Zur Physiologie und Cytologie der Fettbildung bei Endomyces vernalis

Ohne ZusammenfassungD 93.—Als vorläufige Mitteilung erschien: M. Steiner, Ernährung und Fettbildung bei Endomyces vernalis. Ber. d. Deutsch. bot. Ges. 56, 73, 1938.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.     

Structure and mechanism of the magnesium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway

CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel-Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg²⁺, which, by contrast, is a strict requirement for the majority of prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro. Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.     

A simple equation for describing the temperature dependent growth of free-floating macrophytes

Temperature is one of the most important factors determining growth rates of free-floating macrophytes in the field. To analyse and predict temperature dependent growth rates of these pleustophytes, modelling may play an important role. Several equations have been published for describing temperature responses of macrophytes and algae. But they are often complex or are only applicable in a limited range of temperatures. In this paper, we present a simple three-parameter equation for describing the temperature dependent growth rates of pleustophytes. The equation that we developed is tested using results from laboratory growth experiments conducted with three different species of pleustophytes (Lemna minor, Salvinia molesta and Azolla filiculoides). The equation is simple and demonstrates reliable fits (adjusted R² reaching from 0.89 to 0.95). Additionally, our equation primarily uses parameters of biological significance, resulting in estimates of useful cardinal temperatures (minimum and maximum).     

The function of neurofascin155 in oligodendrocytes is regulated by metalloprotease-mediated cleavage and ectodomain shedding

Formation of the paranodal axo-glial junction requires the oligodendrocyte-specific 155-kDa isoform of neurofascin (NF155). Here, we report the presence of two peptides in cultured oligodendrocytes, which are recognized by distinct NF155-specific antibodies and correspond to a membrane anchor of 30 kDa and a 125 kDa peptide, which is shed from the cells, indicating that it consists of the NF155 ectodomain. Transfection of OLN-93 cells with NF155 verified that both peptides originate from NF155 cleavage, and we present evidence that metalloproteases mediate NF155 processing. Interestingly, metalloprotease activity is required for NF155 transport into oligodendrocyte processes supporting the functional significance of NF155 cleavage. To further characterize NF155 cleavage and function, we transfected MDCK cells with NF155. Although ectodomain shedding was observed in polarized and non-polarized MDCK cells, surface localization of NF155 was restricted to the lateral membrane of polarized cells consistent with a role in cell-cell adhesion. Aggregation assays performed with OLN-93 cells confirmed that NF155 accelerates cell-cell adhesion in a metalloprotease-dependent manner. The physiological relevance of NF155 processing is corroborated by the presence of NF155 cleavage products in heavy myelin, suggesting a role of NF155 ectodomain shedding for the generation and/or stabilization of the nodal/paranodal architecture.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.     

IRES-dependent translation of egr2 is induced under inflammatory conditions

Adjusting translation is crucial for cells to rapidly adapt to changing conditions. While pro-proliferative signaling via the PI3K-mTOR-pathway is known to induce cap-dependent translation, stress conditions, such as nutrient deprivation or hypoxia often activate alternative modes of translation, e.g., via internal ribosome entry sites (IRESs). As the effects of inflammatory conditions on translation are only poorly characterized, we aimed at identifying translationally deregulated targets in inflammatory settings. For this purpose, we cocultured breast tumor cells with conditioned medium of activated monocyte-derived macrophages (CM). Polysome profiling and microarray analysis identified early growth response-2 (egr2) to be regulated at the level of translation. Using bicistronic reporter assays, we found that egr2 contains an IRES within its 5′ UTR, which facilitated enhanced translation upon CM treatment. We further provide evidence that the activity of egr2-IRES was induced by IL-1β and p38-MAPK signaling. In addition, we identified several potential IRES trans-acting factors (ITAFs) such as polypyrimidine tract binding protein (PTB) and hnRNP-A1 that directly bind to the egr2-5′UTR. In summary, our data provide evidence that egr2 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment.