Fucoidan-active α-L-fucosidases of the GH29 and GH95 families from a fucoidan degrading cluster of the marine bacterium Wenyingzhuangia fucanilytica.

In present work we provide the bioinformatic and biochemical characterization of six α-L-fucosidases that belong to the 29 and 95 families of glycoside hydrolases (GH) from the fucoidan-degrading locus of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The fucosidases FucWf1GH29, FucWf2GH29, FucWf3GH29 and FucWf6GH29 are relegated to the subfamily A of the GH29 family. The fucosidase FucWf4GH29 bears a distant resemblance to the GH29 and does not belong to either the GH29A or the GH29B subfamilies. Apparently, FucWf4GH29 is the first representative of a new subfamily within the GH29 family of α-L-fucosidases. For the first time the specificity of fucosidases has been studied using a series of fucoidan-related sulfated oligosaccharides. Studied α-L-fucosidases are able to cleave l-fucose from sulfated fucooligosacchrides after their treatment with exo-sulfatases. All studied α-L-fucosidases are cleaving the α-1→3- and α-1→4-linked terminal l-fucose in sulfated fucooligosaccharides. However, only FucWf3GH29 is able to cleave off an α-1→2-linked l-fucose. The fucosidase FucWf5GH95 of the GH95 family is shown to have higher activity on fucoidans than fucosidases of the GH29 family. Supposedly, the α-l-fucosidase FucWf5GH95 participates in fucoidan debranching. The obtained data indicate different roles of fucosidases of the GH29 and GH95 families in the process of fucoidan degradation by the marine bacteria W. fucanilytica CZ1127T.

Effect of Gamma Radiation on Cytogenetic and Growth Endpoints of Allium cepa Seedlings in Long-Term Experiments.

The effect of γ-radiation on the growth and cytogenetic endpoints of Allium cepa L. seedlings in a long period after irradiation in absorbed doses from 1.0 to 10.7 Gy were examined. The chromosome aberration rate was most sensitive to the radiation: it increased immediately after exposure at all doses. In the recovery period (up to 216 h) after irradiation, the chromosome aberration frequency naturally decreased but at the end of experiment in maximum doses remained above the control values. The impact of the initial level of chromosome aberrations on the inhibition of onion roots growth in the long terms after irradiation is discussed.

Discovery of a fucoidan endo-4O-sulfatase: Regioselective 4O-desulfation of fucoidans and its effect on anticancer activity in vitro.

Fucoidans are a class of sulfated fucose-containing bioactive polysaccharides produced by brown algae. The biological effects exhibited by fucoidans are thought to be related to their sulfation. However, the lack of methods for sulfation control does not allow for a reliable conclusion about the influence of the position of certain sulfate groups on the observed biological effects. We identified the gene encoding the endo-acting fucoidan sulfatase swf5 in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. This is the first report on the sequence of fucoidan endo-sulfatase. Sulfatase SWF5 belongs to the subfamily S1_22 of the family S1. SWF5 was shown to remove 4O-sulfation in fucoidans composed from the alternating α-(1→3)- and α-(1→4)-linked residues of sulfated L-fucose but not from fucoidans with the α-(1→3)-linked backbone. The endo-sulfatase was used to selectively prepare 4O-desulfated fucoidan derivatives. It was shown that the 4O-desulfated fucoidans inhibit colony formation of DLD-1 and MCF-7 cells less effectively than unmodified fucoidans. Presumably, 4O-sulfation makes a significant contribution to the anticancer activity of fucoidans.

Revision of Flor's Gene-for-Gene Theory for the Phenomena of Interaction of Cereals Seedlings with Rust Pathogens.

Monopustule isolates of wheat, oats and barley rust pathogens reproduced under different environmental conditions were used to infect experimental samples of these crops. Differences in the types of reactions after infection of one plant genotype with one pathogen genotype reproduced at different temperatures, as well as in the presence of potassium chloride, ammonium nitrate and maleic acid hydrazide indicate the impossibility of explaining the phenomena of plant-pathogen interaction within the framework of Flor's classical gene-for-gene theory. Each gene of the host resistance corresponds to several complementary virulence genes, or to several different alleles of one same gene for virulence.

Prokaryotic community formation on polyethylene films incubated for six months in a tropical soil.

Polyethylene film is one of the most common types of recalcitrant plastic waste materials. Information regarding the fate of plastic films in soil is scarce compared to the fate of plastic films in aquatic environments. The aim of this study was to evaluate the effects of soil fauna and of impregnation of polyethylene films with oil on the colonization of low-density polyethylene films by prokaryotic communities. The field experiment was performed in a monsoon tropical forest (Vietnam). Polyethylene films were incubated in thermally pre-defaunated soil isolated from the surrounding soil by a stainless steel mesh. Three mesh sizes were used, allowing access to different size groups of soil fauna. The diversity, taxonomic structure and co-occurrence patterns in prokaryotic communities were studied using high-throughput sequencing of 16S rRNA gene libraries. The prokaryotic communities that formed on polyethylene films were slightly different from those inhabiting the surrounding soil. Contrary to our expectations, no difference in the diversity of prokaryotes was observed between microcosms with different mesh sizes. Oil impregnation also had only a minor influence on the prokaryotic community structure. Polyethylene films incubated in microcosms with soil appeared to be colonized by various consortia of prokaryotes as a barren and inert surface.

Expression and biochemical characterization of two recombinant fucoidanases from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T.

Genomic analysis of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T revealed the presence of four fucoidanase genes fwf1, fwf2, fwf3, fwf4 that belonged to the glycoside hydrolase family 107 (GH107, CAZy), which is located in one gene cluster putatively involved in fucoidan catabolism. Genes encoding two fucoidanases fwf1 and fwf2 were cloned, and the proteins FWf1 and FWf2 were produced in Escherichia coli cells. The recombinant fucoidanases were purified and the biochemical properties of these enzymes were studied. The amino acid sequences of FWf1 and FWf2 showed 41 and 51% identity respectively with a fucoidanase FcnA from the marine bacterium Mariniflexile fucanivorans, with the established 3D structure. Structures of the oligosaccharides produced during enzymatic hydrolysis of fucoidan by FWf1 and FWf2 have been determined by NMR spectroscopy. Detailed substrate specificities of FWf1 and FWf2 were studied using fucoidans and sulfated fucooligosaccharides with different structures. Both fucoidanases catalyzed hydrolysis of 1→4-glycosidic bonds between sulfated α-l-fucose residues but had different specificities regarding sulfation patterns of the fucose residues in fucoidan molecules. Specific cleavage sites recognizable by the fucoidanases in fucoidan molecules were determined. The obtained results provide new knowledge about differences between specificities of the fucoidanases belonging to the GH107 family.

THE ROLE OF DNA-METHYLTRANSFERASES IN THE LIFE CYCLE OF HEPATITIS B VIRUS AND PATHOGENESIS OF CHRONIC HEPATITIS B.

Chronic hepatitis B is caused by a persistent form of hepatitis B virus, covalently closed circular DNA (cccDNA). Stability of cccDNA is associated with intracellular localization of cccDNA and formation of minichromosome, regulated by epigenetic mechanisms. One of the key mechanisms in epigenetics is methylation of DNA on CpG islands. Expression levels of DNA-methyltransferases (DNMTs) in chronic hepatitis B patients were shown to be upregulated. Nevertheless, the role of DNMTs in the life cycle of HBV and their effects on the cell remain elusive. In this review, we discuss latest achievements on the role of DNMTs in chronic hepatitis B and HBV in vitro models.

[Hepatitis C can be cured: will hepatitis B become next?] / Gepatit S stal izlechim. Gepatit V - sleduiushchii?

Chronic hepatitis B (CHB) and C (CHC) are one of the leading causes of cirrhosis and liver cancer with over a million of people dying annually from their consequences. In Russia CHB and CHC morbidity and related mortality show an upward trend. As a result of recent breakthroughs in antiviral therapeutics CHC became a curable disease. Modern therapeutics effectively suppress viral replication in CHB patients, but withdrawal of antivirals usually results in disease relapse. Loss of HBsAg required for the so called 'functional cure' is a very rare event. Moreover, 'complete cure' when the virus is entirely eliminated from the body is not possible due to a persistent form of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in hepatocytes refractory to modern antivirals. Today, there is a plethora of new promising medications being at different stages of development that target different steps of viral life cycle, including inhibitors of interaction between HBV and its entry receptor NTCP, inhibitors of HBV cccDNA, inhibitors of nucleocapsid assembly, technologies of genome editing (TALENs, CRISPR/Cas etc) and RNA-interference. In addition to direct acting antivirals, there is a number of approaches aimed at enhancement of the innate and adaptive immune responses. In experimental conditions, some of these approaches or their combinations help to achieve functional cure. However, complete elimination of the virus is possible only using technologies of genome editing, capable of specific cccDNA degradation. Nuclease systems are currently at their early stages of development, and there is a long way to prove their efficacy and safety. Nevertheless, highly promising results of the recent years leave no doubt that CRISPR/Cas systems and similar technologies can become the basis of CHB therapy.

[Overexpression of DNA-methyltransferases in persistency of cccDNA pool in chronic hepatitis B]. / Rol' giperékspressii DNK-metiltransferaz v persistentsii kol'tsevoi kovalentno zamknutoi DNK pri khronicheskom gepatite V.

AIM: To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. MATERIAL AND METHODS: Human hepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. RESULTS: DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P<0.005) after transfection of DNMT3A, but is unaltered under DNMT1 treatment. CONCLUSION: DNMT3A regulates the size of cccDNA pool and is important for persistency of HBV infection.

The first results of using the Allium test in estimating the chemical and radiation toxicity of bottom sediments in the Yenisei River.

The first results of the use of the Allium test for estimation of toxicity of bottom sediments in the Yenisei River and the effect of external γ-radiation under laboratory conditions are presented. The effect of stimulation of the onion root growth, i.e., the absence of toxicity was discovered in toxicological experiments using bottom sediments and under external γ-radiation. The stimulating effect of radiation on the growth of onion roots limits the use of the Allium test for testing samples from the Yenisei River ecosystem in the zone subjected to the impact of radioactive discharges from the Mining and Chemical Combine.

[Effect of Echinacea purpurea tincture and its polysaccharide complex on the efficacy of cytostatic therapy of transferred tumors].

Experiments on C57LB/6 mice with transplanted Luis lung carcinoma showed that the officinal Echinacea purpurea preparation did not influence the efficacy of cytostatic therapy. This echinacea preparation did not change the development of metastases and even stimulated the tumor growth. In contrast, a hydrophilic polysaccharide complex isolated from echinacea increased the antitumor and antimetastatic activity of cyclophosphamide.

[Factors influencing the results of osteodensitometry in searching for systemic osteoporosis].

Osteodensitometry (ODM) performed in 485 persons aged 15 to 83 years, referred for ODM due to their having various causes, has demonstrated a significant reduction in bone density in both males and females above 50 years of age. The density of each vertebra individually in patients without cancer and inflammation of the spine is different, even in youth. The difference between the density of the bodies of individual vertebra increases with age. The vertebral density difference is particularly great in osteoplastic metastases. All the processes accompanied by osteogenesis or calcification both within and around the bone increase ODM values excessively. ODM values are decreased by not only osteoporosis, but also by any other bone destruction (tumorous, inflammatory, non-inflammatory), the increased area of a portion under study due to neoplasia of an immature bone, and by intestinal gas at the level of lumbar vertebrae. The older the patient is, the more factors distorting the results of ODM are. The role of each factor is reflected in the present communication. Not only the densitometric, but also X-ray patterns of the area under study should be taken into account to give an objective and final assessment of a specific situation.

First Record of Barley yellow striate mosaic virus and Cereal yellow dwarf virus-RPV Infecting Wheat in Uzbekistan.

A preliminary survey to identify virus diseases affecting wheat in Uzbekistan was conducted during May 2001. The survey covered 12 wheat fields from 2 cereal-growing regions (Tashkent-Angren and Tashkent-Samarkand). A total of 250 wheat samples with symptoms suggestive of virus infection were collected and tested for the presence of nine viruses by tissue-blot immunoassay (TBIA) (1) at the Virology Laboratory of ICARDA, Aleppo, Syria, using the following antisera: monoclonal antibodies for Cereal yellow dwarf virus-RPV (CYDV-RPV) (ATCC PVAS-669 [American Type Culture Collection, Manassas, VA]) and Barley yellow dwarf virus-MAV (BYDV-MAV) (ATCC PVAS-673); and polyclonal antibodies for BYDV-SGV and BYDV-RMV (3); BYDV-PAV, Barley stripe mosaic virus, and Wheat streak mosaic virus (from Virology Laboratory, ICARDA); Wheat dwarf virus (provided by J. Vacke, Research Institute of Crop Production, Prague, Czeck Republic); and Barley yellow striate mosaic virus (BYSMV) isolated from Lebanon (2). The most common virus present was BYDV-PAV (detected in 12% of the 250 samples tested), followed by BYDV-SGV (10.8%), BYSMV (5.6%), BYDV-RMV (2.4%), BYDV-MAV (2%), and CYDV-RPV (1.2%). CYDV-RPV was detected in three fields; one field was 50 km southeast of Tashkent, and the other two fields were between Tashkent and Samarkand. The majority of BYSMV-positive samples originated from the same field, ≈40 km northeast of Samarkand. Field symptoms of BYSMV-infected plants included yellow flag leaf and stunting. All samples that produced a positive reaction to BYSMV-Lebanon antiserum were tested against four other rhabdovirus antisera: BYSMV-Italy, BYSMV-Morocco, Cereal chlorotic mottle virus, and American wheat striate mosaic virus. Serological tests showed that 100% of the samples reacted strongly with BYSMV-Italy and BYSMV-Morocco. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, extracts from BYSMV-infected plants were found to contain 66- and 47-kDa structural proteins, typical of G and N proteins of rhabdoviruses, both of which reacted strongly with BYSMV-Italy antiserum. To our knowledge, this is the first report of BYSMV and CYDV-RPV in Uzbekistan. References: (1) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (2) K. M. Makkouk et al. Plant Dis. 85:446, 2001. (3) G. N. Webby and R. M. Lister. Plant Dis. 76:1125, 1992.