[Show abstract][Hide abstract] ABSTRACT: The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.
Acta Naturae 04/2015; 7(1):87-97. · 1.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two novel mutations in glucokinase (GCK) gene-G to C substitution at -1 position of intron 7 acceptor splice site (c. 864-1G>C) and synonymous substitution c. 666C>G (GTC>GTG, p.V222V) in exon 6--were identified in patients with monogenic diabetes MODY2 (Maturity Onset Diabetes of Young). GCK minigenes with these mutations were constructed. Analysis of splicing products upon transfection of minigenes into human embryonic cell line HEK293 has shown that each of these nucleotide substitutions impair normal splicing. Mutation c.864-1G>C blocks the usage of normal acceptor site which activates cryptic acceptor splice sites within intron 7 and generates aberrant RNAs containing the portions ofintron 7. Synonymous substitution c.666C>G creates novel donor splice site in exon 6 that leads to formation of defective GCK mRNA with deletion of 16 nucleotides of exon 6. Analysis of in vitro splicing of minigenes confirms the inactivating action of novel mutations on glucokinase expression.
[Show abstract][Hide abstract] ABSTRACT: Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of the thermal neutron capture by some non-radioactive elements (e.g., Gds57), which results in subsequent emission of electrons and gamma rays. An effective instrument for delivery of gadolinium into the tumor tissue are the particles of the "rigid" nanostructures (NS) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in such nanostructures may reach 40%. To optimize the process of neutron capture therapy it is very important to investigate possible penetration mechanisms of NS-Gd particles into the tumor cells. In this work, the dynamics of interaction NS-Gd with cultivated chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. It is shown that NS-Gd are able to enter CHO cells. This process begins in about 1 hour after the start ofincubation. After 6 h NS-Gd particles were detected in almost all cells. A further increase of the incubation time does not lead to significant changes in cell morphology, although the number NS-Gd inside the cells increases. The plasma membrane of the cells remains intact. The NS-Gd particles, which entered the cells, remain inside the cells for a long time. The data obtained show that NS-Gd are relatively low-toxic and suggest that the presence of NS-Gd in the tumor cells does not prevent their division. The data obtained are important for improving the efficiency of the neutron capture therapy method.
[Show abstract][Hide abstract] ABSTRACT: Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position −1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.
[Show abstract][Hide abstract] ABSTRACT: Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of thermal neutron capture by some nonradioactive elements (e.g., Gd157), which results in the sub-sequent emission of electrons and gamma rays. An effective instrument for delivering gadolinium into tumor tissue are “rigid” nanostructures (NSs) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in these nanostructures may reach 40%. To optimize the process of neutron capture therapy, it is very important to investigate possible mechanisms of the penetration of NS-Gd particles into tumor cells. In this work, the dynamics of interaction between NS-Gd and cultivated Chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. NS-Gd were shown to be able to enter CHO cells. This process started after about 1 h of incubation. After 6 h, NS-Gd particles were detected in almost all cells. A further increase in the incubation time did not lead to significant changes in cell morphology, although the amount of NS-Gd inside cells continued to increase. The plasma membranes of the cells remained intact. Once entering the cells, NS-Gd particles remained there for a long time. The data show that NS-Gd has relatively low toxicity and suggest that the presence of NS-Gd in tumor cells does not prevent their division. The data are important for improving the efficiency of the method of neutron-capture therapy.
[Show abstract][Hide abstract] ABSTRACT: A diverse collection of 40 derivatives of benzohydroxamic acid (BHAs) of various structural groups were synthesized and tested against hepatitis C virus (HCV) in full-genome replicon assay. Some of these compounds demonstrated an exceptional activity, suppressing viral replication at sub-micromolar concentrations. The compounds were inactive against key viral enzymes NS3, and NS5B in vitro assays, suggesting host cell inhibition target(s). The testing results were consistent with metal coordination by the BHAs hydroxamic group in complex with a target(s). Remarkably, this class of compounds did not suppress poliomyelitis virus (PV) propagation in RD cells indicating a specific antiviral activity of BHAs against HCV.
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.
[Show abstract][Hide abstract] ABSTRACT: Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.
[Show abstract][Hide abstract] ABSTRACT: The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 μg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.
[Show abstract][Hide abstract] ABSTRACT: Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells as well as Kasumi-1 cells from the patient with AML.
[Show abstract][Hide abstract] ABSTRACT: Novel mutation in CYP21A2 gene causing the steroid 21-hydroxylase deficiency - C to G substitution in 7-position ofintron 2 acceptor splice site (c.290-7C>G) was identified. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in the cultured mammalian cells. The mutation impairs the usage of intron 2 acceptor splice site resulting in intron retention.
[Show abstract][Hide abstract] ABSTRACT: First time p53 was found in the complex with viral large T-antigene in the cells transformed by small DNA virus SV40. The cloning of p53 cDNA was done in the beginning of eighties and soon after that the whole p53 gene was cloned. The p53 family is comprised of three genes: TP53,TP63 and TP73, each of which is expressed as a set of structurally and functionally different isoforms. All of them intensively interact with each other forming a united functional network of proteins. In this review we discuss evolution of the p53 family and significance of all its members in embryonic development, reproduction, regeneration, regulation of aging and life span, as well as in the body's defense against cancer. With special attention we review the role of less studied members of the p53 family: p63 and p73, in oncogenesis and tumor progression and show that different isoforms of these proteins might exert a contrary effect on these processes.
[Show abstract][Hide abstract] ABSTRACT: In the present study we have applied the siRNA approach for substantial reduction of AML1-ETO and RUNX1 (K83N) expression, which are frequently found in the leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5'-untranslated region of mRNA for the mutant RUNX1 (K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of the oncogenes following the introduction of shRNAs into cells.
[Show abstract][Hide abstract] ABSTRACT: Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins
in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient
transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in
vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter,
green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery
of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein
complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression
of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.
Key wordsbaculovirus AcMNPV-BacMam virus-hepatitis C virus (HCV)-HCV structural proteins-HCV-like particles-mammalian cells HEK293T-COS-7 and Huh7-Sf9 insect cells
[Show abstract][Hide abstract] ABSTRACT: Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work,
SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian
cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer.
In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels;
the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These
results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.
[Show abstract][Hide abstract] ABSTRACT: Our aim was to investigate how replication protein A (RPA) in a wide range of concentration can regulate the activity of human telomerase. We used an in vitro system based on human cell extracts with or without RPA. It has been shown that removal of RPA leads to loss of telomerase activity and addition of RPA restores telomerase activity and at the same time promotes telomerase processivity. However, high excess of RPA inhibited telomerase processivity and caused the synthesis of relatively short DNA fragments (about 50-100 nucleotides). We assume that, together with other telomere-binding proteins, RPA may take part in activation of telomere overhang elongation by telomerase at a certain stage of a cell cycle as well as in regulation of telomere length.
[Show abstract][Hide abstract] ABSTRACT: An enzymatic assay system was developed to quantify the distribution of recombinant proteins over various cell structures.
The system takes advantage of α-complementation of β-galactosidase. The large ω fragment of β-galactosidase is expressed in
predefined cell structures with the aid of attached protein localization signals. The resulting reporter cell lines are infected
with a second construct expressing a target protein fused with the shorter α fragment of β-galactosidase. The physical proximity
of the two recombinant proteins carrying the β-galactosidase fragments results in the reconstitution of an active enzyme,
and its activity is measured with a plate reader. The recombinant constructs are based on lentiviral vectors and can be rapidly
and efficiently introduced into cells by infection with stocks of lentivirus particles. The efficiency of the system was demonstrated
with the FOXO3A transcription factor, which shuttles between the cytoplasm and nucleus in the model colon carcinoma cell line