Ribonucleases as antiviral agents

Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. The review discusses the well-known RNases that possess established antiviral effects, including both intracellular RNases (RNase L, MCPIP1 protein, and eosinophil-associated RNases) and exogenous RNases (RNase A, BS-RNase, onconase, binase, and synthetic RNases). Attention is paid to two important, but not always obligatory, aspects of molecules of RNases that have antiviral properties, i.e., catalytic activity and ability to dimerize. The hypothetic scheme of virus elimination by exogenous RNases that reflects possible types of interaction of viruses and RNases with a cell is proposed. The evidence for RNases as classical components of immune defense and thus perspective agents for the development of new antiviral therapeutics is proposed. © 2014, Pleiades Publishing, Inc

Secretome of Intestinal bacilli: A natural guard against pathologies

Current studies of human gut microbiome usually do not consider the special functional role of transient microbiota, although some of its members remain in the host for a long time and produce broad spectrum of biologically active substances. Getting into the gastrointestinal tract (GIT) with food, water and probiotic preparations, two representatives of Bacilli class, genera Bacillus and Lactobacillus, colonize epithelium blurring the boundaries between resident and transient microbiota. Despite their minor proportion in the microbiome composition, these bacteria can significantly affect both the intestinal microbiota and the entire body thanks to a wide range of secreted compounds. Recently, insufficiency and limitations of pure genome-based analysis of gut microbiota became known. Thus, the need for intense functional studies is evident. This review aims to characterize the Bacillus and Lactobacillus in GIT, as well as the functional roles of the components released by these members of microbial intestinal community. Complex of their secreted compounds is referred by us as the "bacillary secretome." The composition of the bacillary secretome, its biological effects in GIT and role in counteraction to infectious diseases and oncological pathologies in human organism is the subject of the review. © 2017 Ilinskaya, Ulyanova, Yarullina and Gataullin

Analysis of the intestinal microbiome in colorectal cancer

Aim. To conduct a comparative analysis of the microbiome of biopsies of a tumor and normal intestinal epithelium of patients diagnosed with colorectal cancer and to identify of functional activities of the obtained bacterial isolates that affect the development of the tumor. Methods. The study included 50 patients with malignant neoplasms of the colon: 36 men and 24 women. The mean age of the patients was 64.1±10.2 years. To analyze the microbiota of the biopsies, DNA samples were obtained from the tissue of the unaffected colon mucosa and tumor of the patients. Bacterial 16S rRNA genes fragments were amplified using bar-coded primer bakt_341f. Metagenomic next-generation sequencing was performed using the MiSeq platform (Illumina, USA). The obtained data were processed by bioinformatic methods using the QIIME package. Recognition of microorganisms depending on the morphotype and gram staining of the microflora was carried out using combination differential media and biochemical tests. Statistical analysis was carried out using Microsoft Excel, Service Pack 2 for Office XP, Statistica 6.0 (StatSoft). A comparative analysis was performed with the Student's t-test and the Mann-Whitney test in case of unmet conditions of validity. Alpha diversity of bacterial communities was quantified by the Shannon diversity index and the UniFrac distance for beta diversity analysis. Results. In patients with colorectal cancer, 5 bacterial phyla were isolated, the predominant of which were Firmicutes and Bacteroidetes, while the content of Actinobacteria was low. In addition, a higher number of representatives of Fusobacteria was observed in the tumor tissue compared to the tissue of a healthy mucosa, at a distance of 5 centimeters proximal to the tumor. The results of this study indicate that the microbiome of a tumor and a healthy mucosa fundamentally differ from each other not only in morphotype and gram staining but also in antagonistic, hemolytic and ribonucleolytic activities. Conclusion. Colonization of the tumor by dominant aggressive Gram-negative bacteria leads to significant changes in the tumor microbiome composition compared with normal mucosa, whose representatives are displaced from the damaged epithelium by more aggressive strains

Phylotyping and genotyping of Escherichia coli isolates obtained from patients with colorectal cancer

© 2020, Advanced Scientific Research. All rights reserved. Objective of the research was to phylotype the E. coli isolates from the malignant and adjacent normal epithelium of the rectal mucosa of patients diagnosed with colorectal cancer, identifying and discriminating phylogroups by a set of genes, and also genotyping them by the fimH-gene locus by identifying fimH types. Samples of bacterial cultures isolated from paired biopsy specimens of patients diagnosed with colorectal cancer were subjected to molecular genetic research for species identification, as well as E. coli phylo- and genotyping. By sequencing the 16S rRNA locus, bacterial cultures are identified both as isolates of monocultures of E. coli and as mixed cultures of E. coli and K. pneumonia. The procedure for the phylotyping of E. coli isolates by identifying and discriminating phylogroups by a set of genes established the affiliation of samples of bacterial cultures with phylogroups A and B2. The genotyping procedure for E. coli isolates at the fimH gene locus by identifying the fimH types characterized the studied samples by signs of the third (f-3), fourth (f-4), and seventh (f-7) types, respectively. The strategy for identifying fimH-types during genotyping of E. coli at the fimH-gene locus has been improved, based on the analysis of SNP sequenced DNA sequences, and includes 52 fimH-types, three of which are justified in this paper

Plant materials as a potential source of antitumor agents

Many medicinal plants are used around the world to create new medicinal products. The effectiveness of these products is associated with a complex synergistic interaction of various plant components. In this study, we investigated the extract cytotoxicity of the following five plant species belonging to the Agavaceae (Asparagaceae) family: Sansevieria cylindrical, S. trifasciata, Polianthes tuberosa, Yucca filamentosa, and Furcraea gigantea (var. watsoniana). The plant material was collected in different regions of Egypt. For each plant species, the half maximal inhibitory concentration (IC50) in relation to the human lung adenocarcinoma cell culture (A549) was determined with the help of the IC50 calculator. For the MTT test, we used aqueous solutions of the methanol extracts of S. cylindrical and S. trifasciata in the concentrations of 100, 200, 300, 500, 900, 1300, 1700, and 2000 ?g/mL, as well as P. tuberosa, Y. filamentosa, and F. gigantea in the concentrations of 10, 20, 50, 100, 300, 500, 1000, and 1500 μg/mL. We found that the extracts of S. trifasciata leaves and rhizomes cause no significant effect on the viability of A549 tumor cells in all the studied concentrations. The aqueous solutions of S. cylindrica leaves and rhizome methanol extracts of with increasing concentration reduced the survival of A549 tumor cells more than twice as compared with the control group. The lowest IC50 values were obtained for the extracts of P. tuberosa and F. gigantea leaves: 62.5 and 82 μg/mL, respectively. The results confirm anticarcinogenic potential of the extracts of P. tuberosa and F. gigantea leaves, which is important for development of new phytobiotechnologies aimed at the synthesis of effective medications based on the secondary metabolites of these plants

Crystal structure of binase does not reflect its native conformation

Cytotoxic ribonucleases (RNases) of the T1 family, including binase, the secreted guanyl-preferring RNase of Bacillus pumilus, are considered as promising agents of alternative anticancer chemotherapy. Binase has a selective apoptosis-inducing action against cells expressing oncogenes ras, kit, AML-ETO. The crystal structure of the binase mutant with two-point amino acid substitutions at positions 43 and 81 (Glu43Ala/Phe81Ala) indicates the absence of dimeric forms, which are characteristic for the wild-type binase. We studied the native structural organization of the Glu43Ala/Phe81Ala mutant. It was found that the mutant enzyme, similarly to the wild-type binase, possesses catalytically active dimers of different stability levels identified not only under denaturing gel electrophoresis, but also under native conditions. The results of the study show that binase exists predominantly in the dimeric form, whereas the Glu43Ala/Phe81Ala mutant approximately equally represented by both dimers and monomers formed as a result of the decomposition of unstable dimers. Although the catalytic activity of the mutant with respect to the natural substrate, RNA, was lower, as compared to the wild-type enzyme, it exhibited a 32–35% higher cytotoxicity against human adenocarcinoma cells. The data obtained indicate the contribution of the structural organization of RNases to their cytotoxicity and confirm the significance of the analysis of the native conformation of cytotoxic proteins

Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus: The Impact of Substrate Structure, Metal Ions, and Low Molecular Weight Nucleotide Compounds

© 2020, Pleiades Publishing, Inc. Abstract: Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction

Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus: The Impact of Substrate Structure, Metal Ions, and Low Molecular Weight Nucleotide Compounds

Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction

Bacterial Ribonuclease (Binase) Promotes Decontamination of MDBK Cell Cultures From Bovine Diarrhea Virus

Mammalian cell cultivation is a technique that has played a crucial role in the development of biology, medicine, veterinary medicine, and pharmacology. Cell and tissue cultures are used as model systems in both the study of the mechanisms of pathogenesis of various diseases and the assessment of the effectiveness and toxicity of new drugs. They are frequently employed in the production of vaccines and biopharmaceuticals and play a part in assisted reproductive technologies. One of the factors that disrupt the stability of cell lines, thus affecting the results obtained and limiting the possibility of their application, is the contamination of cell cultures by various microorganisms, including bacteria, fungi, viruses, and mycoplasmas. According to a recent review, the bovine viral diarrhea virus (BVDV) is one of the most frequent and problematic contaminations occurring in animal and human cell cultures. Bacterial ribonuclease (binase) has shown considerable antiviral potential, and its activity against several viruses in vitro and in vivo has been reliably proven. In the present study, we present experimental results that demonstrate the role of bacterial ribonuclease (binase) in the contamination of cultures of bovine cell lines by cytopathogenic BVDV. According to these results, the treatment of infected Madin–Darby bovine kidney (MDBK) cells with binase led to a dose-dependent reduction in the level of viral RNA and infectious titer, thereby confirming the antiviral properties of the enzyme in vitro. Binase in concentrations of 100 to 300 μg/ml, which are nontoxic for cultures of MDBK cells, allowed to reduce the viral titer by 0.77 to 1.57 lg, correspondingly. Our results confirm the feasibility of using binase for the decontamination of cell cultures provided that the optimal modes of binase treatment are applied

Cytotoxic and Apoptosis-Inducing Activity of Plants from the Family Asparagaceae in Relation to Human Alveolar Adenocarcinoma Cells

Cancer is known as the second major mortality cause. The number of new cases is increasing every year. Thus, it is urgent for scientists to search for alternative drugs with selective antitumor action and minimal side effects. It is known that some plant metabolites exhibit antioxidant, cytotoxic, and antitumor activity, while at the same time being less toxic than modern allopathic drugs. In this work, we have investigated the cytotoxic and apoptosis-inducing effects of extracts obtained from plants of the family Asparagaceae on A549 human lung adenocarcinoma cells. The analysis has been performed using flow cytofluorometry. If extracts showed cytotoxicity, the apoptosis-inducing action has been evaluated at the concentration of 50 μg/mL; in other cases, the analyzed concentration range was 50–300 μg/mL. On the basis of the experiments carried out, the following conclusions have been made. Extracts of the leaves and rhizomes of Sansevieria cylindrica and Sansevieria trifasciata do not possess antitumor activity. Extracts of the leaves of Polianthes tuberosa and Furcraea gigantea, which were cytotoxic at high concentrations, cause cell death at 50 μg/mL in the amount of 21.35 ± 1.86 and 15.6 ± 3.23, respectively. Extracts of Polianthes tuberosa bulbs and Yucca filamentosa leaves are able to induce apoptosis at higher concentrations. When the concentration reaches 100 μg/mL, the proportion of apoptotic cells for these plants is 45.76 ± 1.34 and 11.33 ± 0.07, respectively. The number of dead cells at the concentration of 300 μg/mL increased up to 73.33 ± 3.05 and 81.75 ± 4.07. The results have great importance for development of new drugs based on metabolites from these plant extracts