First synthesis of a pentadeuterated 3'-hydroxystanozolol--an internal standard in doping analysis

The first synthesis of 16,16,20,20,20-pentadeuterio-3'-hydroxystanozolol (8) in 26% yield over nine steps is described using moderately priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC-MS or gas chromatography-mass spectrometry (GC-MS).     

Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software

Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community. One major objective is the search for potential biomarkers in complex body fluids like serum, plasma, urine, saliva, or cerebral spinal fluid. For this purpose, efficient visualization of large data sets derived from patient cohorts is crucial to provide clinical experts an interactive impression of the data quality. Additionally, it is necessary to apply statistical analysis and pattern matching algorithms to attain validated signal patterns that may allow for later applications in sample classification. We introduce the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics. ClinProTools is the data interpretation software of the mass spectrometry-based ClinProt solutions for biomarker analysis. ClinProTools performs data pretreatment, visualization, statistics, pattern determination, pattern evaluation, and classification of spectra. This article will focus on ClinProTool's powerful and intuitive visualization options for clinical proteomics applications.     

Inferring the effects of demography and selection on Drosophila melanogaster populations from a chromosome-wide scan of DNA variation

Identifying regions of the Drosophila melanogaster genome that have been recent targets of positive Darwinian selection will provide evidence for adaptations that have helped this species to colonize temperate habitats. We have begun a search for such genomic regions by analyzing multiple loci (about 250) dispersed across the X chromosome in a putatively ancestral population from East Africa and a derived European population. For both populations we found evidence for past changes in population size. We estimated that a major bottleneck associated with the colonization of Europe occurred about 3,500-16,000 years ago. We also found that while this bottleneck can account for most of the reduction in variation observed in the European sample, there is a deficit of polymorphism in some genomic regions that cannot be explained by demography alone.     

Treatment of chronic endometritis in dairy cows with an intrauterine application of enzymes. A field trial

The use of proteolytic enzymes has been established in the non-antibiotic treatment of mastitis in dairy cattle. The objective of this study was to evaluate, if enzymes are efficacious in the treatment of chronic endometritis. In a controlled field trial, cows with vaginal discharge 21-27 days in milk (DIM) were randomly assigned to two treatment groups. Endometritis was classified into three categories, depending on the type of vaginal discharge: clear mucus with flakes of pus (E1), mucopurulent discharge or fluctuating contents in the uterus (E2), and purulent discharge (E3). In group ENZYMES (n=191), cows received an intrauterine treatment with a salve containing the enzymes trypsin (16 mg), chymotrypsin (16 mg), and papain (8 mg). Cows in group PGF (n=225) were treated with 0.5mg of cloprostenol. Cows that did not show any clinical signs of chronic endometritis were regarded as healthy control group (HC, n=699). In groups ENZYMES and PGF, all cows were re-examined 35-41 DIM. In group ENZYMES, cows were re-treated with enzymes if signs of endometritis were found, while in group PGF all cows received a second dose of cloprostenol, regardless of their clinical findings. Cure rate after the first treatment, defined as the absence of vaginal discharge at the re-examinations, was 59.7 and 68.0% in groups ENZYMES and PGF, respectively (P>0.05). Reproductive performance measures showed no significant differences between the two treatment groups. Service rate was significantly lower for ENZYMES and PGF, respectively, compared to HC. Conception rates to all services and percentages of cows pregnant by 250 DIM were significantly lower in group ENZYMES compared to HC, while no further differences were found between PGF and HC. In both treatment groups, cure rate and reproductive performance measures were better for cows categorized E1 or E2, than for cows categorized E3, respectively. Conception rate to all services for cows with endometritis category E1 was higher in group PGF than in group ENZYMES (P<0.05). The results of this field trial suggest that prostaglandin F(2alpha) is still the treatment of choice for chronic endometritis in dairy cattle.     

Heme-dependent rubber oxygenase RoxA of Xanthomonas sp. cleaves the carbon backbone of poly(cis-1,4-Isoprene) by a dioxygenase mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of (16)O(2), (18)O(2), H(2)(16)O, and H(2)(18)O. 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of (18)O-compounds. Incorporation of one (18)O atom in ODTD was found if the cleavage reaction was performed in the presence of (18)O(2) and H(2)(16)O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H(2)(18)O in the presence of (16)O(2) indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of (18)O(2), H(2)(16)O, and alcohol dehydrogenase/NADH, incorporation of two atoms of (18)O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.