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.     

Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally

Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.     

Oxytocin alleviates the neuroendocrine and cytokine response to bacterial endotoxin in healthy men

Oxytocin is a hormone and neurotransmitter found to have anti-inflammatory functions in rodents. Here we used experimental bacterial endotoxinemia to examine the role of exogenous oxytocin administration on innate immune responses in humans. Ten healthy men received, in a randomized, placebo-controlled, crossover design, placebo, oxytocin, LPS, and LPS + oxytocin. Oxytocin treatment resulted in a transient or prolonged reduction of endotoxin-induced increases in plasma ACTH, cortisol, procalcitonin, TNF-alpha, IL-1 receptor antagonist, IL-4, IL-6, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1 (MCP-1), interferon-inducible protein 10, and VEGF. In vitro, oxytocin had no impact on LPS effects in releasing TNF-alpha, IL-6, and MCP-1 in monocytes and peripheral blood mononuclear cells from healthy human donors. In summary, oxytocin decreases the neuroendocrine and cytokine activation caused by bacterial endotoxin in men, possibly due to the pharmacological modulation of the cholinergic anti-inflammatory pathway. Oxytocin might be a candidate for the therapy of inflammatory diseases and conditions associated with high cytokine and VEGF levels.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.