Engineering a heavy atom derivative for the X-ray structure analysis of cyclodextrin glycosyltransferase

Based on a preliminary structural model of cyclodextrin glycosyltransferasefrom Bacillus circulans (EC 2.4.1.19 [EC] ), Ser428 and Ser475 ofthe enzyme were mutated to cysteines in order to produce suitableheavy atom derivatives. Mutant Ser475 - Cys could not be expressedas protein. Mutant Ser428 - Cys was expressed in Escherichiacoli and purified. It crystallized isomorphously and gave riseto a mercury derivative that improved the electron density map.The structural results show that the new mercury-binding siteis in a pocket at the protein surface.     

Highly efficient enzyme-functionalized porous zirconia microtubes for bacteria filtration

In contrast to polymer membranes, ceramic membranes offer considerable advantages for safe drinking water provision due to their excellent chemical, thermal, and mechanical endurance. In this study, porous ceramic microtubes made of yttria stabilized zirconia (YSZ) are presented, which are conditioned for bacteria filtration by immobilizing lysozyme as an antibacterial enzyme. In accordance with determined membrane pore sizes of the nonfunctionalized microtube of ≤200 nm, log reduction values (LRV) of nearly 3 (i.e., bacterial retention of 99.9%) were obtained for bacterial retention studies using gram-positive model bacterium Micrococcus luteus. Immobilization studies of lysozyme on the membrane surface reveal an up to six times higher lysozyme loading for the covalent immobilization route as compared to unspecific immobilization. Antibacterial activity of lysozyme-functionalized microtubes was assessed by qualitative agar plate test using Micrococcus luteus as substrate showing that both the unspecific and the covalent lysozyme immobilization enhance the microtubes' antibacterial properties. Quantification of the enzyme activity at flow conditions by photometric assays reveals that the enzyme activities of lysozyme-functionalized microtubes depend strongly on applied flow rates. Intracapillary feeding of bacteria solution and higher flow rates lead to reduced enzyme activities. In consideration of different applied flow rates in the range of 0.2-0.5 mL/min, the total lysozyme activity increases by a factor of 2 for the covalent immobilization route as compared to the unspecific binding. Lysozyme leaching experiments at flow conditions for 1 h show a significant higher amount of washed-out lysozyme (factor 1.7-3.4) for the unspecific immobilization route when compared to the covalent route where the initial level of antibacterial effectiveness could be achieved by reimmobilization with lysozyme. The presented platform is highly promising for sustainable bacteria filtration.     

Reductive silylation and alkylation of phthalimides

Preparative electrochemical reduction of N-methylphthalimide in dry acetonitrile and in the presence of dimethylsulfate or trimethylchlorosilane lead to bis-lactams by dimerization at the carbonyl group. 1, 3-Bis-trimethylsilyloxy-2-methylisoindole is formed instead using a large excess of silylating agent. An analogous isoindole derivative is obtained from reduction of N-phenylphthalimide. A mechanism accounting for the formation of one or two electron products under different alkylating conditions is discussed.     

Quantifizierende calciumspezies-analytik im wein zur frage der calciumstabilit?t

Zusammenfassung Die Calciumspezies-Stabilität als dekadischer Logarithmus der Komplex-Stabilitätskonstanten nach dem Massenwirkungsgesetz in Wein wird mit Hilfe der Direktpotentiometrie mit Ca-selektiver Elektrode (für die Calciumionenkonzentration) sowie der Photometrie (Ca-Gesamtgehalt) in 16 lager-stabilen Weinen (Ca-Gehalte zwischen 60 und 120 mg/l) mit 3,50±0,20 (Bereich 3,11–3,79) ermittelt. Durch Methodenvergleiche (Photometrie und AAS für Gesamtgehalte sowie Direktpotentiometrie und Ionenaustauschmethode für die Ionenkonzentration) wurden diese Ergebnisse abgesichert. Die Anwendung von Weinbehandlungsverfahren wie der Adsorption phenolischer Stoffe an Polyvinylpolypyrrolidin (PVPP) sowie der Ultrafiltration zur Abtrennung höhermolekularer Eiweißstoffe zeigte keine wesentlichen Veränderungen der Elementspezies-Stabilitäten. Bei einer Entsäuerung mit CaCO₃ oder nach der Doppelsalz-Entsäuerung wurde dagegen nicht nur ein Anstieg der Calciumgesamtgehalte auf 200 bis 535 mg/1 sondern auch eine deutliche Abnahme der Calciumspezies-Stabilität auf Werte von 2,2 bis 2,8 festgestellt. die nach dem Auskristallisieren von Calciumverbindungen nach einigen Wochen wieder Werte um 3 erreicht. Die Lagerstabilität eines Weines im Hinblick auf Calcium kann somit nach der Bestimmung der Calciumspezies-Stabilität beurteilt werden.

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Quantifying analysis of calcium species in wine from the point of view of calcium stability

Summary The stability of calcium species, expressed as the decadic logarithm of the complex stability constant after the chemical equilibrium had been established, was determined to be 3.50 ± 0.20 (range 3.11–3.79) by direct potentiometry with a calcium-selective electrode (for the calcium ion concentration) as well as photometry (total Ca content) in 16 storage-stable wines (Ca contents between 60 and 120 mg/1). By comparing the methods of photometry, AAS (for the total content) and direct potentiometry as well as ion-exchange method for ion concentrations, the results were verified. The treatment of the wine by the adsorption of phenolic substances on polyvinylpolypyrrolidine (PVPP) and by ultrafiltration for the separation of higher molecular proteins showed no significant changes in the stabilities of the calcium species. After deacidification with CaCO₃ or after salt deacidification had been performed twice, not only an increase in the total calcium content occured (200–535 mg/1), but there was also an evident decrease in the stabilities of the calcium species, which had values of 2.2–2.8 and reached values of 3 after crystallization of the calcium species. The storage stability of a wine, with respect to calcium, can be estimated after the determination of the stability of the calcium species.

     

DGAT1 polymorphism in Bos indicus and Bos taurus cattle breeds

As a result of multiple QTL-mapping projects in recent years, a quantitative trait locus for milk fat percentage and milk yield has been described on BTA14. Recent reports name the acyl-CoA : diacylglycerol acyltransferase (DGAT1) gene on BTA14 as a potential candidate gene, with a nonconservative substitution of lysine by alanine (K232A) producing a major effect on milk composition and yield. DGAT1K appears to be the ancestral allele and the K232A substitution probably occurred after the divergence of the Bos indicus and Bos taurus lineages. These findings prompted us to genotype 1748 DNA samples of 38 different Bos taurus and Bos indicus cattle breeds from 13 countries on five continents (Europe, Africa, Asia, North America and South America), to examine the occurrence of the DGAT1 polymorphism and characterize the K232A substitution in cattle breeds of different origins and selected for different purposes (e.g., beef, dairy and dual purpose). Calculating pairwise FST values for pooled subpopulations showed least divergence for Bos indicus breeds with high milk fat percentage. Fixation of DGAT1A was found in some Bos taurus breeds and fixation of DGAT1K in one Bos indicus breed. Breeds of no known organized breeding background from the Near East domestication centre of Bos taurus and taurine African N'Dama cattle were found to possess intermediate frequencies of DGAT1K. While beef breeds tended to harbour higher DGAT1A levels, dairy cattle showed everything from very low levels of DGAT1K to unexpectedly high frequencies of this allele.     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SElM, SElO, and SElN

Staphylococcus aureus remains a leading cause of food-poisoning with substantial impact on public health. Using a multiplex polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), we studied the presence of genes encoding staphylococcal enterotoxin-like (SEl) superantigens sem, sen, and seo, associated with the enterotoxin gene cluster (egc), in 429 clinical Staphylococcus aureus isolates. 294 (68.5%) isolates tested positive for at least one of the three SEl genes. In contrast to the fixed gene combination seg/sei also located on egc, a substantial number of isolates (n = 108) were found to bear only one or two of the genes encoding SElM, SElN, and SElO. Regarding the origin of the S. aureus isolates, a significant difference (P = 0.022) was found for the possession of seo (61.2% of blood isolates versus 42.9% of nasal strains). Also sem (not significantly) was found more common in blood isolates (52.1% versus 40.5%). The survey of the newly described SEl genes sem-seo supports the concept that most clinical S. aureus isolates harbor subsets of pyrogenic toxin superantigens. The potential contribution of seo and sem to the pathogenic potential of S. aureus has to be further evaluated.     

The variability of urinary cotinine levels in young children: implications for measuring ETS exposure.

This study examined the within-subject variability of urinary cotinine levels in young children (aged = 0.6-7.2 years) of smoking parents to determine the number of urine samples needed to provide accurate estimates of exposure to environmental tobacco smoke (ETS) for different time intervals. Secondary analyses were conducted of five independent studies (N = 376), in which multiple urinary cotinine measures had been collected over time periods up to 13 months. Over measurement periods of 4-15 days, the within-subject cotinine levels varied 3-5 times more than would be expected based on measurement error alone. Over 7-13 months, the within-subject variability was 10-20 times higher than would be expected based on the measurement error. Findings indicated that cotinine measures from single urine samples provided highly accurate estimates of only recent exposure (i.e., 2-3 days; rho = 0.99). To achieve similarly precise estimates of the mean cotinine level of an individual child over 4-15 days, up to nine urine samples may be necessary. Up to 12 urine samples may be required to achieve similarly precise estimates of ETS exposure over a 4- to 13-month period. Epidemiologic and clinical research on ETS exposure in children can benefit from multiple urine samples (a) to accurately measure average exposure at the level of the individual child, (b) to describe temporal patterns, (c) to detect incidences of peak exposure that would remain underrecognized if monitoring is limited to a single time point, and (d) to establish stable baseline levels and endpoints based on urine samples collected over clinically relevant time periods.     

Photo-vulcanization using thiol-ene chemistry: Film formation,morphology and network characteristics of UV crosslinked rubber latices

The photo-vulcanization with versatile thiol-ene chemistry represents an innovative approach to crosslink diene-rubber materials both in latex and in solid film state. In this work, the structure of elastomer-based thiol-ene networks and the morphology after film formation are studied in detail using electron microscopic techniques, atomic force microscopy and multiple-quantum solid-state NMR spectroscopy. Additionally, film formation properties and corresponding macroscopic properties of photo-vulcanized natural rubber (NR) latex and its synthetic counterpart, isoprene rubber (IR) latex, are determined in dependence on the curing procedure (pre- and post-vulcanization). The results reveal that thiol-ene cured elastomers comprise homogenously distributed crosslinks with a low amount of short chain defects. Whilst photochemically pre-cured NR latex particles provide coherent films, the film formation and mechanical properties of IR are strongly governed by the crosslink density of the latex particles. In film state, photo-vulcanization promotes narrow crosslink distributions and excellent tensile properties of both NR and IR.