In Vivo Interaction Proteomics Reveal a Novel p38 Mitogen-Activated Protein Kinase/Rack1 Pathway Regulating Proteostasis in Drosophila Muscle

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.     

Comparison of point counts and territory mapping for detecting effects of forest management on songbirds

Point counts are commonly used to assess changes in bird abundance, including analytical approaches such as distance sampling that estimate density. Point‐count methods have come under increasing scrutiny because effects of detection probability and field error are difficult to quantify. For seven forest songbirds, we compared fixed‐radii counts (50 m and 100 m) and density estimates obtained from distance sampling to known numbers of birds determined by territory mapping. We applied point‐count analytic approaches to a typical forest management question and compared results to those obtained by territory mapping. We used a before–after control impact (BACI) analysis with a data set collected across seven study areas in the central Appalachians from 2006 to 2010. Using a 50‐m fixed radius, variance in error was at least 1.5 times that of the other methods, whereas a 100‐m fixed radius underestimated actual density by >3 territories per 10 ha for the most abundant species. Distance sampling improved accuracy and precision compared to fixed‐radius counts, although estimates were affected by birds counted outside 10‐ha units. In the BACI analysis, territory mapping detected an overall treatment effect for five of the seven species, and effects were generally consistent each year. In contrast, all point‐count methods failed to detect two treatment effects due to variance and error in annual estimates. Overall, our results highlight the need for adequate sample sizes to reduce variance, and skilled observers to reduce the level of error in point‐count data. Ultimately, the advantages and disadvantages of different survey methods should be considered in the context of overall study design and objectives, allowing for trade‐offs among effort, accuracy, and power to detect treatment effects.     

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

Complementary strand analysis: a new approach for allelic separation in complex polyallelic genetic systems.

We describe a method, complementary strand analysis (CSA), for separating alleles potentially from any heterozygous genetic locus. Locus specific PCR is performed generating two allelic products. The antisense strands are isolated and hybridised with a sense reference strand to form a chimeric DNA duplex for each allele which is then separated by non-denaturing PAGE. We demonstrate the application of CSA for separation of highly polymorphic HLA-A, -B and -Cw alleles and characterisation of HLA identity in related bone marrow donors and patients. CSA is capable of resolving one nucleotide differences in a DNA fragment nearly as large as a kilobase in length.     

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate

Environmental arsenic poisoning affects roughly 200 million people worldwide. The toxicity and mobility of arsenic in the environment is significantly influenced by microbial redox reactions, with arsenite (As^III) being more toxic than arsenate (As^V). Microbial oxidation of As^III to As^V is known to be regulated by the AioXSR signal transduction system and viewed to function for detoxification or energy generation. Here, we show that As^III oxidation is ultimately regulated by the phosphate starvation response (PSR), requiring the sensor kinase PhoR for expression of the As^III oxidase structural genes aioBA. The PhoRB and AioSR signal transduction systems are capable of transphosphorylation cross‐talk, closely integrating As^III oxidation with the PSR. Further, under PSR conditions, As^V significantly extends bacterial growth and accumulates in the lipid fraction to the apparent exclusion of phosphorus. This could spare phosphorus for nucleic acid synthesis or triphosphate metabolism wherein unstable arsenic esters are not tolerated, thereby enhancing cell survival potential. We conclude that As^III oxidation is logically part of the bacterial PSR, enabling the synthesis of the phosphate analog As^V to replace phosphorus in specific biomolecules or to synthesize other molecules capable of a similar function, although not for total replacement of cellular phosphate.     

P Metabolism in the Bean-Rhizobium tropici Symbiosis

Nodulated legumes require more P than legumes growing on mineral nitrogen, but little is known about the basis for the higher P requirement. Experiments were conducted to determine how Rhizobium tropici responds to P limitation and to understand how P is partitioned between the symbionts under conditions of adequate or limiting P. Free-living R. tropici responds to P stress by increasing P transport capacity and inducing both an acid and an alkaline phosphatase. This P-stress response occurs when the medium P concentration decreases below 1 [mu]M. Both P-stress-inducible phosphatases are found in bacteroids taken from plants growing with adequate P, suggesting that P levels in the symbiosome space is low enough to induce the expression of these enzymes. Bacteroid alkaline phosphatase-specific activity was highest during vegetative growth of the bean plant, but decreased approximately 75% during the host reproductive stages. In hydroponic experiments 32P-tracer studies showed that in vivo rates of P accumulation were significantly higher in bacteroids from P-limited plants compared with those from plants that had been supplied with adequate P. In contrast, label accumulation in leaves was greatest in plants grown with adequate P.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.