Browsing by Author "Baryshev, Mikhail"
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Item Early Cell Cultures from Prostate Cancer Tissue Express Tissue Specific Epithelial and Cancer Markers(2023-02-01) Ryabov, Vladimir M.; Baryshev, Mikhail; Voskresenskiy, Mikhail A.; Popov, Boris V.; Institute of Microbiology and VirologyProstate cancer (PCa) is a widespread oncological disease that proceeds in the indolent form in most patients. However, in some cases, the indolent form can transform into aggressive metastatic incurable cancer. The most important task of PCa diagnostics is to search for early markers that can be used for predicting the transition of indolent cancer into its aggressive form. Currently, there are two effective preclinical models to study PCa pathogenesis: patients derived xenografts (PDXs) and patients derived organoids (PDOs). Both models have limitations that restrict their use in research. In this work, we investigated the ability of the primary 2D prostate cell cultures (PCCs) from PCa patients to express epithelial and cancer markers. Early PCCs were formed by epithelial cells that were progressively replaced with the fibroblast-like cells. Early PCCs contained tissue-specific stem cells that could grow in a 3D culture and form PDOs similar to those produced from the prostate tissue. Early PCCs and PDOs derived from the tissues of PCa patients expressed prostate basal and luminal epithelial markers, as well as cancer markers AMACR, TMPRSS2-ERG, and EZH2, the latter being a promising candidate to mark the transition from the indolent to aggressive PCa. We also identified various TMPRSS2-ERG fusion transcripts in PCCs and PDOs, including new chimeric variants resulting from the intra- and interchromosomal translocations. The results suggest that early PCCs derived from cancerous and normal prostate tissues sustain the phenotype of prostate cells and can be used as a preclinical model to study the pathogenesis of PCa.Item ERp29 triggers a conformational change in polyomavirus to stimulate membrane binding(2005-10-28) Magnuson, Brian; Rainey, Emily K.; Benjamin, Thomas; Baryshev, Mikhail; Mkrtchian, Souren; Tsai, BillyMembrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.Item Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex(2002-05-10) Sargsyan, Ernest; Baryshev, Mikhail; Szekely, Laszlo; Sharipo, Anatoly; Mkrtchian, SourenFolding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated colocalization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.Item A new device‑mediated miniprep method(2022) Baryshev, Mikhail; Merkulov, Dmitrijs; Mironov, Ivan; Institute of Microbiology and VirologySmall-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45 degrees and oscillation frequency of 400 +/- 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45 degrees does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270 degrees, 180 degrees and 90 degrees. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs. Keypoints New devise-mediated plasmid miniprep method (DM) performs all mixing steps without operator intervention. The DM method produces plasmid DNAs free of the dCCC form and significantly reduces the contamination with genomic DNA in the low-copy-number plasmid. DM miniprep plasmids are reliable templates for bisulfite PCR sequencing analysis.Item Oligomerization properties of ERp29, an endoplasmic reticulum stress protein(1998-07-24) Mkrtchiana, Souren; Baryshev, Mikhail; Matvijenko, Olga; Sharipo, Anatoly; Sandalova, Tatyana; Schneider, Gunter; Ingelman-Sundberg, Magnus; Institute of Microbiology and VirologyERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.Item Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter(2022-01) Baryshev, Mikhail; Petrov, Nikolai; Ryabov, Vladimir; Popov, Boris; Institute of Microbiology and VirologyThe retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB, despite being G-418 resistant. However, the consequences of the transient production of exogenous RB had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except −60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.Item Upregulation of the chemokine receptor CCR2B in Epstein-Barr Virus-positive Burkitt lymphoma cell lines with the latency III program(2018-05-03) Kozireva, Svetlana; Rudevica, Zhanna; Baryshev, Mikhail; Leonciks, Ainars; Kashuba, Elena; Kholodnyuk, Irina; Institute of Microbiology and VirologyCCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein-Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.