Biomedical subjects
Publications and source records attributed to O V Morozova.
Quantities of Ureaplasma spp DNA were determined by the methods of competitive and multiplex polymerase chain reactions (PCR). The results obtained by each of the methods were shown to be compatible. It was established that the number of Ureaplasma spp. cells per one epithelial cell in the female cervical canal varied from 0.05 to 10.
We studied the effect of endogenous immunoregulatory myelopeptide-2 on the development of spontaneous and urethane-induced lung adenomas. Long-term treatment with myelopeptide-2 (25 injections) in parallel with urethane poisoning decreased animal mortality caused by this carcinogen. Short-term treatment with myelopeptide-2 decreased the number of spontaneous and urethane-induced lung tumors in mice. Myelopeptide-2 delayed the appearance of urethane-induced tumors irrespective of the number of injections.
The clinical and immunological characteristics of lymphoid tumors were compared in 591 children with acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Comprehensive investigation of a tumor cell by using cytological, morphological, and immunological studies revealed the most significant criteria for differential diagnosis of ALL and NHL in children and showed the specific features of the site of a tumor and the extent of its growth in ALL and NHL in relation to the immunological affiliation of a tumor cell. The predominance of immature forms, such as stem-cell CD34+, pre-pre-B, pre-B and less commonly T-cell forms with almost none peripheral B- and T-cell markers could be immunophenotypically detected in ALL. NHL was, on the contrary, characterized by the prevalence of mature immunological subtypes with peripheral B- and T-cell markers and much less frequently pre-B and pre-T cells and at the same time there was no CD34 antigen in the tumor cells. Anaplastic giant lymphoma was a peculiar type of NHL characterized by the presence of large cells having marked anaplasia and expression on the surface of CD30 antigen. A comprehensive study of lymphoid tumors in children showed that immunophenotyping was of great value, whose results were associated with the specific feature of tumor growth and prognosis, which should be borne in mind while planning antitumor therapy programmes.
Tick-borne encephalitis virus (TBEV) was propagated in porcine embryo kidney (PS) cells until 48 h whereas human kidney (RH) cells maintained the virus persistence during at least 2 months. One of possible reasons of flavivirus chronic infection might be abnormal NS1 gene expression. Immunoblotting with monoclonal antibodies (MAbs) revealed the similarity of the intracellular and secreted NS1 nonstructural glycoprotein size and linear antigenic determinants in both the infected cell lines. However, according to the competitive binding of MAbs with the TBEV NS1 extracellular glycoprotein, its contiguous epitopes differed for acute or persistent infection. To map the TBEV NS1 glycoprotein antigenic determinants its recombinant analogues were used. All the studied MAbs could bind with the full-length NS1 recombinant protein. Deletion of the TBEV NS1 gene internal region resulted in defective NS1d1 protein without the region between 269 and 333 a.a. Lack of NS1d1 binding with 20B4 MAb and diminished binding with 22H8 and 17C3 MAbs permitted to map their antigenic determinants within or nearby deleted region, respectively. Interaction of other MAbs with the NS1 and NS1d1 recombinant proteins did not differ, suggesting that their epitopes were located in the region of N-terminal 268 a.a. or C-terminal 19 a.a. of the TBEV NS1 protein. The second NS1d2 truncated protein contained the first N-terminal 33 a.a. of the TBEV NS1 protein and was able to bind with 29G9 MAb. Taken together the data stand for the differences in the N-terminal structure of the TBEV NS1 multimers secreted from the acute and persistent infected cells whereas the intracellular and secreted monomer processing was the same. The modified NS1 protein oligomers in the RH cellular line might slow virus replication and could result in the TBEV persistence.
The knowledge of virus reproduction is necessary to design new safe drugs for inhibition of infections. Ultra-violet irradiation of virus proteins with labeled virus genome fragments permits to identify specific nucleic acid binding proteins. Affinity modification of enzymes with nucleotide derivatives could help to determine NTP-binding proteins and those involved in viral genome replication. Photoreactive analogues of nucleic acids are among the tools used to detect elongation subunits of replicative complexes. Affinity modification approach has already resulted in successful treatment of virus diseases.
Under phosphate-deficient conditions, B. intermedius, B. pumilus, and B. thuringiensis secrete phosphohydrolases, including phosphomono-, phosphodiesterases, and guanyl-specific ribonucleases which cleave RNA molecules to nucleoside-3'-phosphatases. The enzymes are synthesized by phosphate-starved vegetative cells, which is not associated with sporulation. Using B. subtilis strains with mutation in the regulatory protein genes phoP and phoR, it was shown that these proteins regulate expression of B. intermedius, B. pumilus, and B. thuringiensis ribonuclease genes in B. subtilis cells. Genes of heterologous RNAses were activated in recombinant B. subtilis strains simultaneously with its own PHO regulon genes. Presumably a regulatory system homologous to B. subtilis two-component PhoP-PhoR signal transduction system functions in other representatives of the Bacillus genus.
Expression of hepatocyte-specific genes in slow- and fast-growing hepatocellular mouse carcinomas was studied. The fast-growing poorly differentiated passaged hepatocarcinoma (fHC) originated from the well-differentiated slow-growing variant (sHC). In contrast to the parental hepatocarcinoma, in fHC the expression of the hepatocyte nuclear factor 4 (HNF4), in fHC a key factor responsible for hepatocyte differentiation, and several HNF-4-responsive genes, such as those for transferrin, transthyretin, hepatocyte nuclear factor 1 (HNF1), and serum albumin, was significantly suppressed. The expression of exogenous HNF4 in the fHC cell culture partially restored the expression of hepatocyte marker genes and the appearance of epithelial cell islands in the culture. The described system may serve as a convenient model for further analysis of mechanisms underlying hepatocarcinogenesis and liver tumor progression.
The cDNA encoding influenza virus (A/Udorn/307/72 strain) M2 protein was subcloned into the EBV-based vector pREP9. Three continuous kidney cellular lines of different origin were transfected with recombinant plasmid pREP9-M2. One and 5 months after transfection plasmid DNA rearrangements were detected by means of restriction analysis of recovered plasmids and their hybridization with an influenza-virus-specific radioactive probe. Deletions were the most frequent type of pREP9-M2 mutations. PCR with primers corresponding to cellular genome and plasmid DNA followed by Southern blot analysis with the [(32)P]-labeled M2-fragment allowed host DNA rearrangements to be revealed in transfected cells.
Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89-98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.
The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.
Tick-borne encephalitis virus (TBEV) NS3 gene has been subcloned into the expression vector pcDNAI and expressed in eukaryotic cells. Immunization of mice with the recombinant plasmid pcDNAI-NS3 induced antibodies against NS3 protein but did not protect from viral challenge.
A new label--laccase from the fungus Coriolus hirsutus--was applied for solid-phase enzyme-linked immunosorbent assays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems permit the detection of 2,4-D in concentrations down to 10-20 ng/mL; time of the assays is 1.5-2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.
The antibody (AB) fraction containing sIgA and IgG was isolated from human milk by Protein A-Sepharose chromatography and was shown to possess affinity to DNA-cellulose. Ion-exchange HPLC of these AB on a TSK DEAE-5PW column resulted in the isolation of a fraction containing sIgA and oligonucleotides (ON). Gel-filtration of the AB fraction revealed the presence of ON with length 4-8 nucleotides co-isolating with sIgA. sIgA Preparations purified on DEAE-Fractogel and DNA-cellulose contained lipids which were phosphorylated in the presence of [gamma-32P]ATP. The affinity of HPLC-purified IgG and sIgA to calf thymus DNA, Escherichia coli DNA and total tRNA, and plasmid DNA was demonstrated. IgG was shown to bind to thymus DNA and E. coli DNA, and sIgA was shown to bind to E. coli DNA and tRNA. Nucleic acids of intestinal microflora are supposed to participate in induction of the secretory immune response.
4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.
An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM. The constitutive form of the enzyme was purified 312-fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine. The laccase was found to contain 3.9-4.1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1. Laccase was found to be a pH-stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3.5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.
The largest tick-borne encephalitis virus (TBEV) non-structural protein NS5 (100 kDa) is believed to be involved in RNA replication. The protein is phosphorylated in infected cell extracts in the presence of [gamma-32P]ATP, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using monoclonal antibodies raised against TBEV NS5 protein. Radioactive labeling of NS5 in cellular extracts at an early stage post-infection is higher than at 24 h post-infection. Incubation of immunoprecipitates of NS5 protein with [gamma-32P]ATP in the presence of Mg2+ resulted in the phosphorylation of TBEV NS5 protein and of immunoglobulins. Phosphoamino acid analysis demonstrated that NS5 contains phosphoserine, but not phosphothreonine, or phosphotyrosine.
Dichlorodiphenyltrichloroethane (DDT) administered to CBA female mice provoked a significant uterine weight increase and pseudoestrus comparable to that produced by diethylstilbestrol (DES) used in a positive control.
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