Molecular Virology of Chikungunya Virus.

Chikungunya virus (CHIKV) was discovered more than six decades ago, but has remained poorly investigated. However, after a recent outbreak of CHIK fever in both hemispheres and viral adaptation to new species of mosquitoes, it has attracted a lot of attention. The currently available experimental data suggest that molecular mechanisms of CHIKV replication in vertebrate and mosquito cells are similar to those of other New and Old World alphaviruses. However, this virus exhibits a number of unique characteristics that distinguish it from the other, better studied members of the alphavirus genus. This review is an attempt to summarize the data accumulated thus far regarding the molecular mechanisms of alphavirus RNA replication and interaction with host cells. Emphasis was placed on demonstrating the distinct features of CHIKV in utilizing host factors to build replication complexes and modify the intracellular environment for efficient viral replication and inhibition of the innate immune response. The available data suggest that our knowledge about alphavirus replication contains numerous gaps that potentially hamper the development of new therapeutic means against CHIKV and other pathogenic alphaviruses.

[CD47 receptor as a primary target for cancer therapy].

Recently, a number of new highly efficient antibody-based anticancer therapeutics have emerged. These receptor-binding antibodies have beneficial toxicity profiles associated with relatively mild side effects. Therefore, the search for novel surface proteins that are present on cancer cells and play important metabolic or defensive roles has intensified. Additionally, the therapeutic stimulation of patient's immune system in order to aim its components, specifically, phagocytes and cytotoxic T-lymphocytes, at tumor cells is gaining traction. This review is focused on the CD47 surface receptor, a ubiquitously expressed molecule, which could nevertheless serve as a therapeutic target due to its ability to simultaneously stimulate both natural and adaptive immune response.

[Role of PDLIM4 and c-Src in Breast Cancer Progression].

High heterogeneity is characteristic of oncology diseases, often complicating the choice of optimal anticancer treatment. One cancer type may combine tumors differing in histogenesis, genetic lesions, and mechanism of cell transformation. Differences in the mechanism of cell malignant transformation result in specifics of cancer cell metabolism and sensitivity to various agents, including anticancer treatments. Hence, the molecular subtype of a tumor is essential to know for choosing the optimal therapeutic strategy. The review considers the role actin-associated proteins and tyrosine kinases, in particular, PDLIM4 and Src kinase, play in the formation of pathological signaling pathways.

[Oncotoxic proteins in cancer therapy: mechanisms of action].

Cancer therapeutics based on protein biomolecules that exhibit selective toxic of inhibiting effects towards tumor cells without affecting normal tissue, are gaining extensive attention in cancer research. This heterogenous group of proteins consists of several subgroups, among them, are engineered cancer antigen-specific antibodies that suppress tumor growth by blocking proliferation-inducing receptors, or by direct action of a covalently attached toxin. Another subgroup of anticancer proteins that also represents promising potential therapeutic agents is oncotoxic proteins that can selectively trigger proapoptotic signaling in cancer cells. The oncotoxic proteins target such commonly disturbed processes in tumor calls as enhanced cell proliferation, altered cell-cycle control, deficient apoptotic response, inhibited mitochondrial respiration and activated glycolysis. The introduction of oncotoxic proteins to the clinic might substantially widen and upgrade modern arsenal of anticancer therapeutics.

Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway.

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53-p73 complex as a promising and highly specific potential target for cancer therapy.

Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle.

The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In recent years nanoparticles have been shown to interact with peptides, ranging in size from single amino acids up to proteins. These nanoparticles can be tailor-made with specific functions and properties including bioavailability. To the best of our knowledge, in this study we show for the first time that a tailored titanium oxide nanoparticle interacts specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein. This can be developed potentially into a tool for selective control of viral protein functions.

[A lentivirus vector based assay system for quantitative detection of intracellular translocations of recombinant proteins].

An enzymatic assay system is described that allows quantitative localization within different cellular structures of recombinant proteins. The system is based on alpha-complementation of beta-galactosidase. The large omega-fragment of beta-galactosidase is expressed in predefined cellular structures with the aid of attached protein localization signals. The obtained reporter cell lines are used for the introduction of a second construct that expresses a protein of study fused with a shorter alpha-fragment of beta-galactosidase. Physical proximity of the two recombinant proteins carrying beta-galactosidase fragments results in reconstitution of an active enzyme, and the activity can be measured in a plate reader. The recombinant constructs are based on lentiviral vectors, which allows rapid and efficient introduction of recombinant proteins into cells by infection with stocks of lentiviral particles. The efficiency of the system is demonstrated with transcriptional factor FOXO3A, which is shuttling between cytoplasm and nuclei in model colon carcinoma cell line RKO.

[Transcriptional inhibition of human papilloma virus in cervical carcinoma cells reactivates functions of the tumor suppressor p53].

Inactivation of tumor suppressor p53 accompanies the majority of malignant diseases in humans. Restoration of p53 functions in tumor results in death of cancer cells, which can be used in cancer therapy. In cervical cancer a product of E6 gene of the human papilloma virus promotes accelerated degradation of p53 in proteasome system. Therefore, one of the approaches to reactivation of p53 in cervical carcinoma cells could be the use of small molecules that inhibit functions of viral proteins. By using as a test system human cervical carcinoma cells (HeLa cell line bearing human papilloma virus type 18, HPV-18) with introduced reporter construct that expresses beta-galactosidase under control of a p53-dependent promoter we carried out screening of a library of small molecules to select small molecules capable of reactivating transcriptional activity of p53. We then characterized the effects of two most active compounds in cell lines that differ in the status of p53-dependent signaling pathway. Both of the compounds caused specific activation of p53 in the cell lines expressing HPV-18, to a lesser extent--HPV-16, and do not cause any effect in control p53 negative cells, or in the cells with undisrupted p53 pathway. Activation of p53 in cervical carcinoma cells was accompanied by the induction of the p53-dependent gene CDKN1 (p21), by inhibition of proliferation, and by the induction of apoptosis. Both of the compounds were capable of deep inhibition of transcription from the HPV genome, which apparently was the cause for p53 reactivation in response to decreased expression of the E6 protein. The observed low toxicity for normal cells allows considering these chemical compounds as prototypes for future anticancer drugs.

[Genome-wide lentivector-based pooled shRNA library optimization].

We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.

[Down-regulation of TRIP6 expression induces actin cytoskeleton rearrangements in human carcinoma cell lines].

Structure and dynamics of actin cytoskeleton play a role ih regulation of cell adhesion, spreading and migration. TRIP6 is a LIM domain-containing protein interacting with many actin-associated proteins and in addition modulating activity of certain transcription factors. To study functions of TRIP6 we inhibited its expression in A549 and A431 cells by short interfering RNAs (siRNAs). The TRIP6 knock-down lead to the increased number and length of stress fibers and to the induction of locomotive phenotype. There was observed decreased number and reorganization of focal adhesions revealed by staining for paxillin, and loss of cell to cell adhesions revealed by staining for E-cadherin. The above changes in cell morphology were accompanied by 2-fold increase in the cell motility rate assessed by the wound healing assay. Thus, down-regulation of TRIP6 in the cell lines used results in increase in the features characteristic to malignant transformation of epithelial cells. Possible mechanisms for the observed effects are discussed.

[Intensive therapy for multiple organ dysfunction in neonatal infants after cardiosurgical interventions].

This study was undertaken to evaluate the efficiency of complex intensive therapy for multiple organ dysfunction syndrome (MODS) after cardiosurgical interventions at the resuscitative and intensive care unit of the A. N. Bakulev Research Center of Cardiovascular Surgery, Russian Academy of Medical Sciences. In 2003-2004, MODS developed in 70 (37%) of the neonatal infants operated on the heart and vessels. The babies' age ranged from 6 hours of life to 1 month (8.3 +/- 2.1 days of life, their body weight was from 1.7 to 4.1 kg (3.0 +/- 0.49 kg). All the patients were found to have significant renal and respiratory failures. There were more than 4 (4.1 +/- 0.5) failing vital viscera. The use of phosphodiesterase (III) inhibitors in therapy for acute left ventricular insufficiency significantly improved the performance of the left heart whereas nitric oxide inhalation significantly lowered pulmonary pressure in babies with acute right ventricular insufficiency and improved oxygenation in patients with MODS. The efficiency of nitric oxide inhalation in MODS significantly increased when it was used in combination with endotracheal administration of a surfactant and high-frequency oscillatory ventilation. Peritoneal dialysis effectively replaced renal function when acute renal failure (ARF) developed. Nevertheless, the development of ARF in the pattern of MODS is a marker of high mortality (89% in ARF versus 46% in MODS without ARF).

Tandem arrangement of human genes for interleukin-4 and interleukin-13: resemblance in their organization.

The genes encoding human interleukin-4 (IL-4) and interleukin-13 (IL-13) are located on segment q23-31 of chromosome 5 and encode two multifunctional lymphokines with some common functions. We have cloned 72 kb of human genomic DNA that contain IL-4 and IL-13 and their flanking sequences, and constructed a restriction map of this region. Using Southern analysis, we have shown that IL-13 is located 12 kb 5' to IL-4 and linked in a 'tail-to-head' fashion. We have also determined the complete nucleotide sequence of the DNA fragment (about 4.8 kb) containing IL-13 and its 5' flanking regulatory region (2.1 kb) with a 'CpG island'. We identified potential binding sites for a different transcription factors in the 5' flanking region and in the first intron of IL-13. Comparison of IL-13 and IL-4 revealed considerable similarity in the structural organization of these genes and also many potential binding sites for transcription factors common to both genes: AP1, AP2, AP3, PEA3, HRE, TCF-1, GATA-3 and the interferon-inducible and enhancer elements. These results, along with the similarity in functional activity of IL-4 and IL-13 suggests that their expression may be coregulated.

The human RIL gene: mapping to human chromosome 5q31.1, genomic organization and alternative transcripts.

The ril gene encoding a LIM domain protein of an unknown function was previously identified by differential expression cloning as a candidate tumor suppressor gene in rat fibroblasts (Kiess, M., Scharm, B., Aguzzi, A., Hajnal, A., Klemenz, R., Schwarte-Waldhoff, I., Schafer, R., 1995. Expression of ril, a novel LIM domain gene, is down-regulated in HRAS-transformed cells and restored in phenotypic revertants. Oncogene 10, 61-68). Searching for novel genes on human chromosome 5q31.1 by the cDNA selection technique, we isolated a cDNA clone identical with the cDNA of the human RIL gene (GenBank Accession No. X93510). The human 5q31.1 region is of interest because it contains the cytokine gene cluster and is frequently deleted in the malignant cells of patients with myelodysplasia and myeloid leukemia. Using Southern blot analysis and restriction mapping of genomic YAC (yeast artificial chromosome) and cosmid clones, we located the human RIL gene 240-260 kb telomeric to the IRF1 gene and characterized its genomic structure. PCR analysis indicated the presence of two alternative RIL transcripts in human fetal brain mRNA. The major transcript is identical with the RIL cDNA previously deposited in GenBank and contains seven exons distributed over 14.5 kb of genomic DNA with the two last 3'-exons coding a LIM domain. The minor transcript lacks the sixth exon compared with the major transcript, which leads to the loss of the LIM domain. We also identified two putative transcription start points (tsp) and sequenced the 5'-flanking region of RIL to reveal potential binding sites for transcriptional factors.

Hydroxyl radical generation and DNA strand scission mediated by natural anticancer and synthetic quinones.

Using ESR and spin-trapping techniques, it was found that synthetic 2-dimethylamino-3-chloro-1,4-naphthoquinone and the natural anticancer quinone daunomycin, when added to a system containing purified NADPH-cytochrome P-450 reductase, NADPH, ferric ions, and oxygen, (i) generated hydroxyl radicals and (ii) caused single-strand scission of supercoiled DNA of the plasmic pBR322. Since these two effects of the quinones were correlated to each other, we propose that potential anticancer quinones can be effectively screened by measuring their ability to form hydroxyl radicals in the above system.

Porphyrin-linked oligonucleotides. Synthesis and sequence-specific modification of ssDNA.

Oligonucleotide derivatives bearing hemin and deuterohemin groups were synthesized. The derivatives efficiently react with the complementary nucleotide sequence in ssDNA forming covalent adducts and piperidine-labile sites. In the case of the deuterohemin derivative, some direct cleavage of the target DNA occurs.

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.

Kinetic study of the addressed modification by hemin derivatives of oligonucleotides.

Kinetics of oligonucleotide pd(TGAATGGGAAGA) modification by a hemin derivative of the complementary oligonucleotide pd(TTCCCATT) in the presence of hydrogen peroxide was investigated. The treatment of experimental data permitted to evaluate the association and rate constants at 25 degrees C: Kx = (3.40 +/- 0.38) x 10(5) M-1 (association constant of the reagent with the target), kd = 152 +/- 6 M-1 min-1 (degradation constant of the hemin group of the reagent in a parallel reaction), ko = 51.0 +/- 1.7 M-1 min-1 (target modification constant in the reactive duplex). The modification of DNA is incomplete due to competition of the modification reaction with the degradation of the hemin group of the reagent in a parallel reaction.

Hydroxyl radical generation by oligonucleotide derivatives of anthracycline antibiotic and synthetic quinone.

For the first time the covalent binding of anticancer anthracycline drugs and their potential synthetic analogs to oligonucleotides of different sequences is proposed for obtaining site-specific DNA scission in systems in vitro and in vivo. New compounds such as daunomycin (Dm) and synthetic naphthoquinone (NQ), covalently bound to the heptadeoxynucleotide of pCCAAACA (Dm-pN7) and decadeoxythymidilate (pT10p-NQ), have been obtained. These oligonucleotide derivatives can form specific complexes with complementary oligonucleotide sequences; these compounds and their complementary complexes can be reduced by purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), it has been shown that in aerobic conditions Dm-pN7 and pT10p-NQ are capable of generating OH radicals with and without complementary oligonucleotides. The chemical stability of the compounds in redox reactions has been studied. Oligonucleotide derivatives of natural and synthetic quinones capable of generating OH radicals seem to be a promising tool for site-specific scission of DNA in solution and in cells.

[Effect of bases noncomplementary to the template on the effectiveness of primer interaction with DNA-polymerase alpha from the human placenta]. / Vliianie nekomplementarnykh matritse osnovanii na éffektivnost' vzaimodeistviia praimerov s DNK-polimerazoi alpha iz platsenty cheloveka.

The comparison of the Km and Vmax values for the various primers was carried out. The primers were either completely complementary to the template or contained the non-complementary bases in different positions from the 3'-end. The number of the bases from the 3'-end to the noncomplementary nucleotide but not the primers length was supposed to determine the efficiency of the interaction of the primers containing noncomplementary bases with the enzyme. The Km values for d[(pC) (pT)7] (1.2 microM), d[(pC)3(pT)7] (2.5 microM, d[(pT)2pC(pT)7] (1.4 microM)d[(pT)4pC(pT)5(4.3 microM); d[(pT)7pC(pT)2] (11 microM) are comparable with the Km values for d(pT)7 (1.4 microM); d(pT)5 (4.2 microM) and d(pT)3 (15 mkM), respectively, but not for the decathymidilate d[(Tp)9T] (0.23 microM). The complementary interaction between the first nucleotide from the 3'-end of the primer and the template appear to play the particular role in the interaction of the enzyme with the primer. The Km values for d[(pT)10pC] and d[(pA)9pC] (with the corresponding templates) are 38 and 6 times the ones for d[(Tp)10T] and d(pA)10. However, the Km values for d[(pA)9p(rib)] (0.56 microM) which contains the deoxyribozylurea residue at the 3'-end is practically equal to the Km for d(pA)9 (0.56 microM). The Vmax values for d[(pT)10pC] and d[(pA)9pC] are 1.7 and 2.3 times the values for d[(Tp)10T] and d(pA)10, respectively. The primer affinity decreases, just as its conversion rate increases when the noncomplementary base in the primer is transferred from the 5'-to 3'-end; that results in the rate of primers elongation decrease in total.

[Production and characteristics of seven rat embryo cell lines transformed by adenovirus type 5 and its DNA]. / Poluchenie i kharakteristika semi linii kletok émbriona krysy, transformirovannykh adenovirusom tipa 5 i ego DNK.

Seven cell lines transformed by adenovirus type 5 and its DNA were obtained. It was shown that different cell lines contain the fragments of viral DNA which differ in length and number of copies per DNA of diploid cells. They contain from the left end 6% of the viral DNA to complete or almost complete viral genome. All studied cell lines were sensitive to reinfection with adenovirus type 5. They produced no virus being cocultivated with cell sensitive to the virus. No cell line was able to induce tumors even in immunosuppressed newborn rats. All cell lines formed colonies in soft agar. The level of virus-specific antigens was higher in cells that contained a large part of the viral genome. The methods used did not allow to correlate the biological properties of the transformed cells with the length and the number of copies of the integrated part of the viral genome.