Colocalization of USP1 and РН domain of Bcr­Abl oncoprotein in terms of cronic myeloid leukemia cell rearrangements.

The development of chronic myeloid leukemia (CML) is the result of a reciprocal translocation between chromosomes 9 and 22 due to the emergence of Philadelphia chromosome. The product of this mutation is a hybrid oncoprotein Bcr-Abl. According to the results of mass spectrometric analysis, USP1 protein was identified as a potential candidate for interaction with the PH domain Bcr-Abl oncoprotein. Due to the deubiquitination properties, USP1 protein can prevent proteasomal degradation of Bcr-Abl oncoprotein in a cell and, consequently, contribute to its accumulation, and the progression of the disease. In this work, creating the genetic constructs, we detected the USP1 protein localization in the cell. Also, a nuclear colocalization of USP1 protein with PH domain of Bcr-Abl oncoprotein in HEK293T cells was shown. The results are important for understanding the implications of the Philadelphia chromosome emergence, and the development of new methods for CML treatment, since the recent techniques are not always effective due to the emergence of numerous mutations that cause drug resistance and relapse of the disease.

Nuclear localization of BCR and cortactin indicates their potential role in regulation of actin branching in nucleus.

AIM: To study cellular localization of full-length breakpoint cluster region (BCR), Pleckstrin homology domain of BCR and cortactin and determine whether they can coexist in cell nucleus. MATERIALS AND METHODS: HEK293T cell line was transfected with pECFP-BCR, pEGFP-PH and pmTagRFP-N1-CTTN using polyethyleneimine. Live cells were imaged in cell culture dishes with glass coverslip attached to the bottom with Leica SP8 STED 3D confocal microscope in the environmental chamber. Obtained images were processed and analyzed with Fiji software. RESULTS: We identified colocalization of full-length BCR and cortactin in nucleus of cell undergoing terminal phase of cell division. We did not observe nuclear localization of cortactin in non-dividing cell. Both Pleckstrin homology domain and full-length BCR exhibited cytoplasmic as well as nuclear localization. CONCLUSIONS: Colocalization of BCR with cortactin in cell nucleus indicates their potential role in regulation of actin network allowing for the maintenance of nuclear architecture and DNA integrity.

Natural interferon alpha/beta-producing cells link innate and adaptive immunity.

Innate immune responses to pathogens critically impact the development of adaptive immune responses. However, it is not completely understood how innate immunity controls the initiation of adaptive immunities or how it determines which type of adaptive immunity will be induced to eliminate a given pathogen. Here we show that viral stimulation not only triggers natural interferon (IFN)-alpha/beta-producing cells (IPCs) to produce vast amounts of antiviral IFN-alpha/beta but also induces these cells to differentiate into dendritic cells (DCs). IFN-alpha/beta and tumor necrosis factor alpha produced by virus-activated IPCs act as autocrine survival and DC differentiation factors, respectively. The virus-induced DCs stimulate naive CD4(+) T cells to produce IFN-gamma and interleukin (IL)-10, in contrast to IL-3-induced DCs, which stimulate naive CD4(+) T cells to produce T helper type 2 cytokines IL-4, IL-5, and IL-10. Thus, IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-alpha/beta, and subsequently triggering adaptive T cell-mediated immunity by differentiating into DCs. IPCs constitute a critical link between innate and adaptive immunity.

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells.

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.

Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens.

Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells.

Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1-mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4-dependent manner. Thus, it is important to understand how the development of IL-4- versus interferon (IFN)-gamma-producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500-70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-gamma, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4(+)IFN-gamma(-) NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4(-)IFN-gamma(+) NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4(+)IFN-gamma(-) NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-gamma.

Inhibition of USP1, a new partner of Bcr-Abl, results in decrease of Bcr-Abl level in K562 cells.

AIM: To analyze interaction of ubiquitin specific peptidase 1 (USP1) with Bcr-Abl and to assess the relation between USP1 functional activity and Bcr-Abl expression in K562 chronic myeloid leukemia cells. MATERIALS AND METHODS: The interaction between USP1 and Bcr-Abl in K562 cells was analyzed by co-immunoprecipitation, Western blot analysis, and confocal microscopy. RESULTS: A direct interaction between Bcr-Abl oncoprotein and USP1 protein in K562 cells was established by co-immunoprecipitation. Immunofluorescence analysis and confocal microscopy revealed that Bcr-Abl/USP1 protein complex is formed in the cell nucleus. The inhibition of USP1 protein activity by ML323 reduced the level of Bcr-Abl oncoprotein in K562 cells. CONCLUSIONS: USP1 protein has been identified as a new protein partner of Bcr-Abl oncoprotein in chronic myeloid leukemia. The relationship between the functional activity of USP1 protein and the level of Bcr-Abl oncoprotein has been demonstrated, suggesting that the targeted inhibition of USP1 activity could be a challenging approach for reducing Bcr-Abl expression.

The nature of the principal type 1 interferon-producing cells in human blood.

Interferons (IFNs) are the most important cytokines in antiviral immune responses. "Natural IFN-producing cells" (IPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4(+)CD11c- type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.

[Antiherpetic effect of RNA-tilorone molecular complex in cell culture].

The authors studied the antiviral effect of an interferonogenic yeast RNA-tilorone molecular complex (MC) compared to the Virolex, videly used antiherpetic drug, and standard interferon (IFN) alpha/beta inducer poly(I)poly(C) in Vero cells culture infected with herpes simplex type I virus (HSV-1). The tilorone contained by MC has been shown to be twice less toxic and twice more active against HSV than its free molecules. The value of chemotherapeutic index (CI) of Virolex in experiments with Vero cells reaches 2500, CI poly(I)poly(C) and MC being 324; the last value meets the requirements for promising drugs.

HIV-1 reverse transcriptase inhibitor design using artificial neural networks.

Artificial neural networks were used to analyze and predict the human immunodeficiency virus type 1 reverse transcriptase inhibitors. The training and control sets included 44 molecules (most of them are well-known substances such as AZT, dde, etc.). The activities of the molecules were taken from literature. Topological indices were calculated and used as molecular parameters. The four most informative parameters were chosen and applied to predict activities of both new and control molecules. We used a network pruning algorithm and network ensembles to obtain the final classifier. Increasing of neural network generalization of the new data was observed, when using the aforementioned methods. The prognosis of new molecules revealed one molecule as possibly very active. It was confirmed by further biological tests.

Production and characterization of cross-reactive monoclonal antibodies to sweet proteins.

We report on the characterization of a panel of eight murine monoclonal antibodies induced by immunization with the sweet protein monellin and which display cross-reactivity with thaumatin, another sweet natural molecule. Cross-reactivity was determined based on direct binding assays and antigen competition experiments. Interestingly, half of antibodies bound thaumatin more than monellin, the immunizing antigen. The relatedness between the binding sites of the eight monoclonal antibodies was assessed by cross-competition of the binding of monellin to individual monoclonal antibodies. Antibodies that bound in a similar way monellin and thaumatin were on average the best inhibitors. All antibodies also bound Sp1, a genetically-engineered single chain monellin. These studies demonstrate that the immune repertoire encodes for specificities that are common to monellin and thaumatin despite the fact that the two molecules share little, if any, structural homology. These results are discussed in relation to the use of cross-reactive antibodies to generate anti-receptor antibodies via idiotypic immunization.

[The role of the components of the cyclic nucleotide system in N-nitrosodiethylamine-induced hepatic carcinogenesis in rats]. / Rol' nekotorykh komponentov sistemy tsiklicheskikh nukleotidov pri gepatokantserogeneze, indutsirovannom N-nitrozodiétilaminom, u krys.

The content of cyclic nucleotides and activity cAMP- and cGMP-dependent PDE-ases have been studied in the rat liver and activity of NaF stimulated adenylate cyclase was studied in plasma membranes of the rat liver at late stages (2-5.5 months) of hepatocarcinogenesis induced by NDEA. A considerable decrease in the basal adenylate cyclase activity and NaF stimulated adenylate cyclase activity in plasma membranes of hepatomas in comparison with norm has been observed. During the hepatocarcinogenesis it was observed a decrease in the cAMP/cGMP ratio, an increase of the cAMP-dependent PDE-ase and a decrease in activity of cGMP-dependent PDE-ase in the rat hepatomas have been found.

[Interrelation between the phospholipid composition of rat liver plasma membranes and adenylate cyclase activity in the early stages of liver carcinogenesis induced by nitrosodiethylamine]. / Vzaimosviaz' mezhdu izmeneniem fosfolipidnogo sostava plazmaticheskikh membran pecheni krys i aktivnost'iu adenilattsiklazy na rannykh stadiiakh gepatokantserogeneza, indutsirovannogo nitrozodiétilaminom.

The content of total phospholipids, the level of cholesterol and individual phospholipids and the activity of basal as well as of NaF-stimulated adenylate cyclase (AC) (EC 4.6.1.1.) in plasma membranes of the rat liver are studied at the initial stages of nitrosodiethylamine-induced hepatocarcinogenesis. Already by the 2nd week a substantial increase in the phospholipid content and a decrease in the cholesterol content are observed. By the 4th week of the experiment the cholesterol content is normalized. In this period the phospholipid content increases sharply and the basal and NaF-stimulated adenylate cyclase activity lowers significantly as compared with the normal level. In the process of hepatocarcinogenesis changes in individual phospholipids and particularly in phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin are observed.

[The role of cyclic nucleotides in the development of resistance to metastases after treatment with immunomodulators]. / Uchastie tsiklicheskikh nukleotidov v formirovanii soprotivliaemosti organizma razvitiiu metastazov pod vliianiem immunomoduliatorov.

It is shown that zymosan, prodigiosan, tiloron and levamisol have a different effect on the level of cyclic nucleotides and the cAMP/cGMP correlation in the lungs, thymus and spleen of mice during metastatic spreading of the Lewis carcinoma. A more pronounced antimetastatic effect after administration of immunomodulators is accompanied by a more considerable increase in the cAMP/cAMP coefficient in immunocompetent organs. There exists a close negative correlation between the increase in the cAMP/cGMP coefficient in the thymus and spleen, on the one hand, and inhibition of the metastatic spreading in the lungs under the effect of levamisol and zymosan, on the other.

Virus-inhibitory effect of a yeast RNA-tilorone molecular complex in cell cultures.

The virus-inhibitory activity of a molecular complex (MC) of tilorone and yeast RNA was studied in vitro on three virus-cell systems: vesicular stomatitis virus (VSV) - murine fibroblast L929 cells, Venezuelan equine encephalittis virus (VEEV) - swine embryo kidney (SEK) cells and encephalomyocarditis virus (EMCV) - established piglet testicular (EPT) cells. In all these systems the MC exerted an antiviral effect similar to that of polynucleotide interferon (IFN) inducers such as poly(I)-poly(C), larifan and ridostin. The antiviral effect of the MC was similar when the compound was applied before or after virus adsorption to cells. The MC may be regarded as a perspective antiviral agent of common use.

[Study of the effect of parotitis vaccine on DNA repair in human lymphocytes]. / Izuchenie vliianiia parotitnoi vaktsiny na reparativnyi sintez DNK vlimfotsitakh cheloveka.

The effect of parotitis vaccine virus (strain L-3) on the DNA repair synthesis induced by 4-nitroquinoline-1-oxide has been studied. The efficiency of the repair synthesis depends on individual properties of the human body, viral multiplicity and concentration of the mutagen. A two-fold increase in DNA repair synthesis was obtained after infection of cells with low viral multiplicity (0.001 HADU50 per cell) and using 2.5 x 10(-7) M concentration of the mutagen A ten-fold increase in mutagen concentration affecting the infected cells was accompanied by the inhibition of DNA repair synthesis. Lymphocytes from children studied 7 days after vaccination by the attenuated virus did not reveal any changes in DNA repair synthesis as compared with the cells from nonvaccinated children.

[Fluorescent-cytochemical study of the action of benz(a)pyrene on subcutaneous connective tissue cells during malignant transformation in vitro]. / Fluorestsentno-tsitokhimicheskoe izuchenie deistviia benz(a)pirena na kletki podkozhnoi soedinitel'noi tkani v protssesse malignizatsii vne organizma.

Alterations in the secondary DNA structure of cell nuclei of the rat's subcutaneous connective tissue exposed to benz(a)pyrene (BP) action have been revealed using the Roshlay fluorescent-cytochemical method in cultivating in vitro. Alterations in the luminescence of the acridin orange complex with DNA, i.e. displacement towards the red region of spectrum, indicates the changes in the secondary structure of DNA. The strongest action on the primary cultures were noted after the injection of carcinogen (on the 3rd day of explanation). The toxical effect was lower after a repeated BP injection (on the 10th and 20th days of explanation). Cells undergone malignization and transformation, due to the BP action on the primary explantates, were more resistant to carcinogen action than the normal cells. However, the spontaneously malignized cells were more sensitive to the BP action than the cells transformed under the carcinogen action.

[Study of anti-HIV activity of the yeast RNA-tilorone molecular complex]. / Izuchenie anti-VICh-aktivnosti molekuliarnogo kompleksa drozhzhevaia RNK-tiloron.

Anti-HIV activity of the molecular complex forming during interaction between yeast RNA and tilorone was studied in vitro on the models of acute and chronic infection. Addition of this agent to the cells infected with HIV-1/IIIB and HIV-1/BRU decreased the virus reproduction controlled by assessing the viability of cells, syncytium production, and accumulation of p24 antigen in culture medium. The authors hypothesize that the detected anti-HIV effect is due to its capacity to produce type I interferon and direct antiviral action.

[The interferon reaction of the leukocytes and the natural killer activity in patients with acute viral hepatitis B undergoing interferon treatment]. / Interferonovaia reaktsiia leikotsitov i aktivnost' estestvennykh killerov u bol'nykh ostrym virusnym gepatitom B pri lechenii interferonom.

The interferon response of leukocytes (IRL) alpha and gamma as well as the functional activity of natural killers (NK) were studied in patients with the verified diagnosis of acute viral hepatitis B (AVH-B) treated with leukinferon (a preparation of natural interferon-alpha and cytokines) and reaferon (recombinant alpha 2 interferon). The effect of therapy with LF and RF in patients with AVH-B manifested by an increase in the functional activity of NK up to complete normalization after termination of the course of therapy. In the group of patients treated with LF as a result of therapy IRL-alpha increased by the end of the treatment and in the convalescence period. The observed activation of NK and the IF-alpha system under the effect of IF preparations in patients with AVH-B correlated with enhanced elimination of HBsAg.