Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.     

The temperature sensitivity of soil: microbial biodiversity,growth, and carbon mineralization

Microorganisms drive soil carbon mineralization and changes in their activity with increased temperature could feedback to climate change. Variation in microbial biodiversity and the temperature sensitivities (Q₁₀) of individual taxa may explain differences in the Q₁₀ of soil respiration, a possibility not previously examined due to methodological limitations. Here, we show phylogenetic and taxonomic variation in the Q₁₀ of growth (5–35 °C) among soil bacteria from four sites, one from each of Arctic, boreal, temperate, and tropical biomes. Differences in the temperature sensitivities of taxa and the taxonomic composition of communities determined community-assembled bacterial growth Q₁₀, which was strongly predictive of soil respiration Q₁₀ within and across biomes. Our results suggest community-assembled traits of microbial taxa may enable enhanced prediction of carbon cycling feedbacks to climate change in ecosystems across the globe.Subject terms: Biogeochemistry, Ecosystem ecology, Soil microbiology     

A proteomics approach to the study of absorption, distribution, metabolism, excretion, and toxicity.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.     

Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein

The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300–14305). We now show that CpeS1 can bind PCB, as assayed by Ni²⁺ chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower (∼1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1–PCB complex.     

Engineered bakers yeast as a sensitive bioassay indicator organism for the trichothecene toxin deoxynivalenol

The aim of this study was to increase the sensitivity of Saccharomyces cerevisiae towards trichothecene toxins, in particular to deoxynivalenol (DON), in order to improve the utility of this yeast as a bioassay indicator organism. We report the construction of a strain with inactivated genes (PDR5, PDR10, PDR15) encoding ABC transporter proteins with specificity for the trichothecene deoxynivalenol, with inactivated AYT1 (encoding a trichothecene-3-O-acetyltransferase), and inactivated UBI4 and UBP6 genes. Inactivation of the stress inducible polyubiquitin gene UBI4 or the ubiquitin protease UBP6 increased DON sensitivity, the inactivation of both genes had a synergistic effect. The resulting pdr5 pdr10 pdr15 ayt1 ubp6 ubi4 mutant strain showed 50% growth inhibition at a DON concentration of 5 mg/l under optimal conditions. The development of a simple two step assay for microbial DON degradation in 96 well microtiter format and its testing with the DON detoxifying bacterium BBSH 797 is reported.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.     

Comparative evaluation of three molecular typing methods in their applicability to differentiate Lactobacillus strains with human origin

We isolated a total of 49 strains of lactic acid bacteria from the faeces of healthy donors. The species in that group were determined as L. plantarum (11 strains), L. casei (11 strains), L. rhamnosus (seven strains), L. fermentum (seven strains), L. gasseri (six strains), L. delbrueckii ssp. lactis (four strains), L. salivarius (two strains), and L. acidophilus (one strain). Genotyping at strain level was performed using random amplification of polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE) with endonucleases ApaI and XhoI and amplified fragment length polymorphism (AFLP) with enzymes XhoI and TaqI. The main objective was the comparison of three molecular typing techniques: AFLP, PFGE and RAPD in their applicability to determine the genetic diversity among the isolates. RAPD was the easiest, comparatively rapid and fairly strain discriminative tool. PFGE was the most laborious method but producing the most stable profiles with satisfactory discriminatory power. AFLP proved to be the most discriminative approach for typing of the strains. AFLP could differentiate strains with the same PFGE profiles. Therefore, AFLP successfully could replace the labor consuming PFGE. The specially developed AFLP and PFGE proved very high potential to evaluate the strain diversity of Lactobacillus spp. with human origin.     

Stress generation in the tension wood of poplar is based on the lateral swelling power of the G-layer

The mechanism of active stress generation in tension wood is still not fully understood. To characterize the functional interdependency between the G-layer and the secondary cell wall, nanostructural characterization and mechanical tests were performed on native tension wood tissues of poplar (Populus nigra x Populus deltoids) and on tissues in which the G-layer was removed by an enzymatic treatment. In addition to the well-known axial orientation of the cellulose fibrils in the G-layer, it was shown that the microfibril angle of the S2-layer was very large (about 36 degrees). The removal of the G-layer resulted in an axial extension and a tangential contraction of the tissues. The tensile stress-strain curves of native tension wood slices showed a jagged appearance after yield that could not be seen in the enzyme-treated samples. The behaviour of the native tissue was modelled by assuming that cells deform elastically up to a critical strain at which the G-layer slips, causing a drop in stress. The results suggest that tensile stresses in poplar are generated in the living plant by a lateral swelling of the G-layer which forces the surrounding secondary cell wall to contract in the axial direction.