Tracking Protein S‐Fatty Acylation with Proteomics

Recently, Hang and co‐workers developed “acyl‐PEG exchange” (APE), which allows the investigation of protein S‐fatty acylation with mass‐tag labelling and gel electrophoresis, methods that are accessible to any biochemistry laboratory.     

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post‐translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C‐terminal “follower” sequence, rather than the canonical N‐terminal “leader” sequence. We report herein the structural and biochemical characterization of BotP, a leucyl‐aminopeptidase‐like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N‐terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn²⁺ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro‐ mycin.     

Shock Consolidation of Aluminium Powder with SiC fiber reinforcement

For the explosive compaction of Al 2024 alloy powder the influence of E/M ratio and detonation velocity of the explosive as well as the influences of powder microhardness and initial density are discussed. With a choice of parameters that gives a fully compacted aluminium sample the results of embedding single SiC fibers are described and a procedure to avoid fiber cracking due to shock loads is derived. First results for the compaction of samples with 38% fiber content are presented.     

Measurements of the Refractive Index Increment on Hydroxyethyl Starch as a Basis for Absolute Molecular Weight Determinations

Solutions of hydroxyethyl starch are used as a blood plasma substitute. If their physiological efficiency is to be optimized, they need to be accurately characterized in terms of their molecular weight and its distribution. The absolute determination of molecular weight and molecular weight distribution by means of light scattering require a knowledge of the refractive index increment. Although numerous investigations of the refractive index increment of hydroxyethyl starch have already been published, the results vary significantly due to the use of different samples and the choice of different measuring parameters. There was therefore an urgent need to examine the extent to which the refractive index increment depends on molecular parameters, the experimental method used and the type of processing. Here it was found that different sample preparations result in different contents of solid matter, so that an exact determination of the quantity is required. Hydroxyethyl starches in the molecular weight range of about Mw = 200.000 g/mol and varying degrees of substitution between DS 0.38 and 0.50 which are regarded as optimal for clinical use give a refractive index increment of dn/dc^633nm = 0.146 = 0.005 cm³/g (solvent: H₂O/0.02% NaN₃; T = 25°C).     

119Sn-NMR spektroskopische Untersuchungen an Tributylzinnderivaten aromatischer Sulfonsäuren

¹¹⁹Sn-NMR Spectroscopic Investigations of Tributyltin Derivatives of Aromatic Sulfonic Acids . Tributyltin derivatives of nine aromatic sulfonic acids were synthesized and investigated by means of ¹¹⁹Sn-n.m.r. spectroscopy. ¹¹⁹Sn-chemical shifts and ¹J (Sn, C) coupling constants in dependence on temperature and concentration were applied to study the association processes of the substances in solution. In CDCl₃ as a solvent a monomeric/polymeric equilibrium of the substances was demonstrated, while CD₃OD as a donor solvent forms five-coordinated complexes with the tin compounds.     

Graphenated Ceramic Particles as Functional Fillers for Nonisocyanate Polyhydroxyurethane Composites

Graphenation of corundum and silicon carbide filler particles simultaneously improves mechanical properties and electrical conductivity of nonisocyanate polyhydroxyurethanes (NIPU) composites prepared by amine cure of polyfunctional cyclic carbonates. Typically, the ceramic fillers coated with either glucose, polydopamine, or graphite oxide (GO) are thermolyzed to produce an ultrathin graphene shell around the ceramic core, as verified by transmission electron microscopy. As compared to a blend of corundum particles with the thermally reduced graphite oxide (TRGO) nanofiller, graphenation of corundum with GO at a similar total carbon content significantly improves the Young’s modulus (7000 MPa, +184%) of trimethylolpropane glycidylether carbonate (TMPGC) cured with diethylenetriamine (DETA). Moreover, up to 30 wt% of the graphenated corundum filler is uniformly dispersed, whereas a few percent of neat TRGO account for intolerable high viscosity. Furthermore, NIPU composites containing graphenated ceramic fillers exhibit electrical conductivities of up 2.58 × 10^?5 S m^?1 well below the percolation threshold of neat TRGO in the same NIPU matrix. Hence, the graphenation of inorganic particles represents a facile and universal synthetic route toward tailoring functional fillers and combines the two worlds of functionalized graphene and inorganic fillers in an economic way by eliminating the tedious syntheses and handling typical for graphene nanofillers.     

A microarray for monitoring the production of mycotoxins in food

A microarray, which covers most of the known relevant mycotoxin biosynthesis genes, has been developed. The microarray carries oligonucleotides of the fumonisin, the aflatoxin, the ochratoxin, the trichothecene (type A and B) and the patulin biosynthesis pathways. For trichothecene producing Fusaria the biosynthesis cluster of trichothecene producing Fusarium sporotrichioides (type A) and of Gibberrella zeae (type B, teleomorph of F. graminearum) have been spotted. The aflatoxin cluster carries oligonucleotides specific for Aspergillus flavus. The ochratoxin pattern is specific for ochratoxin A producing Penicillia, the fumonisin cluster is specific for G. moniliformis (teleomorph of F. verticillioides) and the patulin genes have been obtained from Penicillium expansum. The microarray is designed in a way that newly identified pathway genes can be added easily at any time. The microarray was used to detect the activation of all gene clusters under conditions conducive for mycotoxin biosynthesis. According to the results the obtained signals were specific under the hybridization conditions used and only insignificant cross-hybridizations occurred. The microarray was used to demonstrate differences in mycotoxin pathway gene expressions after growth on various media for trichothecene and ochratoxin A biosynthesis. It was used further to study and compare the expression kinetics of the trichothecene biosynthesis genes of Fusarium on different trichothecene supporting media. An expression pattern indicative for trichothecene biosynthesis could be identified.     

Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides

Structured triacylglycerides with medium-chain fatty acids (caprylic acid) in sn1- and sn3-positions and a long-chain unsaturated fatty acid (oleic or linoleic acid) in the sn2-position of glycerol (MLM) were synthesized by lipase catalysis in a two-step process. First, pure 2-monoacylglycerides (2-MG) were synthesized by alcoholysis of triacylglycerides (triolein, trilinolein, or peanut oil) in organic solvents with 1,3-regiospecific lipases (from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus). The 2-MG were purified by crystallization and obtained in up to 71.8% yield. These 2-MG were esterified in a second reaction with caprylic acid in n-hexane to form almost pure MLM. For 2-MG obtained from peanut oil, the final product contained more than 90% caprylic acid in the sn1- and sn3-positions, whereas the sn2-position was composed of 98.5% unsaturated long-chain fatty acids. Reaction conditions for both steps were optimized with respect to source and immobilization of lipase, water activity, and solvent.     

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[³H]glycerol/diacyl[³H]glycerol ratio was observed. The distribution of [1-¹⁴C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-¹⁴C]EPA was oxidized to a much greater extent than [1-¹⁴C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.