Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.     

The identification of 1,3-oxazolidine-2-thiones and 1,3-thiazolidine-2-thiones from the reaction of glucose with benzyl isothiocyanate

The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid-phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3-trihydroxypropyl)-1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b]oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione. The identity of the last compound was secured by X-ray crystal structure data.     

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.     

Physiological role of macrophage inflammatory protein-3 alpha induction during maturation of intestinal macrophages

Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3alpha, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3alpha protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3alpha. FACS of purified IMAC clearly indicated expression of MIP-3alpha in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3alpha protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3alpha during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3alpha is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3alpha expression could be involved in the recruitment of CD45R0+ cells into the lamina propria.     

Identification of the tRNA-binding protein Arc1p as a novel target of in vivo biotinylation in Saccharomyces cerevisiae

Biotin is an essential cofactor of cell metabolism serving as a protein-bound coenzyme in ATP-dependent carboxylation, in transcarboxylation, and certain decarboxylation reactions. The involvement of biotinylated proteins in other cellular functions has been suggested occasionally, but available data on this are limited. In the present study, a Saccharomyces cerevisiae protein was identified that reacts with streptavidin on Western blots and is not identical to one of the known biotinylated yeast proteins. After affinity purification on monomeric avidin, the biotinylated protein was identified as Arc1p. Using 14C-labeled biotin, the cofactor was shown to be incorporated into Arc1p by covalent and alkali-stable linkage. Similar to the known carboxylases, Arc1p biotinylation is mediated by the yeast biotin:protein ligase, Bpl1p. Mutational studies revealed that biotinylation occurs at lysine 86 within the N-terminal domain of Arc1p. In contrast to the known carboxylases, however, in vitro biotinylation of Arc1p is incomplete and increases with BPL1 overexpression. In accordance to this fact, Arc1p lacks the canonical consensus sequence of known biotin binding domains, and the bacterial biotin:protein ligase, BirA, is unable to use Arc1p as a substrate. Arc1p was shown previously to organize the association of MetRS and GluRS tRNA synthetases with their cognate tRNAs thereby increasing the substrate affinity and catalytic efficiency of these enzymes. Remarkably, not only biotinylated but also the biotin-free Arc1p obtained by replacement of lysine 86 with arginine were capable of restoring Arc1p function in both arc1Delta and arc1Deltalos1Delta mutants, indicating that biotinylation of Arc1p is not essential for activity.     

ImmunoTrack: the novel antibody-based technology for tracing in animal health

This paper describes a novel antibody-based livestock movement control tool and method of meat allocation, both in livestock husbandry as well as during the meat-processing chain. Immuno Track fulfills diverse prerequisites and meets regulatory demands which are substantial for a successful monitoring technology: (i) the induction of long-lasting antibody responses detectable onsite throughout the whole mast period of pigs, (ii) a single immunization injection with protein derivatives is sufficient to evoke a strong epitope-specific antibody response, and (iii) the complete degradation of the protein markers after the antibody response has been triggered in meatproducing animals such as cattle or pigs. There are diverse fields of application for the Immuno-Track marker technology, such as in quality meat programs, as compliance markers for animal vaccines or as a tool for verification of origin. Combination of this monitoring technology with the husbandry and identification databases for cattle and pigs within the European Community will lead to greater transparency in meat production, thereby regaining consumers' trust in concomitant structures of the meat-producing industry.     

Mitotic activation of the kinase Aurora-A requires its binding partner Bora

The protein kinase Aurora-A is required for centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis. Here, we describe the identification of Bora, a conserved protein that is required for the activation of Aurora-A at the onset of mitosis. In the Drosophila peripheral nervous system, bora mutants have defects during asymmetric cell division identical to those observed in aurora-A. Furthermore, overexpression of bora can rescue defects caused by mutations in aurora-A. Bora is conserved in vertebrates, and both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro. In interphase cells, Bora is a nuclear protein, but upon entry into mitosis, Bora is excluded from the nucleus and translocates into the cytoplasm in a Cdc2-dependent manner. We propose a model in which activation of Cdc2 initiates the release of Bora into the cytoplasm where it can bind and activate Aurora-A.     

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Strategies for the conservation of endangered freshwater pearl mussels (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.): a synthesis of Conservation Genetics and Ecology

Freshwater pearl mussels (Margartifera margaritifera L.) are among the most critically threatened freshwater bivalves worldwide. The pearl mussel simultaneously fulfils criteria of indicator, flagship, keystone and umbrella species and can thus be considered an ideal target species for the process conservation of aquatic ecosystem functioning. The development of conservation strategies for freshwater pearl mussels and for other bivalve species faces many challenges, including the selection of priority populations for conservation and strategic decisions on habitat restoration and/or captive breeding. This article summarises the current information about the species’ systematics and phylogeny, its distribution and status as well as about its life history strategy and genetic population structure. Based on this information, integrative conservation strategies for freshwater mollusc species which combine genetic and ecological information are discussed. Holistic conservation strategies for pearl mussels require the integration of Conservation Genetics and Conservation Ecology actions at various spatial scales, from the individual and population level to global biodiversity conservation strategies. The availability of high resolution genetic markers for the species and the knowledge of the critical stages in the life cycle, particularly of the most sensitive post-parasitic phase, are important prerequisites for conservation. Effective adaptive conservation management also requires an evaluation of previous actions and management decisions. As with other freshwater bivalves, an integrative conservation approach that identifies and sustains ecological processes and evolutionary lineages is urgently needed to protect and manage freshwater pearl mussel diversity. Such research is important for the conservation of free-living populations, as well as for artificial culturing and breeding techniques, which have recently been or which are currently being established for freshwater pearl mussels in several countries.