Dynamic of apoptosis of cells in duodenal villi infected with <Emphasis Type=Italic>Eimeria acervulina</Emphasis> in broiler chickens

The aim of our study was to evaluate the parasite — host interactions at apoptosis level. We studied histopathological changes and time course of apoptosis in the duodenum during Eimeria acervulina infection. One-day-old broiler chicks were randomly allocated into two equal groups. At the age of two weeks the first group was experimentally infected with a pure suspension of sporulated E. acervulina oocysts. The second group served as a negative control. Tissue samples from the upper part of duodenum were obtained at 0.5, 1, 2, 3, 4, 5 and 6 days post infection. Biopsies of duodenum were studied immunohistochemically using DeadEnd™ Colometric TUNEL System for apoptosis detection in duodenal mucosa. Number of parasites in duodenal epithelium was also investigated. Our experimental results demonstrate: (i) macroscopic and histopathological changes in epithelium detected mainly in proximal segment of duodenum in infected groups; (ii) the number of developmental stages of E. acervulina (DSEA) during our trial increased, reaching the maximum 5 days post infection (dpi) (332.2 ± 16.12) (mean ± SEM), whereas the amount of DSEA declined significantly as late as 6 dpi (124.6 ± 3.91); (iii) the highest apoptosis level was recorded in initiatory 0.5 dpi (13.2 ± 1.02) and on the end of parasite development cycle after 5 dpi (12.6 ± 1.36). Finally, results showed that there was a period of inhibition of apoptosis during infection by E. acervulina.     

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.     

Role of proliferating cell nuclear antigen interactions in the mismatch repair-dependent processing of mitotic and meiotic recombination intermediates in yeast

The mismatch repair (MMR) system is critical not only for the repair of DNA replication errors, but also for the regulation of mitotic and meiotic recombination processes. In a manner analogous to its ability to remove replication errors, the MMR system can remove mismatches in heteroduplex recombination intermediates to generate gene conversion events. Alternatively, such mismatches can trigger an MMR-dependent antirecombination activity that blocks the completion of recombination, thereby limiting interactions between diverged sequences. In Saccharomyces cerevisiae, the MMR proteins Msh3, Msh6, and Mlh1 interact with proliferating cell nuclear antigen (PCNA), and mutations that disrupt these interactions result in a mutator phenotype. In addition, some mutations in the PCNA-encoding POL30 gene increase mutation rates in an MMR-dependent manner. In the current study, pol30, mlh1, and msh6 mutants were used to examine whether MMR-PCNA interactions are similarly important during mitotic and meiotic recombination. We find that MMR-PCNA interactions are important for repairing mismatches formed during meiotic recombination, but play only a relatively minor role in regulating the fidelity of mitotic recombination.     

Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

The dinuclear iron-oxo ferroxidase center of Pyrococcus furiosus ferritin is a stable prosthetic group with unexpectedly high reduction potentials

Recombinant ferritin from Pyrococcus furiosus expressed in Escherichia coli exhibits in EPR monitored redox titrations a mixed valence (Fe(3+)-Fe2+) S=1/2 signal at intermediate potentials that is a high-resolution homolog of the ferroxidase signal previously described for reconstituted horse spleen apo-ferritin. P. furiosus reconstituted apo-ferritin reduced to intermediate potentials exhibits the same mixed-valence signal, which integrates to close to one spin per subunit. The reduction potentials of +210 and +50 mV imply that the iron dimer is a stable prosthetic group with three redox states.     

Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae

Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.     

The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli

Twin-arginine translocation (Tat) systems allow the translocation of folded proteins across biological membranes of most prokaryotes. In proteobacteria, a TatBC complex binds Tat substrates and initiates their translocation after recruitment of the component TatA. TatA and TatB belong to one protein family, but only TatB forms stable complexes with TatC. Here we show that TatB builds up TatA-like modular complexes in the absence of TatC. This TatB ladder ranges from about 100 to over 880 kDa with 105+/-10 kDa increments. TatC alone can form a 250 kDa complex which could be a scaffold that can recruit TatB to form defined TatBC complexes.     

Essential‐Oil Variability of Juniperus deltoides R.P.Adams along the East Adriatic Coast – How Many Chemotypes Are There?

The composition of the essential oils isolated from twigs of ten Juniperus deltoides R.P . Adams populations from the east Adriatic coast was determined by GC‐FID and GC/MS analyses. Altogether, 169 compounds were identified, representing 95.6–98.4% of the total oil composition. The oils were dominated by monoterpenes (average content of 61.6%), which are characteristic oil components of species of the Juniperus section. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, comprising on average 46.78% of the essential oils. Statistical methods were deployed to determine the diversity of the terpene classes and the common terpenes between the investigated populations. These statistical analyses revealed the existence of three chemotypes within all populations, i.e., a α‐pinene, limonene, and limonene/α‐pinene type.