Straits decreased most between 2000 and 2010, where NANI and NAPI declined by 25 and 40%, respectively, and corresponding fluxes of TN and TP declined 31 and 18%, respectively. For the Baltic Proper, NAPI was relatively unchanged between 2000 and 2010, while riverine TP fluxes decreased 25%, due possibly to lagged effects of fertilizer reduction resulting from socio-political changes in the early 1990s or improvements in sewage treatment capabilities. For most regions, further reductions in NANI and NAPI could be achieved by more efficient production and greater substitution of manure for imported mineral fertilizers.     

Penaeid and carid community changes in the St Lucia estuarine lake system,South Africa,under low water level,extended closed periods and marine reconnection conditions

Penaeid and carid communities were assessed in St Lucia over a wide range of saline conditions, water level conditions and mouth states, including a six-month marine connection and various connections to the Mfolozi Estuary. Samples were collected biannually in spring and autumn from November 2004 to May 2012 using seine nets. Seven penaeid and seven carid species were recorded, of which Palaemon pacificus, P. peringueyi, Penaeus indicus and Metapenaeus monoceros were the most abundant. The decline from 2004 to 2007 in both species recorded and densities of freshwater, estuarine and marine species during the initial closed period was linked to increased salinity, lack of recruitment and reduced estuarine surface area. Increased wave action and high seas following Cyclone Gamede opened the mouth in March 2007 after a 57-month closed period, resulting in recruitment of marine species, predominantly P. indicus, followed by a decline in densities after reclosure in August 2007. Increases in marine and freshwater species were evident after Mfolozi flood connections during 2008, 2009 and 2010. Highest densities were recorded after breaching in 2007 and after flood connections, highlighting the importance of a marine link to maintain the recruitment of penaeid species.     

The influence of climate on average nitrogen export from large watersheds in the Northeastern United States

The flux of nitrogen in large rivers in North America and Europe is well explained as a function of the net anthropogenic inputs of nitrogen to the landscape, with on average 20 to 25% of these inputs exported in rivers and 75 to 80% of the nitrogen retained or denitrified in the landscape. Here, we use data for average riverine nitrogen fluxes and anthropogenic inputs of nitrogen over a 6-year period (1988–1993) for 16 major watersheds in the northeastern United States to examine if there is also a climatic influence on nitrogen fluxes in rivers. Previous studies have shown that for any given river, nitrogen fluxes are greater in years with higher discharge, but this can be interpreted as storage of nitrogen in the landscape during dry years and flushing of this stored nitrogen during wet years. Our analyses demonstrate that there is also a longer-term steady-state influence of climate on riverine nitrogen fluxes. Those watersheds that have higher precipitation and higher discharge export a greater fraction of the net anthropogenic inputs of nitrogen. This fractional export ranges from 10 to 15% of the nitrogen inputs in drier watersheds in the northeastern United States to over 35% in the wetter watersheds. We believe this is driven by lower rates of denitrification in the wetter watersheds, perhaps because shorter water residence times do not allow for as much denitrification in riparian wetlands and low-order streams. Using mean projections for the consequences of future climate change on precipitation and discharge, we estimate that nitrogen fluxes in the Susquehanna River to Chesapeake Bay may increase by 3 to 17% by 2030 and by 16 to 65% by 2095 due to greater fractional delivery of net anthropogenic nitrogen inputs as precipitation and discharge increase. Although these projections are highly uncertain, they suggest a need to better consider the influence of climate on riverine nitrogen fluxes as part of management efforts to control coastal nitrogen pollution.     

Effect of phosphatidylethanolamine on the properties of phospholipid-apolipoprotein complexes

Plasma high density lipoproteins (HDL) are synthesized in intestinal mucosal cells and hepatocytes and are secreted into the blood. Factors influencing the structure and function of these HDL, such as lipid and protein composition, are poorly understood. It appears, however, that intracellular, discoidal HDL are enriched, relative to plasma HDL, in phosphatidylethanolamine (PE), a phospholipid known to generate unusual, nonbilayer structures of putative physiological significance. Although incubation of dimyristoylphosphatidylcholine (DMPC) with apolipoprotein A-I at the gel-liquid crystalline phase transition temperature results in the spontaneous formation of lipid-protein complexes, the presence of proportionately small amounts of PE prevents the formation of such complexes, suggesting that PE profoundly alters the phase properties of the phospholipid bilayers. However, by using a detergent-mediated method for the formation of PE-rich model nascent HDL from phospholipids and apolipoprotein A-I, lipid-protein complexes containing as much as 75% DLPE could be formed, thus demonstrating that the presence of PE causes a kinetic, rather than a thermodynamic, barrier to spontaneous complex formation. The products contained a DLPE:DMPC molar ratio similar to that of the initial incubation mixture; however, as the mole percentage of DLPE increased, the products became less heterogeneous, the buoyant density of the products increased, and the Stokes diameter of the products decreased. Similar results were obtained when dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) were employed in lieu of DLPE. Electron microscopy of complexes containing DLPE and DMPC at a 1:1 molar ratio showed that these particles possessed a discoidal, bilayer morphology similar to that seen with complexes containing only phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

95% of basidiospores fall within 1 m of the cap: a field-and modeling-based study

Plant establishment patterns suggest that ectomycorrhizal fungal (EMF) inoculant is not found ubiquitously. The role of animal vectors dispersing viable EMF spores is well documented. Here we investigate the role of wind in basidiospore dispersal for six EMF species, Inocybe lacera, Laccaria laccata, Lactarius rufus, Suillus brevipes, Suillus tomentosus and Thelephora americana. Basidiospores adhered to microscope slides placed on three 60 cm transects radiating from sporocarps. Morphological characteristics of species as well as average basidiospore volume were recorded. Number of basidiospores was quantified at specific distances to produce actual dispersal gradients. We found a negative exponential decay model using characteristics for each species fit the field data well. The 95% modeled downwind dispersal distance of basidiospores was calculated for each species. The 95% modeled downwind dispersal distance increased with increasing cap height and decreasing basidiospore volume for the species sampled, with 95% of basidiospores predicted to fall within 58 cm of the cap. Differences in anatomical characteristics of EMF species influence how far basidiospores are dispersed by wind. We discuss the role of wind dispersal leading to patterns of EMF establishment during primary succession.     

Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase.