Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non-toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an alpha-motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone-rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m-Carboxycinnamic acid bis-Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS-275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood-brain barrier, oral administration may allow deceleration of progressive alpha-motoneurone degeneration by epigenetic SMN2 gene activation.     

Mycobacterial infections in free-living cervids in Germany (2002-2006)

We examined 1,022 free-living roe deer, red deer, and fallow deer for mycobacteria in Germany, 2002-2006. Retropharyngeal lymph nodes and other tissues were processed for culture and isolates were identified with the use of polymerase chain reaction and DNA sequencing. Mycobacteria were found in 18.3% of deer, with Mycobacterium avium in 14.8%. Other atypical mycobacteria were detected in 5.3%. Members of the Mycobacterium tuberculosis complex were not detected.     

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.     

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit ³H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A₁, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A₁ pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

The Escherichia coli SRP Receptor Forms a Homodimer at the Membrane

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Increased Soil Frost Versus Summer Drought as Drivers of Plant Biomass Responses to Reduced Precipitation: Results from a Globally Coordinated Field Experiment

Reduced precipitation treatments often are used in field experiments to explore the effects of drought on plant productivity and species composition. However, in seasonally snow-covered regions reduced precipitation also reduces snow cover, which can increase soil frost depth, decrease minimum soil temperatures and increase soil freeze–thaw cycles. Therefore, in addition to the effects of reduced precipitation on plants via drought, freezing damage to overwintering plant tissues at or below the soil surface could further affect plant productivity and relative species abundances during the growing season. We examined the effects of both reduced rainfall (via rain-out shelters) and reduced snow cover (via snow removal) at 13 sites globally (primarily grasslands) within the framework of the International Drought Experiment, a coordinated distributed experiment. Plant cover was estimated at the species level, and aboveground biomass was quantified at the functional group level. Among sites, we observed a negative correlation between the snow removal effect on minimum soil temperature and plant biomass production the next growing season. Three sites exhibited significant rain-out shelter effects on plant productivity, but there was no correlation among sites between the rain-out shelter effect on minimum soil moisture and plant biomass. There was no interaction between snow removal and rain-out shelters for plant biomass, although these two factors only exhibited significant effects simultaneously for a single site. Overall, our results reveal that reduced snowfall, when it decreases minimum soil temperatures, can be an important component of the total effect of reduced precipitation on plant productivity.