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A fundamental difference between somatic nuclei (macronuclei) of ciliates and cell nuclei of higher eukaryotes is that the macronuclear genome is a huge number (up to tens or hundreds of thousands) of gene-sized (0.5-25 kb) or subchromosomal (up to 2000 kb) minichromosomes. Electron microscopy shows that macronuclear chromatin usually looks like chromatin bodies or fibrils 200-300 nm thick in the interphase. However, the question of how many DNA molecules are contained in an individual chromatin body remains open. The organization of chromatin in macronuclei was studied in the ciliates Didinium nasutum and three Paramecium sp, which differ in pulsed-field gel electrophoresis (PFGE) karyotype, and compared with the model of topologically associated domains (TADs) of higher eukaryotic nuclei. PFGE showed that the sizes of macronuclear DNAs ranged from 50 to 1700 kb, while the majority of the molecules were less than 500 kb in length. A comparative quantitative analysis of the PFGE and electron microscopic data showed that each chromatin body contained one minichromosome in P. multimicronucleatum in the logarithmic growth phase, while bodies in the D. nasutum macronucleus contained two or more DNA molecules each. Chromatin bodies aggregated during starvation, when activity of the macronuclei decreased, leading to an increase of chromatin body size or the formation of 200- to 300-nm fibrils of several chromatin bodies. A model was proposed to explain the formation of such structures. In terms of topological characteristics, macronuclear chromatin bodies with subchromosomal DNA molecules were found to correspond to higher eukaryotic TADs.

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.

The data of transmission electron microscopy (TEM) on morphology of M. tuberculosis H37Rv bacterial cells treated with four analogues of pyrimidine nucleosides with different substituents at 5 position of base are presented. We showed that the growth of M. tuberculosis H37Rv cells effectively inhibited by each of these compounds. This process is accompanied with the accumulation of lipid intracellular vacuole-like inclusions in the cells, appearance of deep protrusions and indentations on the surface, partial and/or complete destruction of the three-layered cell envelope. The exact molecular mechanism of action of 5-substituted pyrimidine nucleosides on M. tuberculosis cells remains to be proved. However, one can suggest that mechanism of action for these compounds is related either to their direct interactions with bacteria cell walls or to interactions with enzymes participating in the process of cell wall formation.

Cobalt(II) salts are synthesized from saturated monocarboxylic acids: formic, butyric, valerianic, caproic, enanthic, and caprylic acids. The resulting compounds are characterized by elemental analysis, IR spectroscopy, thermogravimetry, and differential scanning calorimetry. As a result of the thermal decomposition of the synthesized carboxylates and cobalt acetate, cobalt-containing nanocomposites are obtained. The obtained nanocomposites are studied by elemental analysis, IR spectroscopy, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, and X-ray phase analysis. Magnetic studies of the obtained nanocomposites are carried out.

The degradation of most intracellular proteins is a dynamic and tightly regulated process performed by proteasomes. To date, different forms of proteasomes have been identified. Currently the role of non-constitutive proteasomes (immunoproteasomes (iPs) and intermediate proteasomes (intPs)) has attracted special attention. Here, using a CRISPR-Cas9 nickase technology, four cell lines: histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma, and hepatocarcinoma were modified to express proteasomes with mCherry-tagged β5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the expression of the chimeric gene in modified cells is under the control of endogenous regulatory mechanisms and is increased following IFN-γ and/or TNF-α stimulation. Fluorescent proteasomes retain catalytic activity and are distributed within the nucleus and cytoplasm. RNAseq reveals marginal differences in gene expression profiles between the modified and wild-type cell lines. Predominant metabolic pathways and patterns of expressed receptors were identified for each cell line. Using established cell lines, we demonstrated that anti-cancer drugs Ruxolitinib, Vincristine and Gefitinib stimulated the expression of β5i-containing proteasomes, which might affect disease prognosis. Taken together, obtained cell lines can be used as a platform for real-time studies of immunoproteasome gene expression, localization of iPs and intPs, interaction of non-constitutive proteasomes with other proteins, proteasome trafficking and many other aspects of proteasome biology in living cells. Moreover, the established platform might be especially useful for fast and large-scale experiments intended to evaluate the effects of different conditions including treatment with various drugs and compounds on the proteasome pool.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions—from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.

Extracellular cell matrices deposited by cells stimulate cell proliferation. However, their generation is cumbersome and time consuming. We show here that controlled fixation of fibronectin layers after coating culture vessels significantly enhances expansion of murine and human mesenchymal stem cells (MSCs) and, to a lesser extent, primary fibroblasts. In contrast, fibronection fixation did not stimulate proliferation of established cancer cell lines. Fixed vitronectin or collagen IV layers also enhanced proliferation of murine MSCs. Thus, controlled formaldehyde fixation of layers formed by fibronectin or some other extracellular matrix components represents a simple and reproducible way to enhance proliferation of primary cells.

The efficiency of magnetic labeling with L-Lys-modified Fe

Pediatric cancers represent a wide variety of different tumors, though they have unique features that distinguish them from adult cancers. Receptor tyrosine kinases KIT and TrkA functions in AML and NB, respectively, are well-characterized. Though expression of these receptors is found in both tumors, little is known about KIT function in NB and TrkA in AML. By combining gene enrichment analysis with multidimensional scaling we showed that pediatric AMLs with t(8;21) or inv16 and high KIT expression levels stand out from other AML subtypes as they share prominent transcriptomic features exclusively with KIT-overexpressing NBs. We showed that AML cell lines had a predominant expression of an alternative TrkAIII isoform, which reportedly has oncogenic features, while NB cell lines had dominating TrkAI-II isoforms. NB cells, on the other hand, had an abnormal ratio of KIT isoforms as opposed to AML cells. Both SCF and NGF exerted protective action against doxorubicin and cytarabine for t(8;21) AML and NB cells. We identified several gene sets both unique and common for pediatric AML and NB, and this expression is associated with KIT or TrkA levels. NMU, DUSP4, RET, SUSD5, NOS1, and GABRA5 genes are differentially expressed in NBs with high KIT expression and are associated with poor survival in NB. We identified HOXA10, BAG3, and MARCKS genes that are connected with TrkA expression and are marker genes of poor outcome in AML. We also report that SLC18A2, PLXNC1, and MRPL33 gene expression is associated with TrkA or KIT expression levels in both AML and NB, and these genes have a prognostic value for both cancers. Thus, we have provided a comprehensive characterization of TrkA and KIT expression along with the oncogenic signatures of these genes across two pediatric tumors.

Tyrosine hydroxylase (TH) - the specific marker of the catecholamine synthesizing cells, has been detected in the pancreatic endocrine, acinar and ductal cells in some mammals. In the developing mouse pancreas, TH+ cells are often contained glucagon and shared some features that are characteristics for differentiated endocrine cells. As it was shown in experimental studies on mice, a non-neural catecholaminergic pathway appears to modulate pancreatic endocrine precursor and insulin producing cell neogenesis. Our aim was to evaluate whether TH+ cells are present in the developing human pancreas. We have analyzed pancreatic autopsies from 34 human fetuses (10-37 gestational weeks (g.w.)) using double immunofluorescent labeling with antibodies to TH and either insulin or glucagon. Rare TH+ cells were found in the pancreas of all fetuses from 10th to 21th g.w., which were not detected later in development. At early stages (10-12 g.w.), such cells were located among the epithelial cells of primitive ducts, often in close proximity to the endocrine cell clusters, or were associated with the TH+ nerve bundles. In the pancreas of fetuses from 14th to 21th g.w., TH+ cells were observed in the nerve bundles, nerve ganglia and in the neuro-inular complexes type 1 (endocrine cells integrated with the neurons and nerve fibers). Some of the TH+ cells simultaneously contained glucagon. However, the majority of the TH+ cells were negative for insulin or glucagon. Thus, we first detected TH+ cells in the human fetal pancreas. These cells may be differentiating endocrine cells, because some of the TH+ cells were located in the ductal epithelium and rarely contained glucagon. Similarly with mice, non-neural catecholamines may be involved in the endocrine cells differentiation during human pancreas development. Disclosure Y. Krivova: None. A. Proshchina: None. D. Otlyga: None. O. Leonova: None. V. Popenko: None. V. Barabanov: None. S. Saveliev: None. Funding Russian Foundation for Basic Research (18-015-00146)

Objective . To estimate performance characteristics and diagnostic value of immunofluorescent islet cell antibody (ICA) assay, immunoradiometric glutamic acid decarboxylase antibody (GADA) assay, and ELISA tyrosine phosphatase IA-2 antibody (IA-2A) and insulin antibody (IA) assays. Research Design and Methods . Antibodies were tested in 438 children and adolescents with newly diagnosed diabetes mellitus (DM) type 1, and in 891 subjects without DM type 1. ICA were determined by the classic indirect immunofluorescent method recommended by the Juvenile Diabetes Foundation International, GADA were determined with the Immunotech IRMA Anti-GAD kit, and IA-2A and IA were determined with Medizym Anti-IA2 and Orgentec Anti-Insulin ELISA kits, respectively. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the tests were estimated with contingency tables. Diagnostic accuracy was estimated from areas under receiver operating curves (AUC). Results . ICA test was of the greatest diagnostic value (Se=88%, Sp=96%, PPV=96%, NPV=94%, AUC=0,94), followed by IA-2A (Se=66%, Sp=98%, PPV=98%, NPV=59%, AUC=0,82) and GADA (Se=73%, Sp=84%, PPV=75%, NPV=83%, AUC=0,79). IA test exhibited a very low Se (4,3%) and lacked diagnostic accuracy (AUC=0,5). Conclusions . We recommend to use ICA, IA-2A and GADA tests surveyed in our study for diagnosis of DM type 1 and differential diagnosis of DM. We don’t recommend IA testing with an Orgentec Anti-Insulin ELISA kit for usage in clinical practice.

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1], [2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants., Highlights • Baculovirus expression system in mammalian and insect cells for. • Glycosylation at certain sites of HCV genotype 1b. • Part of glycosylation sites of E1, E2 HCV important for folding of the proteins involved in the formation of viral particles.

The efficiency of magnetic labeling with L-Lys-modified Fe3O4 magnetic nanoparticles (MNPs) and the stability of magnetization of rat adipose-derived mesenchymal stem cells, lineage-negative (Lin(-)) hematopoietic progenitor cells from mouse bone marrow and human leukemia K562 cells were studied. For this purpose, covalent modification of MNPs with 3-aminopropylsilane and N-di-Fmoc-L-lysine followed by removal of N-protecting groups was carried out. Since the degree of hydroxylation of the surface of the starting nanoparticles plays a crucial role in the silanization reaction and the possibility of obtaining stable colloidal solutions. In present work we for the first time performed a comparative qualitative and quantitative evaluation of the number of adsorbed water molecules and hydroxyl groups on the surface of chemically and physically obtained Fe3O4 MNPs using comprehensive FTIR spectroscopy and thermogravimetric analysis. The results obtained can be further used for magnetic labeling of cells in experiments in vitro and in vivo.

A series of novel 5&prime, norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 &mu, g/mL (mc2155) and a MIC99 of 6.7&ndash, 67 &mu, g/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28&ndash, 61 &mu, g/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50&ndash, 60 &mu, g/mL), as well as two virulent strains of M. tuberculosis, a laboratory strain H37Rv (MIC99 20&ndash, 50 &mu, g/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20&ndash, g/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Different types of stress including heat shock may induce genomic instability, due to the derepression and amplification of mobile elements (MEs). It remains unclear, however, whether piRNA-machinery regulating ME expression functions normally under stressful conditions. The aim of this study was to explore the features of piRNA expression after heat shock (HS) exposure in Drosophila melanogaster. We also evaluated functioning of piRNA-machinery in the absence of major stress protein Hsp70 in this species. We analyzed the deep sequence data of piRNA expression after HS treatment and demonstrated that it modulates the expression of certain double-stranded germinal piRNA-clusters. Notable, we demonstrated significant changes in piRNA levels targeting a group of MEs after HS only in the strain containing normal set of hsp70 genes. Surprisingly, we failed to detect any correlation between the levels of piRNAs and the transcription of complementary MEs in the studied strains. We propose that modulation of certain piRNA-clusters expression upon HS exposure in D. melanogaster occurs due to HS-induced altering of chromatin state at certain chromosome regions.

The formation of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules, handled by positively charged superparamagnetic cobalt ferrite nanoparticles, as well as action of these nanoparticles on DNA dispersion, are considered. The binding of magnetic nanoparticles to the linear double-stranded DNA in solution of high ionic strength (0.3 M NaCl) and subsequent phase exclusion of these complexes from polyethylene glycol-containing solutions lead to their inability to form dispersions, whose particles do possess the spatially twisted arrangement of neighboring double-stranded DNA molecules. The action of magnetic nanoparticles on DNA dispersion (one magnetic nanoparticle per one double-stranded DNA molecule) results in such "perturbation" of DNA structure at sites of magnetic nanoparticles binding that the regular spatial structure of DNA dispersion particles "blows up"; this process is accompanied by disappearance of both abnormal optical activity and characteristic Bragg maximum on the small-angle X-ray scattering curve. Allowing with the fact that the physicochemical properties of the DNA liquid-crystalline dispersion particles reflect features of spatial organization of these molecules in chromosomes of primitive organisms, it is possible, that the found effect can have the relevant biological consequences.

Pulsed-field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short "gene-sized" DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80-265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.

The effect of gold (Au) nanoparticles with an average size of about 2 nm on double-stranded DNA cholesteric liquid-crystal dispersion (CLCD) particles has been studied. Treatment of DNA CLCD by Au nanoparticles results in two effects: a “disturbance” of the spatial structure of dispersion particles and the induction of the cholesteric → nematic phase transition, as well as the formation of 40.5–53.0 nm linear clusters of Au nanoparticles between neighboring DNA molecules. The efficiency of formation of these clusters and their size depend on the solution properties. Clusters of Au nanoparticles can crosslink neighboring DNA molecules, thus forming “rigid” DNA CLCD particles. The average size of rigid DNA CLCD particles is 450–500 nm, and their height does not exceed 300 nm. Thus, the effect of Au nanoparticles on DNA CLCD leads to the formation of nanobiomaterial in which clusters of Au nanoparticles are formed between DNA molecules fixed in the spatial structure of dispersion particles. This nanobiomaterial has new physicochemical properties (such as a lack of abnormal optical activity and the presence of linear clusters of Au nanoparticles in the structure of DNA CLCD particles, via which the interaction between neighboring DNA molecules is implemented); as a result, it differs from standard nanobiomaterials based on double-stranded DNA molecules.

In adult skin, hair follicles cyclically self-renew in a manner that recapitulates embryonic hair follicle morphogenesis. The most common pathology of hair in adults is alopecia, which is hair loss to different extent. There are a number of murine models of alopecia including spontaneous mutations. In the present study, we worked with double homozygouswe/we wal/walmice which demonstrate symptoms closely resembling human alopecia. Using whole-mount preparations of epidermis of E18.5 embryos we show that hair follicle defects can be revealed as early as during embryonic morphogenesis in these mutants. The number of hair follicles was reduced almost 1.5-fold in mutant skin. The shape of the early stage small follicles was altered in mutant animals as compared to control ones. Additionally, follicles of mutant embryos were wider at the point of conjunction with interfollicular epidermis. We believe that the mutant mice studied represent a fascinating model to address the problem of hair loss. We demonstrated alterations in the morphogenesis of embryonic hair follicle inwe/we wal/waldouble homozygous mice developing alopecia postnatally. We suppose that incorrect morphogenesis of hair follicles during embryogenesis is closely related to alopecia in the adult life. Unveiling the mechanisms involved in altered embryogenesis may elucidate the pathogenesis of alopecia.

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.

The localization of the P185HER2 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185HER2 receptor is a marker of breast and ovarian tumors; it is also considered to be a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, firstly, to study the distribution of P185HER2 in cancer cells, secondarily, to remove P185HER2 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185HER2 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185HER2 domain fused with two molecules of barnase (cytotoxic RNAase from Bacillus amyloliquefaciens) and its specific inhibitor, barstar. Fluorescent microscopy showed that the module 4D5 scFc-dibarnase:barstar is efficient for identifying P185HER2 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185HER2. The localization of P185HER2 in human ovarian carcinoma cells (SKOV-3) and breast carcinoma cells (BT-474) was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185HER2 is distributed unequally on the cell surface with preferential localization on protrusions or close to their bases and at contacts between protrusions and the cell membrane. At 37°C, P185HER2 is internalized through coated pits and vesicles and concentrates in endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in BT-474 cells.

Gold (Au) nanoparticles of diameter ~2 nm (but not 5 or 15 nm) were capable of incorporating effectively into quasi-nematic layers of particles of cholesteric liquid-crystalline dispersions (CLCD) formed by double-stranded nucleic acids of various families [B-form DNA, A-form poly(I) × poly(C)]. A study of the properties of CLCD particles treated with Au nanoparticles by various physicochemical methods demonstrated that incorporation of Au nanoparticles into quasi-nematic layers of these particles resulted in two effects. First, it caused rearrangement of the spatial cholesteric structure of LC dispersion particles that was accompanied by a decrease in the amplitude of the anomalous band in the circular dichroism spectrum of the corresponding initial CLCD. Second, it induced Au cluster formation within the LC particles that was accompanied by the appearance of a surface plasmon resonance band in the visible spectral region. It was possible that these structural effects were responsible for the genotoxicity of the Au nanoparticles.

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

Overexpression of the P185(HER2) protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185(HER2) in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185(HER2) domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185(HER2) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185(HER2) occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185(HER2) caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185(HER2) was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar-gold complex can be used as a new immunodetection system for exploring the P185(HER2) distribution.

Viral hepatitis C is a dangerous, widespread human disease. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited, and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances is an issue of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis, which determines the correct assembly of HCV virions. Derivatives of a glucose analog deoxynojirimycin (DNJ) act as an α-glucosidase inhibitor and can impair the assembly of structural proteins and HCV particle formation. In the present work, the effects of alkylated DNJ derivatives, N-pentyl-DNJ and N-benzyl-DNJ, on HCV morphogenesis were studied in a model system of insect cells that produce three viral structural proteins with the formation of virus-like particles. It was shown that DNJ derivatives impair the intracellular N-glycosylation of HCV envelope glycoproteins. At the concentration of 1 mM, these substances cause an increase in the levels of gpE1 and gpE2 glycoproteins and a decrease in their electrophoretic mobility, apparently due to the inhibition of α-glucosidase in the endoplasmic reticulum and the accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latter with calnexin results in the formation of unproductive dimers and blocks the productive assembly of virus-like particles.

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.

Amino acid sequence of the drosophila retrovirus MDG4 (gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated the ability of each product to form virus-like particles in the bacterial cell, i.e., in the absence of eukaryotic cell factors. The results obtained showed that both variants of the gypsy protein Gag form globular particles in the bacterial cell.

Both human and other mammalian genomes contain a number of closely linked gene pairs transcribed in opposite directions. Bioinformatic analysis suggests that up to 10% of human genes are arranged in this way. This work reports cloning of a human genome fragment that separates two head-to-head oriented genes located at 2p13.1 and encoding hypothetical proteins with unknown functions: CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. The intergenic region CCDC142-TTC31 overlaps with a CpG island and contains a number of potential binding sites for transcription factors. This fragment functions as a bidirectional promoter in the system of luciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. Vectors containing oppositely oriented genes of two fluorescent proteins: green (EGFP) and red (DsRed2), separated by a fragment of the CCDC142-TTC31 intergenic region, were constructed. Transfection of HEK293 cells with these vectors resulted in simultaneous expression of both fluorescent proteins. The promoter activity was also determined for truncated versions of the intergenic region. The minimal promoter fragment contained Inr, BRE, and DPE elements characteristic for TATA-less promoters. Thus, a novel bidirectional promoter was cloned from the human genome; it can be used for simultaneous constitutive expression of two different genes in human cells.

Cell-to-cell movement of Potato mop-top virus (PMTV) is mediated by three virus-encoded ‘triple gene block’ (TGB) proteins termed TGBp1, TGBp2 and TGBp3. TGBp1 binds virus RNAs to form viral ribonucleoprotein complexes (vRNPs), the transport form of viral genome. TGBp2 and TGBp3 are necessary for intracellular delivery of TGBp1-containing vRNPs to plasmodesmata. To analyze subcellular localization and transport of TGBp1 we used a single binary vector for agrobacterium-mediated co-expression of PMTV TGBp1 fused to green fluorescent protein and TGBp2/TGBp3. At two days post infiltration (dpi) TGBp1 was found in the nucleus and in association with microtubules (MTs). Similar localization pattern was revealed in cells expressing GFP-TGBp1 alone after particle bombardment. At 3 dpi, in addition to the nucleus and MTs, TGBp1 was detected in numerous granular bodies located both along the MTs and at the cell wall. The latter structures co-localized with plasmodesmata-associated callose depositions. At 4 dpi, GFP-TGBp1 was detected in cell wall-associated bodies and also in residual MTs, the nucleoplasm and large perinuclear inclusions resembling aggresomes. Therefore GFP-TGBp1 association with MTs preceded to its localization to plasmodesmata. Disassembly of cell MTs by colchicine prevented GFP-TGBp1 targeting to plasmodesmata and the MT-dependent aggresome formation. Deletion analysis also revealed a correlation between TGBp1 microtubule association and plasmodesmata targeting. We propose that TGBp1 interaction with MTs may be important for the formation of vRNP bodies destined for the transport to plasmodesmata as well as degradation of the excessive TGBp1.

Au-nanoparticles (size about 2 nm, but not 5 or 15 nm) are capable of effectively incorporating into quasinematic layers of particles of cholesteric liquid-crystalline dispersion formed by double-stranded nucleic acid molecules of various families (DNA and poly(I)xpoly(C)). This Au-size-dependent process is accompanied by a decrease in amplitudes of abnormal bands in the CD spectra specific to initial cholesteric liquid-crystalline dispersions and simultaneously by an appearance of plasmon resonance band in visible absorption spectrum. The study of properties of particles of cholesteric liquid-crystalline dispersion treated with Au-nanoparticles by means of various physico-chemical methods demonstrates that incorporation of Au-nanoparticles into quasinematic layers of these particles results in two effects: i) it facilitates reorganization of the spatial cholesteric structure of particles, and ii) it induces the formation of Au-clusters in the content of particles. It is not excluded that these effects represent a possible reason for genotoxicity of Au-nanopar- ticles.

The formation and physico-chemical properties of biomaterial, based on double-stranded (ds) DNA molecules and bearing high concentration of gadolinium, is described. This “rigid” biomaterial demonstrate a few unique properties: (i) the ds DNA molecules forming complexes with gadolinium are fixed in the spatial structure of “rigid” particles, (ii) an abnormal negative band in the circular dichroism spectrum permits to follow the formation of this biomaterial; (iii) local concentration gadolinium in the content of biomaterial can reach 40%. These properties show that we are dealing with a novel type of biomaterial strongly enriched by gadolinium. This opens a gateway for practical application of this biomaterial for neutron-capture reactions. A first attempt to apply this material for neutron-capture reaction in combination with neutron generator of thermal neutron flux was performed. Positive result obtained at destruction of CHO cells allows one to state that the advantages of this biomaterial are a simple manipulation with it, a possibility to adjust its gadolinium content, long-term stability of its physico-chemical properties, as well as a reduced cost of neutron-capture experiment.

A method for investigating the distribution of therapeutic and diagnostic substances inside nanoparticles was developed for spherical amorphous nanoparticles (SANPs). SANPs were generated from the lupan triterpenoids of birch bark (LTs). This method was based on the sequence of short exposures of SANPs in a special solvent. The thin surface layers of the main material and the material of substances incorporated into the SANPs were dissolved. The distribution of three different substances in the SANPs were analyzed by determining the optical density of the solutions containing material extracted from SANPs. It was shown that the distribution of therapeutic and diagnostic substances in SANPs depended on their solubility in the solution saturated with the main SANPs material and their mass fraction.

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Delta(5)-3beta-hydroxycholenic, Delta(5)-3beta-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

The distribution of the putative protein products of gene hcs2 in giant command neurons of the parietal ganglia of the terrestrial snail Helix lucorum has been studied using light- and electron-microscopic immunocytochemistry. The product of the hcs2 gene is a hybrid protein belonging to the EF-hand family of Ca2+-binding proteins and is a precursor of several neuropeptides. Polyclonal antibodies to neuropeptides CNP3 and CNP4 and the C-terminal Ca2+-binding domain of the precursor protein have been used to determine their intracellular localization. The targets for all three types of antibodies have been found in cytoplasmic secretory granules. The label (colloidal gold) density in the secretory granules is two times higher in the case of neuropeptides CNP3 and CNP4 than in the case of the Ca2+-binding domain. Thus, a specific association between the putative products of the hcs2 gene and the cell secretory apparatus has been demonstrated. This agrees with the earlier hypothesis that hcs2 products may serve as neurotransmitters or neuromodulators.