Characterization of Class V DyP-Type Peroxidase SaDyP1 from Streptomyces avermitilis and Evaluation of SaDyPs Expression in Mycelium

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.     

The 5N neutral particle beamline of the U-70 accelerator (Institute for High Energy Physics)

The 5N neutral particle beamline at the U-70 accelerator (Institute for High Energy Physics), which has been designed to produce a high-intensity neutron beam with the highest possible energy, is described. The average neutron energy is ∼51 GeV, and the beam intensity is up to 10⁷ neutrons per spill. For several years, this beam has been used to investigate charmed particles and narrow baryon resonances in neutron-nucleus interactions and search for exotic multiquark states with the aid of the EXCHARM spectrometer. Original Russian Text A.N. Aleev, V.A. Arefiev, A.A. Aseev, Yu.G. Basha, V.P. Balandin, A.P. Bugorsky, T.S. Grigalashvili, B.N. Guskov, A.A. Zhuravlev, V.N. Zapolsky, A.I. Zinchenko, I.M. Ivanchenko, N.N. Karpenko, M.N. Kapishin, V.D. Kekelidze, D.A. Kirillov, I.G. Kosarev, N.A. Kuzmin, M.F. Likhachev, A.L. Ljubimov, D.T. Madigozhin, A.N. Maximov, N.A. Molokanova, A.N. Morozov, F.N. Novoskoltsev, Yu.K. Potrebenikov, Yu.P. Petukhov, V.E. Simonov, V.N. Spaskov, G.T. Tatishvili, P.Z. Hristov, I.P. Yudin (EXCHARM Collaboration), 2009, published in Pribory i Tekhnika Eksperimenta, 2009, No. 3, pp. 5–12.     

Preparation of ZrF4-NaF-BaF2-LaF3-YbF2 glasses with YbF2 substitution for BaF2 (LaF3)

We have prepared glasses and semicrystalline phases with a green color in the systems ZrF₄(53.5)-NaF(20)-BaF₂(20)-LaF₃(6.5 ? x)-YbF₂(x) (0 ≤ x ≤ 6.5 mol %) (I) and ZrF₄(53.5)-NaF(20)-BaF₂(20 ? x)-LaF₃(6.5)-YbF₂(x) (0 ≤ x ≤ 20 mol %) (II). Thermal analysis results demonstrate that, in both systems, the glass transition temperature of the samples containing ≤3 mol % YbF₂ lies in the range 200–250°C, their heating curves show two or three crystallization events (at 320–340, 380–460, and 415–490°C), and their melting points range from 460 to 490°C. Increasing the YbF₂ content of the glasses to 4 mol % and above (system I) has no effect on their glass transition temperature, reduces the temperature of the first crystallization event from 340 to 305°C, and produces extra peaks in the range 545–600°C, above the major melting peak, which can be accounted for by a nonequilibrium state of the glasses. Ytterbium difluoride substitution for barium difluoride (10 to 20 mol %) (system II) leads to the formation of semicrystalline phases and increases the glass-transition (305°C), crystallization (470–515°C), and melting (570–690°C) temperatures. The IR spectra of such samples show, in addition to a so-called “featureless continuum” (～500 cm^?1), absorption bands characteristic of the Yb-F bond. Electronic spectra also confirm that the glasses contain both divalent and trivalent ytterbium.     

Photochemical modulation of DNA conformation by organic dications

A group of azobenzene derivatives containing two quaternary ammonium groups with various intercharge distances between them was synthesised and used to control photochemically the conformation of genomic DNA by switching the distance between cationic ammonium groups in the dications. It was found that isomerisation of either dication from the trans form to cis resulted in an increase in the dication's efficiency for DNA compaction; this is associated with a decrease in intercharge distance between ammonium groups and leads to a better match of the binder's cationic groups to adjacent phosphate groups of DNA. Ammonium dications have several important advantages over the photosensitive surfactant type of diazobenzene reported earlier: they can be used at significantly lower (>100-fold) concentrations than photosensitive surfactants, and DNA conformation control can be performed over a broader concentration range of dications. The influence of intercharge distance in photosensitive dications on photo-induced DNA binding discrimination is discussed, and the molecular mechanism is proposed.     

Opportunity for interaction? A naturalistic observation study of dual-earner families after work and school.

Everyday patterns of interaction can strengthen or undermine bonds between family members. This naturalistic observation study focused on an understudied facet of family life: opportunities for interaction among dual-earner family members after work and family members’ responses to these opportunities. Thirty dual-earner couples and their children were observed and video-recorded in their homes throughout two weekday afternoons and evenings. Two interaction opportunities were analyzed: (1) the behavior of family members toward a parent returning home from work and (2) the physical proximity of family members throughout the evening. Three main findings emerged. Women, who tended to return home before men, were greeted with positive behavior and reports of the day’s information from family members. Men, in contrast, returned home later in the day and received positive behavior or no acknowledgment from family members distracted by other activities. Throughout the evening, mothers spent more time with children whereas fathers spent more time alone. Couples were seldom together without their children. The implications of observed interaction patterns and the contribution of naturalistic observation methods to the study of family relationships are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca²⁺-binding protein recoverincontains four potential EF-hand Ca²⁺-binding sites, of whichonly two, the second and the third, are capable of binding calciumions. We have studied the effects of substitutions in the second,third and fourth EF-hand sites of recoverin on its Ca²⁺-bindingproperties and some other characteristics, using intrinsic fluorescence,circular dichroism spectroscopy and differential scanning microcalorimetry.The interaction of the two operating binding sites of wild-typerecoverin with calcium increases the protein's thermal stability,but makes the environment around the tryptophan residues moreflexible. The amino acid substitution in the EF-hand 3 (E121Q)totally abolishes the high calcium affinity of recoverin, whilethe mutation in the EF-hand 2 (E85Q) causes only a moderatedecrease in calcium binding. Based on this evidence, we suggestthat the binding of calcium ions to recoverin is a sequentialprocess with the EF-hand 3 being filled first. Estimation ofCa²⁺-binding constants according to the sequential binding schemegave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^–1 for thirdand second EF-hands, respectively. The substitutions in theEF-hand 2 or 3 (or in both the sites simultaneously) do notdisturb significantly either tertiary or secondary structureof the apo-protein. Amino acid substitutions, which have beendesigned to restore the calcium affinity of the EF-hand 4 (G160D,K161E, K162N, D165G and K166Q), increase the calcium capacityand affinity of recoverin but also perturb the protein structureand decrease the thermostability of its apo-form.     

Relative Frequencies of PAX6 Mutational Events in a Russian Cohort of Aniridia Patients in Comparison with the World’s Population and the Human Genome

Genome-wide sequencing metadata allows researchers to infer bias in the relative frequencies of mutational events and to predict putative mutagenic models. In addition, much less data could be useful in the evaluation of the mutational frequency spectrum and the prevalent local mutagenic process. Here we analyzed the PAX6 gene locus for mutational spectra obtained in our own and previous studies and compared them with data on other genes as well as the whole human genome. MLPA and Sanger sequencing were used for mutation searching in a cohort of 199 index patients from Russia with aniridia and aniridia-related phenotypes. The relative frequencies of different categories of PAX6 mutations were consistent with those previously reported by other researchers. The ratio between substitutions, small indels, and chromosome deletions in the 11p13 locus was within the interval previously published for 20 disease associated genomic loci, but corresponded to a higher end due to very high frequencies of small indels and chromosome deletions. The ratio between substitutions, small indels, and chromosome deletions for disease associated genes, including the PAX6 gene as well as the share of PAX6 missense mutations, differed considerably from those typical for the whole genome.     

Uterine Deletion of Bmal1 Impairs Placental Vascularization and Induces Intrauterine Fetal Death in Mice

Recently, it was demonstrated that the expression of BMAL1 was decreased in the endometrium of women suffering from recurrent spontaneous abortion. To investigate the pathological roles of uterine clock genes during pregnancy, we produced conditional deletion of uterine Bmal1 (cKO) mice and found that cKO mice could receive embryo implantation but not sustain pregnancy. Gene ontology analysis of microarray suggested that uterine NK (uNK) cell function was suppressed in cKO mice. Histological examination revealed the poor formation of maternal vascular spaces in the placenta. In contrast to WT mice, uNK cells in the spongiotrophoblast layer, where maternal uNK cells are directly in contact with fetal trophoblast, hardly expressed an immunosuppressive NK marker, CD161, in cKO mice. By progesterone supplementation, pregnancy could be sustained until the end of pregnancy in some cKO mice. Although this treatment did not improve the structural abnormalities of the placenta, it recruited CD161-positive NK cells into the spongiotrophoblast layer in cKO mice. These findings indicate that the uterine clock system may be critical for pregnancy maintenance after embryo implantation.     

Visualizing Intramolecular Dynamics of Membrane Proteins

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.