Перегляд за Автор "Boretsky, Yuriy"

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Aspartate aminotransferase from an alkalophilic Bacillus contains an additional 20 - amino acid extension at its fu nctionally i mportant N - terminus
(1996) Battchikova, Natalia; Koivulehto, Marianne; Denesyuk, Alexander; Ptitsyn, Leonid; Boretsky, Yuriy; Hellman, Jukka; Korpela, Timo; Борецький, Юрій
Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (Mr 47,439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.
Correction of the functional state of 5 - 9 - grade students at rural schools selected for special medical groups due to articular manifestations of connective tissue dysplasia in Ukraine
(2017) Tymochko-Voloshyn, R.; Trach, Volodymyr; Boretsky, Yuriy; Dyka, M.; Тимочко-Волошин, Роксолана; Трач, Володимир; Борецький, Юрій; Дика, М.
The most frequent signs of connective tissue dysplasia and its links with a general morbidity of pupils were summarized. Health problems and some means for physical education of Ukrainian rural school pupils that were classified to special medical group due to connective tissues dysplasia are described in the article. Based on obtained data we tried to develop a complex approach that can be used during school year to stimulate moving activi ty and to improve a functional state of cardio - vascular and respiratory systems of special medical groups pupils possessing joint hypermobility caused by connective tissue dysplasia. Then evaluation of the developed approach was done by studying pupil’s heart rate and applying different tests such as Stange test, Hench test , indexes of Ruffier and Robinson, vegetative index of Kerdo. As a result, positive influence of the developed program on a physical state of pupils enrolled was confirmed. Thus, the de veloped program can be used in Ukrainian rural schools to teach pupils exhibiting connective tissue dysplasia in order to compensate their lack of physical activity and improve their functional state thus preventing morbidity and development of complications.
Creatinine is a biochemical marker for assessing how untrained people adapt to fitness training loads
(2020-05-25) Chernozub, Andrii; Potop, Vladimir; Korobeynikov, Georgiy; Carmen Timne, Olivia; Dubachinskiy, Oleg; Ikkert, Oksana; Briskin, Yuriy; Boretsky, Yuriy; Korobeynikova, Lesia; Бріскін, Юрій; Борецький, Юрій
To study the peculiarities of changes in creatinine concentration in blood serum of untrained men during the prolonged usage of training loads different in volume and intensity, and to determine the value of this biochemical marker for the assessment of adaptive body changes during fitness training.
Deficiency in frataxin homolog ue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation
(2009) Pynyaha, Yuriy; Boretsky, Yuriy; Fedorovych, Daria; Fayura, Lubov; Levkiv, Andriy; Ubiyvovk, Vira; Protchenko, Olha; Philpott, Caroline; Sibirny, Andriy; Борецький, Юрій
Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Dyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Dyfh1 mutant was *3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Dyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.
Development of a transformation system for gene knock - out in the flavinogenic yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Pynyaha, Yuriy; Boretsky, Volodymyr; Kutsyaba, Vasyl; Protchenko, Olga; Philpott, Caroline; Sibirny, Andriy; Борецький, Юрій
Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5′-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib− Ura+ phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3–12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3′ end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8–0.9 kb sequences homologous to the target gene.
Identification of an ARS element and development of a high efficiency transformation system for Pichia guilliermondii
(1999) Boretsky, Yuriy; Voronovsky, Andriy; Liuta-Tehlivets, Oksana; Hasslacher, Meinhard; Kohlwein, Sepp D.; Shavlovsky, Georgiy M.; Борецький, Юрій
An Autonomously Replicating Sequence ele- ment adjacent to the RIB1 gene encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identi®ed by transformation experiments. Detailed se- quence analysis unveiled two potential ARS elements located 5¢ and 3¢ of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like se- quence 3¢ to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 104±105 transformants/lg of DNA to a pUC19-derived plasmid in P. guilliermondii. The PgARS element also conferred autonomous replication to hybrid plasmids in this host. Based on this element a series of Escherichia coli shuttle vectors for e cient transformation of the ¯avinogenic yeast P. guilliermondii was developed.
Identification of the genes a¡ecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis
(2011) Boretsky, Yuriy; Pynyaha, Yuriy; Boretsky, Volodymyr; Fedorovych, Dariya; Fayura, Lyubov; Protchenko, Olha; Philpott, Caroline; Sibirny, Andriy
Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Dvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Dfra1-45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Dvma1-17 and Dfes1-77 knockout strains could not grow at 37 1C in contrast to the wild-type strain and the Dfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 1C. Although the Dfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Dvma1-17 and Dfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.
Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli
(2013) Fayura, Lyubov; Boretsky, Yuriy; Pynyaha, Yuriy; Wheatley, Denys; Sibirny, Andriy; Борецький, Юрій
Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl -d-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30–34 U/mg for 12 months when stored at 4 ◦C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.
Improving the efficiency of plasmid transformation in Shewanella oneidensis MR-1 by removing ClaI restriction site
(2014) Rachkevych, Nazarii; Sybirna, Kateryna; Boyko, Solomiya; Boretsky, Yuriy; Sibirny, Andriy; Борецький, Юрій
Here we demonstrate that elimination of ClaI restriction site from the sequence of a plasmid DNA increases the efficiency of transformation of Shewanella oneidensis MR-1 significantly. To achieve reliable transformation of S. oneidensisMR-1 plasmids either lacking ClaI site or isolated from primary transformants of S. oneidensis should be used.
Molecular Cloning of the GTP - Cyclohydrolase Structural Gene RIB1 of Pichia guilliermondii involved in riboflavin biosynthesis
(1995) Liauta - Teglivets, O.; Hasslache, M.; Boretsky, Yuriy; Kohlwein, S.; Shavlovskii, G.
The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase 11, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38 71 1 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type I1 from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiue. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits. The sequence of the RlBl gene of P. guilliermondii was submitted to the EMBL sequence database and is accessible under Accession Number 249093.
Molecular Cloning of the GTP-Cyclohydrolase Structural Gene RIB1 of Pichia guiZZiermondii Involved in Riboflavin Biosynthesis
(1995) Liauta-Teglivets, O.; Hasslacher, M.; Boretsky, Yuriy; Konlwein, S.; Shavlovskii, G.; Борецький, Юрій
The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase 11, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38 71 1 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type I1 from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiue. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits. The sequence of the RlBl gene of P. guilliermondii was submitted to the EMBL sequence database and is accessible under Accession Number 249093.
Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Protchenko, Olga; Prokopiv, Tetiana; Mukalov, Igor; Fedorovych, Daria; Sibirny, Andriy
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.
Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Protchenko, Olga; Prokopiv, Tetiana; Mukalov, Igor; Fedorovych, Daria; Sibirny, Andriy; Борецький, Юрій
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.
Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities
(2013) Prokopiv, Tetyana; Fedorovych, Dariya; Boretsky, Yuriy; Sibirny, Andriy; Борецький, Юрій
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the nonflavinogenic yeast Saccharomyces cerevisiae.
Pichia guilliermondii
(2009) Sibirny, Andriy; Boretsky, Yuriy; Борецький, Юрій
Pichia guilliermondii (asporogenous strains of this species are designated as Candida guilliermondii ) is the model organism of a group so named “ flavinogenic yeasts ” capable of riboflavin oversynthesis during starvation for iron. Besides, some strains of this species efficiently convert xylose to xylitol, an anti-caries sweetener. However, there are also pathogenic C. guilliermondii strains. This species has been used for studying enzymology of riboflavin synthesis due to overproduction of participating enzymes and intermediates under iron-limiting conditions as well as for identification of genes of negative and positive action involved in such a regulation. Besides, P. guilliermondii was used for identification and studying the properties of the systems for active transport of riboflavin in the cell (riboflavin permease) and out of the cell (riboflavin “ excretase ” ). The genetic line of P. guilliermondii with high fertility has been selected and the methods of classic genetics (hybridization and analysis of meiotic segregation) have been developed. More recently, tools for molecular genetic studies of P. guilliermondii have been developed which include collection of host strains, vectors with recessive and dominant markers, several transformation protocols including that for gene knock out. Recently, the genome of this yeast species was sequenced and become publicly available ( http://www.broad.mit.edu ).
Positive selection of mutants defective in transcriptional repression of riboflavin synthesis by iron in the flavinogenic yeast Pichia guilliermondii
(2005) Boretsky, Yuriy; Kapustyak, Kostyantyn; Fayura, Lyubov; Stasyk, Oleh; Stenchuk, Mykola; Bobak, Yaroslav; Drobot, Lyudmyla; Sibirny, Andriy; Борецький, Юрій
It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.
Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics
(2015) Stasyk, Oleh; Boretsky, Yuriy; Gonchar, Mykhailo; Sibirny, Andriy
Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed.
Restoration of the Wild-Type Phenotype in Pichia guilliermondii Transformants
(2002) Pinyaga, Yu.; Prokopiv, T.; Petrishin, A.; Khalimonchuk, O.; Protchenko, O.; Fedorovich, D.; Boretsky, Yuriy; Борецький, Юрій
Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB1 of this enzyme. Experiments showed that the presence of the ARS element and the promoter region of this gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondii are discussed.
The Pleiotropic Nature of rib80, hit1 , and red6 Mutations Affecting Riboflavin Biosynthesis in the Yeast Pichia guilliermondii
(2007) Fayura, Lyubov; Fedorovych, Daria; Prokopiv, Tetiana; Boretsky, Yuriy; Sibirny, Andriy
The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1 , and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6 ) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe–S cluster proteins as aconitase and flavocytochrome b2, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.
The response to iron deprivation in Saccharomyces cerevisiae : expression of siderophore - based systems of iron uptake
(2002) Philpott, C.; Protchenko, О.; Kim, Y .; Boretsky, Yuriy; Shakoury - Elizeh, M.; Борецький, Юрій
The budding yeast Saccharomyces cerevisiae responds to growth in limiting amounts of iron by activating the transcription factor Aftlp and expressing a set of genes that ameliorate the effects of iron deprivation. Analysis of iron-regulated gene expression using cDNA microarrays has revealed the set of genes controlled by iron and Aftlp. Many of these genes are involved in the uptake of siderophore-bound iron from the environment. One family of genes, FIT1, FIT2 and FIT3, codes for mannoproteins that are incorporated into the cell wall via glycosylphosphatidylinositol anchors. These genes are involved in the retention of siderophore-iron in the cell wall. Siderophorebound iron can be taken up into the cell via two genetically separable systems. One system requires the reduction and release of the iron from the siderophore prior to uptake by members of the Fre family of plasma-membrane metalloreductases. Following reduction and release from the siderophore, the iron is then taken up via the high-affinity ferrous transport system. A set of transporters that specifically recognizes siderophore- iron chelates is also expressed under conditions of iron deprivation. These transporters, encoded by ARNI, ARN2/ TAFl, ARN3/SIT1 and ARN4IENB1, facilitate the uptake of both hydroxamate- and catecholate-type siderophores. The Arn transporters are expressed in intracellular vesicles that correspond to the endosomal compartment, which suggests that intracellular trafficking of the siderophore and/or its transporter may be important for uptake.