Nucleotide positions in the hypervariable V4 and V9 regions of the small subunit (SSU)-rDNA locus are normally difficult to align and are usually removed before standard phylogenetic analyses. Yet, with next-generation sequencing data, amplicons of these regions are all that are available to answer ecological and evolutionary questions that rely on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window algorithm as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European nearshore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.
The protein binding of nifedipine in concentrations up to 1200 ng ml-1 has been measured in serum, pure human albumin solution and pure human alpha 1-acid glycoprotein (AAG) solutions by ultrafiltration. The drug was extensively bound in serum from four healthy volunteers with a mean (+/- s.d.) fraction bound of 0.992 +/- 0.008. In albumin solution (40 g litre-1) the mean (+/- s.d.) fraction bound 0.970 +/- 0.012, was not significantly different (P greater than 0.05) from that in serum, suggesting that albumin is the major, but not necessarily the only, binding protein for nifedipine in serum. The binding of nifedipine in solutions of AAG was proportional to the AAG concentration and ranged from 0.514 +/- 0.059 to 0.755 +/- 0.035 in solutions containing 50 and 150 mg % AAG, respectively. Binding of nifedipine in all protein solutions was linear.
Esterase 1 F was isolated from mouse serum and purified by ion-exchange chromatography, isoelectrofocusing, and molecular sieve chromatography. It is considered to be a glycoprotein with an apparent molecular weight of 75000. The equivalent weight (= 77000 x g/mol) was estimated by titration of the catalytic site with diethyl p-nitrophenyl phosphate. The Michaelis constant K , and the catalytic constant k,,, of the enzyme for 4-nitrophenyl hexanoate were determined. Esterase I F is characterized by its ability to split a wide spectrum of substrates and its relatively low turnover rates towards the substrates tested. It belongs to the isozyme system of carboxylesterase (EC 3.1
Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10) genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8) GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 10(5) GU/mL for MS2, 5.3 × 10(3) GU/mL for ΦX174, and 1.5 × 10(2) GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.
The heredity and linkage of gene loci were established for two different enzymes with esterproteolytic activity from mouse submandibular gland: protease A and protease E. Based upon strain distribution and biochemical properties of the two esterproteases, the existence of two corresponding structural loci is proposed: Prt-4 (protease A) and Prt-5 (protease E). Prt-4 and Prt-5 proved to be different from Tam-1. From a four-point-cross, the gene order Gpi-1-(Tam-1, Prt-4, Prt-5)-c is suggested. Thus a gene cluster was shown to exist on chromosome 7 coding for esterproteases, all of which are controlled by testosterone.
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