Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG

Growth and B-Phycoerythrin Production of Red Microalga <em>Porphyridium purpureum</em> (Porphyridiales, Rhodophyta) under Different Carbon Supply

Red microalga Porphyridium purpureum (Bory) Drew is a well-known object of biotechnology due to its unique ability to synthesize a wide range of biologically active compounds. Enough minerals in an accessible form in a medium are a prerequisite for maintaining a high growth rate of P. purpureum. Carbon is the main element of microalgal biomass and is a component of all organic compounds. The work aimed to study the morphological features of cells and the accumulation and production of B-phycoerythrin and total protein in P. purpureum biomass in different ways of supplying CO2 into the culture. In Variant 1, CO2 was directly injected into a gas–air mixture (2–3 percent v/v) used for culture bubbling via capillary. In Variant 2, the air was supplied to the culture through the aquarium sparger. Variant 3 was like the first one but without the additional introduction of carbon dioxide. The application of the method for sparging atmospheric air led to a significant increase in both the productivity of the P. purpureum and the rate of protein and B-phycoerythrin synthesis in comparison with growing it using the air without spraying (two-and-a-half times, five times, and more than eight times, respectively). Moreover, there were significant changes in the morphological structure of P. purpureum cells, which were visualized both by microscopy and by changes in the color of the culture. Based on the experimental data obtained, the variants for the carbon supply experiment were ranked as follows: Variant 1 is better than Variant 2 and Variant 3. The use of atomization as a technological method made it possible to speed up the transfer of carbon dioxide from the air to the medium, which helped to keep the growth rate of P. purpureum biomass and B-phycoerythrin accumulation high

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.clos

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive &quot;damages&quot; were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase beta using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were pet-formed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg2+ whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair. (c) 2007 Elsevier B.V. All rights reservedclos

Features of the Bioconversion of Pentacyclic Triterpenoid Oleanolic Acid Using Rhodococcus Actinobacteria

The ability of actinobacteria of the genus Rhodococcus to transform oleanolic acid (OA), a plant pentacyclic triterpenoid, was shown for the first time using bioresources of the Regional Specialized Collection of AlkanotrophicMicroorganisms (IEGM; WDCM #768;www.iegmcol.ru). The most promising strains (R.opacus IEGM 488 and R.rhodochrousIEGM 285) were selected, and these catalyzed80% bioconversion of OA (0.5 g/L) in the presence of n-hexadecane (0.1% v/v) for seven days. The process of OA bioconversion was accompanied by a gradual decrease in the culture medium pH. Adaptive responses of bacterial cells to the OA effects included the formation of compact cellular aggregates, a marked change in the surface-to-volume ratio of cells, and a significant increase in the Zeta potential values. The results demonstrated that the process of OA bioconversion was catalyzed by membrane-bound enzyme complexes. Participation of cytochrome P450-dependent monooxygenases in the oxidation of the OA moleculewas confirmedusing specific inhibitors. The obtained data expand our knowledge on the catalytic activity of actinobacteria of the genus Rhodococcus and their possible use as biocatalysts for the bioconversion of complex hydrophobic compounds. The results can also be used inthe searchfor promising OA derivatives to be used in the synthesis of biologically active agents. Keywords: bioconversion, oleanolic acid, Rhodococcus, biologically active compound

Impact of Multimedia Components in Children Magazines on Psychological Profile: Case of Russian Online Editions

The article analyzes the use of various multimedia components and reveals changes in the personal characteristics of children with varying degrees of Internet addiction, including those caused by the abundance of multimedia components in online publications, based on the analysis of multimedia components often found in Russian online magazines for children. All the analyzed media are classified into two categories: representing a copy of a printed magazine without any video materials and online games; an online magazine containing games, videos and graphic elements without any text content. The abundant use of multimedia components affects the level of child's Internet addiction that has an influence, in its turn, on personal psychological profile. The abundance of multimedia components in online magazines for children of different types causes an increase in the level of Internet addiction that affects, in its turn, child's personality

Species-Specific Proteins in the Oviducts of Snail Sibling Species: Proteotranscriptomic Study of Littorina fabalis and L. obtusata

SIMPLE SUMMARY: Genitalia and reproduction-associated proteins are often species-specific and might evolve rapidly. The situation in which the morphology of the reproductive system is the only difference between two or several closely related species has been reported on multiple occasions. Nevertheless, the reasons for such rapid divergence of the reproductive system is still poorly investigated. To shed some light on the issue, we performed a transcriptomic and proteomic comparison of pallial oviducts from the two sibling species of gastropods Littorina obtusata and L. fabalis. The main identified differences were associated with three functional groups of genes: transposable elements, which enhance genome variation and promote the evolution of new genes, receptor proteins potentially involved in friend or foe recognition, and various enzymes. We hypothesize that these functional groups reflect both the mechanism (transposable elements) and the directions (friend or foe recognition and reproductive physiology) of the rapid evolution of the reproductive system. ABSTRACT: Genus Littorina subgenus Neritrema (Mollusca, Caenogastropoda) includes the “obtusata” group of closely related species (Littorina obtusata and L. fabalis). The anatomy of the adult reproductive system (pallial oviduct) is the only reliable feature used for species identification in females of these species. Reproductive system anatomy and reproduction-associated proteins often diverge between sibling species. Despite being of high evolutionary interest, the molecular basis of this divergence remains poorly understood. We performed proteotranscriptomic comparison of oviducts of L. obtusata and L. fabalis by RNA-seq on Illumina HiSeq 2500 and two-dimensional protein electrophoresis (2D DIGE) with MS/MS identification of the species-specific proteins. The interspecies differences in the oviduct were associated with (1) metabolic proteins reflecting overall physiological differences between L. obtusata and L. fabalis, (2) receptor proteins, and (3) transcripts related to transposable elements (TEs). Various receptors identified may recognize a wide variety of ligands from pathogen-associated molecular patterns to specific carbohydrates on the sperm surface. Therefore, these may participate in immune defense as well as in sperm storage and regulation. Species-specificity of multiple TE sequences (coding for reverse transcriptase and ribonuclease H) may indicate the important role of these genomic elements in the Littorina species divergence, which has not been reported previously

Hotspot and conservation gap analysis of endemic vascular plants in the Altai Mountain Country based on a new global conservation assessment

The Altai Mountains of Central and North Asia are biologically rich and comprise a wide range of ecosystems and phytogeographical regions. According to the latest checklist, a total of 321 endemic vascular plant species, including 217 endemic and 104 subendemic taxa, have been recognized in the Altai Mountain Country (AMC). In this study, we conducted species risk assessment, distribution evaluation and conservation gap analysis for the endemic vascular flora of the AMC. The conservation status of 217 endemic species was assessed at the global level using the ConR package. As a result, 197 species were evaluated as potentially threatened, of which 101 are critically endangered (CR), 72 species are endangered (EN), and 24 species are vulnerable (VU). The remaining 20 species were evaluated as not threatened. Furthermore, the AMC was divided into 350 grid cells, with a grid cell size of 50 × 50 km2, for the spatial assessments of the endemic vascular plants. A total of 2657 unique georeferenced occurrences of endemic species were found and analyzed with three endemism indices, species richness (SR), weighted endemism (WE), and corrected weighted endemism (CWE), to quantify geographic patterns and centers of endemism across the whole AMC. The results showed that the endemic species are spread across 186 grid cells and distributed unevenly within the AMC. According to the conservation gap analysis, the main hotspots of endemism (i.e., SR and WE indices) were found at high elevations in the Russian Altai, while the CWE points to the Kazakh Altai as a hotspot, and many such hotspots are currently afforded no formal protections