Vol 52, No 4 (2018)

With the advance of sequencing technology, the number of sequenced plant genomes has been rapidly increasing. However, understanding of the gene function in these sequenced genomes lags far behind; as a result, many coding plant sequences in public databases are annotated as proteins with domains of unknown function (DUF). Function of a protein family DUF810 in rice is not known. In this study, we analysed seven members of OsDU810 (OsDUF810.1–OsDUF810.7) family with three distinct motifs in rice Nipponbare. By phylogenetic analysis, OsDUF810 proteins fall into three major groups (I, II, III). Expression patterns of the seven corresponding OsDUF810 protein-encoding genes in 15 different rice tissues vary. Under drought, salt, cold and heat stress conditions and ABA treatment, the expression of OsDUF810.7 significantly increases. Overexpression of this protein in E. coli lead to a significant enhancement of catalase (CAT) and peroxidase (POD) activities, and improved bacterial resistance to salt and drought.

Gene therapy for AIDS based on RNA interference (RNAi) is currently looked upon as a promising alternative to conventional antiretroviral chemotherapy. The high variability of HIV-1 is the main challenge in developing new approaches to AIDS therapy. To date, about 18 million HIV-1 infected individuals receive antiretroviral therapy worldwide. As of 2017, about 44% of individuals with AIDS received antiretroviral therapy in Russia. Since the RNAs used for efficient RNAi and the corresponding targets in the viral transcript should be perfectly complementary to each other, it is necessary to continuously monitor the nucleotide sequences of clinical HIV-1 isolates obtained from blood and cells of naïve patients and patients receiving antiretroviral therapy. Comprehensive analysis of the mutation frequencies in the viral genome is only possible with deep sequencing approaches. The present paper reports on an analysis of the mutation frequencies in six 100 bp genome regions in clinical HIV-1 isolates obtained from blood plasma of four Russian AIDS patients who have been receiving antiretroviral therapy for several years. These regions contain efficient RNAi targets. The average frequencies of all possible transversions and transitions within the RNAi targets and in their proximity have been estimated. It has been demonstrated that reverse transcriptase inhibition decreases the frequency of a number of reverse mutations. It has been found that mutations in RNAi targets are rarer (5–75 times lower than the mutation frequency for different nucleotide substitutions) than in the adjacent sequences. Our findings speak in favor of these conservative targets for developing new approaches to gene therapy of AIDS.

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na⁺-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca²⁺-ATPases of SERCA type (subfamily P2A); (3) H⁺-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation- transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na⁺-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na⁺-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na⁺ transport.

One of the end-products of ROS-induced peroxidation, malondialdehyde (MDA), induces the cross-links in proteins, which leads to perturbation of the physiological functions of cells and contributes to abnormal biological regulation and various disorders. Taurine (2-aminoethanesulfonic acid, Tau) aids in adjusting normal physiological functions to confer stress resistance. The protective effects of Tau against MDA stress in vitro or in vivo were reported previously. In this study, we had investigated the protective effects of taurine on viability, oxidative stress levels and mitochondrial biogenesis in mouse muscle C2C12 cells undergoing MDA induced stress. We show that the treatment with 100 μM MDA leads to increase in cell oxidative stress levels, inhibition of mitochondrial biogenesis and the reduction of the cell survival rates. The pretreatment with 0.1 μM taurine reduced MDA-induced death rate via inhibition of oxidative stress, restoration of mitochondrial functions of the mitochondrial membrane potential (MMP) and ATP production. In MDA stress, the pre-treatment with 0.1 μM taurine leads to upregulation of the factors of mitochondrial biogenesis. These observations suggest that the cytoprotective effects of taurine may be due to an induction of mitochondrial biogenesis.

Glycans and anti-glycan antibodies (AGAs) are essential for infiltration of inflammatory cells in various allergies. The glycocalyx structure of the cells is modified during disease progression, and this modification is possible to evaluate by assessment of AGAs. A printed glycan array with 55 immobilized glycans and immobilized antibodies to IgG, IgA, and IgM was used to study the changes in AGA profiles in bronchial asthma (BA). Levels of antibodies to certain glycans in BA patients statistically differed from levels in healthy donors (p < 0.0007 by the Mann–Whitney test); the glycan set included 6Su-6`-SiaLec, Sia LeX, Sia6Htype2; Tαα, Manβ1-4GlcNAc, and Manα1-4Manβ. The obtained results help to better understand the mechanisms of the cell-mediated immune response in bronchial asthma and other types of allergic reactions.

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

The mistletoe lectin viscumin (MLI) is a ribosome-inactivating protein from Viscum album widely used in cancer therapy. Its antitumor properties are due to its immunomodulating action, previously demonstrated in experiments involving intravenous, subcutaneous, and oral administration of viscumin. To investigate whether viscumin has a cytotoxic effect on the intestinal epithelium, its safety was assessed using (i) impedance spectroscopy to measure the integrity of the colorectal adenocarcinoma Caco-2 cell monolayer after exposure to viscumin and (ii) a novel technique of determining the portion of viscumin-inactivated ribosomes. It was shown that inactivation of at least 20% of the ribosomes within 6 h did not lead to disruption of the Caco-2 cell monolayer or alter the physicochemical parameters of enterocyte membranes.

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

Cytostatic colchicine is widely used in the treatment of Familial Mediterranean fever, but it has several side effects. For finding new, more effective drugs with higher affinity and diminishside effects we carried out virtual screening of potential inhibitors of the main target of colchicine, the polymerization of tubulin by evaluating affinity 25745 compounds, structurally related to the colchicine. We have identified 11 commercially available compounds with higher affinity to tubulin. Compounds with highest binding scores include trimethoxybenzene and its derivatives; these compounds bind to the same site in similar orientation. Information provided can form the basis for design of new cytostatics.

Polysaccharides influence concentration and purity of extracted DNA. Here we present rapid and efficient protocol for DNA extraction from samples rich in polysaccharides. The technique has been developed using cultures of Schizophyllum commune and involves a modification of known Cetyltrimethyl Ammonium Bromide (CTAB) protocol. To remove polysaccharides, Polyethylene Glycol (PEG) 8000 was added during DNA precipitation. Genomic DNA obtained with the CTAB-PEG method had high integrity, with average fragment size >30 kb, the concentration higher than 100 ng/μL, and the yield more than 30 μg/g. Presented technique is suitable for DNA extraction from fungi, bacteria, archaea or even mollusks with high polysaccharide content.