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Moscow, Russia

Goptar I.A.,Moscow State University | Shagin D.A.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Shagina I.A.,Evrogen JSC | Mudrik E.S.,Evrogen JSC | And 7 more authors.
Insect Biochemistry and Molecular Biology

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP. © 2013. Source

Shemiakina I.I.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Ermakova G.V.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Cranfill P.J.,Florida State University | Baird M.A.,Florida State University | And 13 more authors.
Nature Communications

Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues. © 2012 Macmillan Publishers Limited. All rights reserved. Source

Shcherbo D.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Shemiakina I.I.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Ryabova A.V.,RAS A.M. Prokhorov General Physics Institute | Luker K.E.,University of Michigan | And 11 more authors.
Nature Methods

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability. © 2010 Nature America, Inc. All rights reserved. Source

Shagina I.,Evrogen JSC | Bogdanova E.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Mamedov I.Z.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Lebedev Y.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | And 2 more authors.

An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at baseline levels. Thus, DSN normalization applied to C 0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants. Source

Serebrovskaya E.O.,Nizhny Novgorod State Medical Academy | Gorodnicheva T.V.,Evrogen JSC | Ermakova G.V.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | Solovieva E.A.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | And 6 more authors.
Biochemical Journal

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo. © The Authors Journal compilation © 2011 Biochemical Society. Source

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