Making a single-chain four-helix bundle for redox chemistry studies

The construction and characteristics of the stable and well-structured alpha(4)W protein are described. The 117-residue, single-chain protein has a molecular weight of 13.1 kDa and is designed to fold into a four-helix bundle. Experimental characterization of the expressed and purified protein shows a 69.8 +/- 0.8% helical content over a 5.5-10.0 pH range. The protein is thermostable with a T(M) > 355 K and has a free energy of unfolding as measured by chemical denaturation of -4.7 kcal mol(-1) at 25 degrees C and neutral pH. One-dimensional (1D) proton and 2D (15)N-HSQC spectra show narrow, well-dispersed spectral lines consistent with a uniquely structured alpha-helical protein. Analytical ultracentrifugation and NMR data show that the protein is monomeric over a broad protein concentration range. The 324 nm emission maximum of the unique Trp-106 is consistent with a sequestered position of the aromatic residue. Additionally, differential pulse voltammetry characterization indicates an elevated peak potential for Trp-106 when the protein is folded (pH range 7.0-8.5) relative to partly unfolded (pH range 11.4-13.2). The oxidation of Trp-106 is coupled to proton release as shown by a 53 +/- 3 mV/pH unit dependence of the peak potential over the 7.0-8.5 pH range.     

Evaluation of methods for measuring particulate matter emissions from gas turbines

The project SAMPLE evaluated methods for measuring particle properties in the exhaust of aircraft engines with respect to the development of standardized operation procedures for particulate matter measurement in aviation industry. Filter-based off-line mass methods included gravimetry and chemical analysis of carbonaceous species by combustion methods. Online mass methods were based on light absorption measurement or used size distribution measurements obtained from an electrical mobility analyzer approach. Number concentrations were determined using different condensation particle counters (CPC). Total mass from filter-based methods balanced gravimetric mass within 8% error. Carbonaceous matter accounted for 70% of gravimetric mass while the remaining 30% were attributed to hydrated sulfate and noncarbonaceous organic matter fractions. Online methods were closely correlated over the entire range of emission levels studied in the tests. Elemental carbon from combustion methods and black carbon from optical methods deviated by maximum 5% with respect to mass for low to medium emission levels, whereas for high emission levels a systematic deviation between online methods and filter based methods was found which is attributed to sampling effects. CPC based instruments proved highly reproducible for number concentration measurements with a maximum interinstrument standard deviation of 7.5%.     

Laccase-mediated michael addition of 15N-sulfapyridine to a model humic constituent

Chemical incorporation of sulfonamide antimicrobials into natural organic matter may represent an important process influencing the fate of these synthetic, primarily agents in soil and sediment environments. We previously demonstrated that a fungal peroxidase mediates covalent coupling of sulfonamide antimicrobials to model humic constituents; reactions with the 2,6-dimethoxyphenol syringic acid produced Schiff bases (Bialk et al. Environ. Sci. TechnoL 2005, 39, 4436-4473). Here, we show that fungal laccase-mediated reaction of sulfapyridine with the orthodihydroxyphenol protocatechuic acid yields a Michael adduct. We synthesized 15N-enriched sulfapyridine to facilitate determination of the covalent linkage(s) formed between sulfapyridine and protocatechuic acid by NMR spectroscopy. 1H-(15)N heteronuclear multiple bond correlation experiments and tandem mass spectrometry demonstrated that the sulfapyridine anilinic nitrogen engaged in a Michael addition reaction to oxidized protocatechuic acid to form an anilinoquinone. Michael adducts are more stable than the previously reported imine linkages between sulfonamides and 2,6-dimethoxyphenols. Michael addition to quinone-like structures in soil organic matter is expected to diminish the mobility and biological activity of sulfonamide antimicrobials.     

Age-Related Shifts in Leaf Chemistry of Clonal Aspen (Populus tremuloides)

Developmental changes in plant structure and function can influence both mammalian and arthropod feeding preferences for many woody plant species. This study documents age-related changes that occur in the leaf chemistry of trembling aspen (Populus tremuloides Michx., Salicaceae) and discusses implications for the herbivore community and ecosystem processes. We collected leaves from replicate ramets from six age classes (1–25+ yr) in each of seven aspen clones growing in south central Wisconsin, USA. Chemical analyses were conducted to determine concentrations of condensed tannins, phenolic glycosides (salicortin and tremulacin), nitrogen, starch, and soluble sugars. Each variable differed significantly among clones and among age classes. On average, condensed tannin concentrations doubled in the first five years and then remained fairly constant among older age classes. Combined phenolic glycoside (salicortin + tremulacin) concentrations were high in the youngest ramets (ca. 19%) and decreased sharply with age. Developmental changes in tannin, salicortin, and tremulacin concentrations exceeded those of nitrogen and carbohydrates. Developmental shifts of this magnitude, and the age-related tradeoff that occurs between condensed tannins and phenolic glycosides, are likely to have significant influence on the herbivore community of aspen and may influence leaf litter decomposition and nutrient cycling.     

Korrosionsinhibitoren für die Instandsetzung chloridverseuchter Stahlbetonbauten

Der Einsatz von Inhibitoren wird weltweit als technisch und wirtschaftlich interessante Alternative zur konventionellen Instandsetzung von Stahlbetonbauten betrachtet, da der Betonabtrag erheblich reduziert oder sogar ganz wegfallen kann. In diesem Aufsatz werden die Resultate von praxisnahen Laboruntersuchungen vorgestellt (aktive, d. h. laufende Bewehrungskorrosion, unterschiedliche Bewehrungsüberdeckung, unterschiedlicher Chloridgehalt im Untergrund bzw. im “Altbeton”, Makroelementwirkung). Keines der drei geprüften Systeme war in der Lage, die Korrosionsgeschwindigkeit zu reduzieren oder sogar zu stoppen. Corrosion Inhibitors for the Rehabilitation of Chloride Contaminated Reinforced Concrete Structures The use of inhibitors is considered worldwide as a technically and economically interesting alternative to the conventional repair of reinforced concrete structures since the removal of concrete can be considerably reduced or completely omitted. In this contribution the results of practical laboratory investigations are presented (active, e.g. ongoing corrosion, different concrete cover depths and chloride contents of the substrate or “old concrete”, macroelement effect). None of the three tested systems was able to reduce the corrosion rate or to stop the corrosion.     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

Stability of arsenic compounds in seafood samples during processing and storage by freezing

In this study, the stability of arsenic compounds in fresh and frozen samples of raw, boiled and fried Atlantic cod (Gadhus morhua), Atlantic salmon (Salmo salar) and blue mussel (Mytilus edulis) were examined. Results show that the total arsenic concentrations of the fresh Atlantic cod and Atlantic salmon samples were not different from the frozen samples within the same seafood type. For blue mussel, the total arsenic concentration decreased significantly after storage. Inorganic arsenic was found only in blue mussels and, importantly, no significant increase of inorganic arsenic was observed after processing or after storage by freezing. The content of tetramethylarsonium ion was generally low in all samples types, but increased significantly in all fried samples of both fresh and frozen seafood. Upon storage by freezing, the arsenobetaine content was reduced significantly, but only in the samples of blue mussels.     

全球电子废物的研究(下)

(续接本刊2007年第11期第42页) 　　3中国、印度和南非的电子废物管理及评述 　　3.1发展中国家和转型期国家面临的特殊问题 　　上面已经提到了部分发展中国家和转型期国家面临的特殊问题,总结如下: 　　①尽管国内人均电子废物的产生量仍相对较小,但对于人口大国而言,如中国和印度,电子废物的绝对产生量大(Empa,2005). 　　……     

Sharing morphemes without sharing meaning: Production and comprehension of German verbs in the context of morphological relatives.

We investigated the impact of derived German verbs on the production and recognition of morphologically related simple verbs. In order to disentangle effects of morphological, semantic, and phonological relatedness, target verbs were combined (e.g., z?hlen – to count) with four context verbs: Two morphologically related context verbs that were either semantically transparent (verz?hlen – to miscount) or semantically opaque (erz?hlen – to tell), a semantically related (rechnen – to calculate) and a phonologically related (z?hmen – to tame) context verb. Morphologically related complex verbs reduced picture naming latencies as well as lexical decision latencies. Semantically related verbs did not show any reliable effects. In production, morphological facilitation was almost four times larger than phonological facilitation. In comprehension, pure form overlap produced inhibition. We argue that in German, production and comprehension processes operate on morphologically decomposed lexical form representations. Independent from semantic transparency, complex verbs are broken down into their morphemes during comprehension and are assembled during production. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Blood Cell Responses Following Heavy Alcohol Consumption Coincide with Changes in Acute Phase Reactants of Inflammation,Indices of Hemolysis and Immune Responses to Ethanol Metabolites

Aberrations in blood cells are common among heavy alcohol drinkers. In order to shed further light on such responses, we compared blood cell status with markers of hemolysis, mediators of inflammation and immune responses to ethanol metabolites in alcohol-dependent patients at the time of admission for detoxification and after abstinence. Blood cell counts, indices of hemolysis (LDH, haptoglobin, bilirubin), calprotectin (a marker of neutrophil activation), suPAR, CD163, pro- and anti-inflammatory cytokines and autoantibodies against protein adducts with acetaldehyde, the first metabolite of ethanol, were measured from alcohol-dependent patients (73 men, 26 women, mean age 43.8 ± 10.4 years) at baseline and after 8 ± 1 days of abstinence. The assessments also included information on the quantities of alcohol drinking and assays for biomarkers of alcohol consumption (CDT), liver function (AST, ALT, ALP, GGT) and acute phase reactants of inflammation. At baseline, the patients showed elevated values of CDT and biomarkers of liver status, which decreased significantly during abstinence. A significant decrease also occurred in LDH, bilirubin, CD163 and IgA and IgM antibodies against acetaldehyde adducts, whereas a significant increase was noted in blood leukocytes, platelets, MCV and suPAR levels. The changes in blood leukocytes correlated with those in serum calprotectin (p < 0.001), haptoglobin (p < 0.001), IL-6 (p < 0.02) and suPAR (p < 0.02). The changes in MCV correlated with those in LDH (p < 0.02), MCH (p < 0.01), bilirubin (p < 0.001) and anti-adduct IgG (p < 0.01). The data indicates that ethanol-induced changes in blood leukocytes are related with acute phase reactants of inflammation and release of neutrophil calprotectin. The studies also highlight the role of hemolysis and immune responses to ethanol metabolites underlying erythrocyte abnormalities in alcohol abusers.