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.     

Parallel evolution of histophagy in ciliates of the genus Tetrahymena

Background  

Current evidence suggests that lepidopteran baculoviruses may be divided into two phylogenetic groups based on their envelope fusion proteins. One group utilizes gp64, a low pH-dependent envelope fusion protein, whereas the other employs a protein family (e.g. LD130 in the Lymantria dispar nucleopolyhedrovirus) unrelated to gp64, but that is also low pH-dependent. Database searches with members of the LD130 protein family often record significant levels of homology to envelope proteins from a number of insect retrovirus-like transposable elements of the gypsy class. In this report, the significance of the homology between these two types of envelope proteins is analyzed.     

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.     

Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.     

4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

Background  

The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei.     

Isoflavonoids in the Rutaceae family: 1. Fortunella obovata, Murraya paniculata and four Citrus species

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.     

An efficient protein complex purification method for functional proteomics in higher eukaryotes

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.