Biochemistry (Moscow) (v.81, #9)

Role of ceramide in apoptosis and development of insulin resistance by D. I. Kuzmenko; T. K. Klimentyeva (913-927).
This review presents data on the functional biochemistry of ceramide, one of the key sphingolipids with properties of a secondary messenger. Molecular mechanisms of the involvement of ceramide in apoptosis in pancreatic β-cells and its role in the formation of insulin resistance in pathogenesis of type 2 diabetes are reviewed. One of the main predispositions for the development of insulin resistance and diabetes is obesity, which is associated with ectopic fat deposition and significant increase in intracellular concentrations of cytotoxic ceramides. A possible approach to the restoration of tissue sensitivity to insulin in type 2 diabetes based on selective reduction of the content of cytotoxic ceramides is discussed.
Keywords: ceramide; sphingolipids; apoptosis mediator; insulin resistance; diabetes

The road to optogenetics: Microbial rhodopsins by E. G. Govorunova; L. A. Koppel (928-940).
Optogenetics technology (using light-sensitive microbial proteins to control animal cell physiology) is becoming increasingly popular in laboratories around the world. Among these proteins, particularly important are rhodopsins that transport ions across the membrane and are used in optogenetics to regulate membrane potential by light, mostly in neurons. Although rhodopsin ion pumps transport only one charge per captured photon, channelrhodopsins are capable of more efficient passive transport. In this review, we follow the history of channelrhodopsin discovery in flagellate algae and discuss the latest addition to the channelrhodopsin family, channels with anion, rather than cation, selectivity.
Keywords: rhodopsins; ion channels; neurons; membrane potential; light

Nucleolar methyltransferase fibrillarin: Evolution of structure and functions by M. Y. Shubina; Y. R. Musinova; E. V. Sheval (941-950).
Fibrillarin is one of the most studied nucleolar proteins. Its main functions are methylation and processing of pre-rRNA. Fibrillarin is a highly conserved protein; however, in the course of evolution from archaea to eukaryotes, it acquired an additional N-terminal glycine and arginine-rich (GAR) domain. In this review, we discuss the evolution of fibrillarin structure and its relation to the functions of the protein in prokaryotes and eukaryotes.
Keywords: fibrillarin; nucleolus; methyltransferase; GAR domain

AP endonuclease 1 as a key enzyme in repair of apurinic/apyrimidinic sites by N. S. Dyrkheeva; N. A. Lebedeva; O. I. Lavrik (951-967).
Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5′-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3′-hydroxyl group and a 5′-deoxyribose phosphate moiety. APE1 exhibits 3′-phosphodiesterase, 3′-5′-exonuclease, and 3-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system.
Keywords: human apurinic/apyrimidinic endonuclease 1 (APE1); AP site; base excision repair; protein–DNA interactions; protein–protein interactions

35S promoter methylation in kanamycin-resistant kalanchoe (Kalanchoe pinnata L.) plants expressing the antimicrobial peptide cecropin P1 transgene by T. V. Shevchuk; N. S. Zakharchenko; S. V. Tarlachkov; O. V. Furs; O. V. Dyachenko; Y. I. Buryanov (968-971).
Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.
Keywords: Kalanchoe pinnata ; transgenic plants; antimicrobial peptide cecropin P1; DNA methylation; 35S cauliflower mosaic virus 35S RNA promoter; kanamycin; neomycin phosphotransferase II

Molecular genetic markers of intra- and interspecific divergence within starfish and sea urchins (Echinodermata) by N. B. Petrov; I. P. Vladychenskaya; A. L. Drozdov; O. S. Kedrova (972-980).
A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1–5.8S rDNA–ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions–Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster–were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1–5.8S rDNA–ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.
Keywords: molecular evolution; population; speciation; Echinodermata; cytochrome oxidase subunit I gene; internal transcribed spacers

Plastid genome of Seseli montanum: Complete sequence and comparison with plastomes of other members of the Apiaceae family by T. H. Samigullin; M. D. Logacheva; E. I. Terenteva; G. V. Degtjareva; C. M. Vallejo-Roman (981-985).
This work reports the complete plastid (pt) DNA sequence of Seseli montanum L. of the Apiaceae family, determined using next-generation sequencing technology. The complete genome sequence has been deposited in GenBank with accession No. KM035851. The S. montanum plastome is 147,823 bp in length. The plastid genome has a typical structure for angiosperms and contains a large single-copy region (LSC) of 92,620 bp and a small single-copy region (SSC) of 17,481 bp separated by a pair of 18,861 bp inverted repeats (IRa and IRb). The composition, gene order, and AT-content in the S. montanum plastome are similar to that of a typical flowering plant pt DNA. One hundred fourteen unique genes have been identified, including 30 tRNA genes, four rRNA genes, and 80 protein genes. Of 18 intron-containing genes found, 16 genes have one intron, and two genes (ycf3, clpP) have two introns. Comparative analysis of Apiaceae plastomes reveals in the S. montanum plastome a LSC/IRb junction shift, so that the part of the ycf2 (4980 bp) gene is located in the LSC, but the other part of ycf2 (1301 bp) is within the inverted repeat. Thus, structural rearrangements in the plastid genome of S. montanum result in an enlargement of the LSC region by means of capture of a large part of ycf2, in contrast to eight Apiaceae plastomes where the complete ycf2 gene sequence is located in the inverted repeat.
Keywords: plastid genome; genome comparative analysis; Seseli montanum ; Apiaceae

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding αand β-subunits of protocatechuate 3,4-dioxygenase and to two genes of theR. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.
Keywords: Rhodococcus wratislaviensis G10; Rhodococcus opacus 1CP; benzoate; catechol 1,2-dioxygenase; protocatechuate 3,4-dioxygenase; biodegradation genes

Cell wall glycopolymers of type strains from three species of the genus Actinoplanes by G. M. Streshinskaya; A. S. Sashkov; E. M. Tul’skaya; S. N. Senchenkova; A. S. Dmitrenok; N. F. Piskunkova; O. V. Bueva; L. I. Evtushenko (999-1012).
The structures of cell wall glycopolymers from the type strains of three Actinoplanes species were investigated using chemical methods, NMR spectroscopy, and mass spectrometry. Actinoplanes digitatis VKM Ac-649T contains two phosphate-containing glycopolymers: poly(diglycosyl-1-phosphate) →6)-α-D-GlcpNAc-(1-P-6)-α-D-GlcpN-(1→ and teichoic acid →1)-sn-Gro-(3-P-3)-β-[β-D-GlcpNAc-(1→2]-D-Galp-(1→. Two glycopolymers were identified in A. auranticolor VKM Ac-648T and A. cyaneus VKM Ac-1095T: minor polymer–unsubstituted 2,3-poly(glycerol phosphate), widely abundant in actinobacteria (Ac-648T), and mannan with trisaccharide repeating unit →2)-α-D-Manp-(1→2)-α-D-Manp(1→6)-α-D-Manp-(1→(Ac-1095T). In addition, both microorganisms contain a teichuronic acid of unique structure containing a pentasaccharide repeating unit with two residues of glucopyranose and three residues of diaminouronic acids in D-manno- and/or D-gluco-configuration. Each of the strains demonstrates peculiarities in the structure of teichuronic acid with respect to the ratio of diaminouronic acids and availability and location of O-methyl groups in glucopyranose residues. All investigated strains contain a unique set of glycopolymers in their cell walls with structures not described earlier for prokaryotes.
Keywords: Actinoplanes ; cell wall; teichoic acids; glycosyl-1-phosphate polymers; teichuronic acids; mannan; NMR spectroscopy

Identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain by O. A. Akimova; L. V. Kapilevich; S. N. Orlov; O. D. Lopina (1013-1022).
Prolonged exposure of different epithelial cells (canine renal epithelial cells (MDCK), vascular endothelial cells from porcine aorta (PAEC), human umbilical vein endothelial cells (HUVEC), cervical adenocarcinoma (HeLa), as well as epithelial cells from colon carcinoma (Caco-2)) with ouabain or with other cardiotonic steroids was shown earlier to result in the death of these cells. Intermediates in the cell death signal cascade remain unknown. In the present study, we used proteomics methods for identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain. After exposure of Caco-2 human colorectal adenocarcinoma cells with 3 μM of ouabain for 3 h, the protein interacting in complex with Na+,K+-ATPase was coimmunoprecipitated using antibodies against the enzyme α1-subunit. Proteins of coimmunoprecipitates were separated by 2D electrophoresis in polyacrylamide gel. A number of proteins in the coimmunoprecipitates with molecular masses of 71-74, 46, 40-43, 38, and 33-35 kDa was revealed whose binding to Na+,K+-ATPase was activated by ouabain. Analyses conducted by mass spectroscopy allowed us to identify some of them, including seven signal proteins from superfamilies of glucocorticoid receptors, serine/threonine protein kinases, and protein phosphatases 2C, Src-, and Rho-GTPases. The possible participation of these proteins in activation of cell signaling terminated by cell death is discussed.
Keywords: Na+,K+-ATPase; ouabain; coimmunoprecipitation; 2D-PAGE; mass spectrometry

Interaction of telomeric retroelement HeT-A transcripts and their protein product gag in early embryogenesis of Drosophila by I. A. Olovnikov; V. V. Morgunova; A. A. Mironova; M. Y. Kordyukova; E. I. Radion; O. M. Olenkina; N. V. Akulenko; A. I. Kalmykova (1023-1030).
The telomere is a nucleoprotein complex at the ends of linear chromosomes that protects them from fusion and degradation. The telomere consists of telomeric DNA, a protective protein complex and telomeric RNA. Biogenesis of telomeric transcripts in development is still far from being understood. Drosophila telomeres are elongated by a transposition of specialized telomeric retrotransposons that encode proteins. Using transgenic constructs encoding tagged telomeric protein, we found that transcripts of Drosophila telomeric element HeT-A bind Gag-HeT-A protein encoded by these transcripts. Maternal HeT-A transcripts and Gag-HeT-A form ribonucleoprotein granules around centrosomes, centers of microtubule organization, during blastoderm formation, upon disruption of telomere silencing during oogenesis. The specific localization of HeT-A RNA is dependent on microtubules since disruption of microtubules caused delocalization of HeT-A transcripts. This transgenic system is a valuable model for the study of telomeric RNA biogenesis.
Keywords: telomere; development; retrotransposon HeT-A ; Drosophila ; telomeric RNA