Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

Functionalized Fullerene Increases NF-κB Activity and Blocks Genotoxic Effect of Oxidative Stress in Serum-Starving Human Embryo Lung Diploid Fibroblasts.

The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1-1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2-0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.

RSH ANTIOXIDANTS INCREASE INTRACELLULAR ROS LEVEL AND PROLIFERATION OF SP2/0 MYELOMA CELLS IN SERUM-FREE MEDIUM.

At present research we have demonstrated the link between introducing of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) into serum-free medium composition, reactive oxygen species (ROS) generation and mouse myeloma SP2/0-SF cells proliferative activity. The presence of these compounds in the medium changed the pattern of ROS-activity in cells by concentration-dependent manner, and affected their proliferative characteristics. The optimal value of the proliferative activity was related to 0.2 mM for both thyols and not depended from the thyol-compound nature. Further increasing up from the found concentration optimum lead to growth inhibition with different expression for N-acetylcysteine and glutathione.

Modern principles of classification and development of nutrient media for culturing of human and animal cells.

Analysis of the main principles of classification and development of nutrient media used for culturing of human and animal cells in biology and medicine is presented. The key moments of induction and regulation of mitogenic cascades and their differences in cells of continuous lines, diploid cells, and primary cultures are discussed. Some variants of classification of nutrient media for various cell culture types are described. Peculiarities of composition and the prospects of using serum-free media are discussed. Practical results obtained by the authors in the development of diverse purpose nutrient media are presented.

[New feasibility of using a tissue culture technique in diagnostic oncomorphology in case of neuroblastoma and breast cancer].

The paper gives the results of investigating the cultured tissues from patients with breast cancer (BC) and those with neuroblastoma, by applying both the traditional studies and molecular genetic techniques (FISH). The cultures from the patients with neuroblastoma represented as three cell types--N, S, and I. There are certain similarities between the cell composition in the culture medium and the histological structure of the tumor. Mature neoplasms of the ganglioneuroma type contain S-type cells only while intermediate type I cells are a predominant element of immature neuroblastomas. The presence of a considerable number of I cells in the explants appears to suggest a poor prognosis even when the tumor has a comparatively mature structure as a whole. The results of FISH on explant cells presented a means of detecting a broad range of points that are not recorded on histologic specimens--amplification on the metaphase plate, localization of an amplified product, amplification on one chromosome, and double acentric chromosomes. The results of cultivation of BC cells are difficult to systematize although they offer the advantage of performing the FISH reaction. The authors recommend that the tissue culture method should be more extensively used in the day-to-day diagnostic work of pathology laboratories.

[Clinical features of acute cerebral blood circulation disorders in young patients with non-differential dysplasia of the connective tissue].

One hundred young patients (aged 17-45 years) with acute cerebral blood circulation disorders (ACBCD) have been studied. Phenotypic signs of non-differential dysplasia of the connective tissue (NDDCT) have been found in 50 patients (the main group) and other 50 patients had no clinical signs of NDDCT (the comparison group). Patients underwent a complex clinico-neurological, instrumental and laboratory study. In young patients with NDDCT, transient ischemic attacks were observed in 14%, ischemic strokes in 66%, hemorrhagic strokes in 20%. Risk factors characteristic of young patients with ACBCD have been singled out. The higher content of free oxyproline, the distinct positive dynamics (the reduction of clinical symptoms severity during the acute phase of stroke) and the significantly better restoration of lost functions were observed in patients with NDDCT compared to patients without NDDCT.

[Influenza virus reproduction in the MDCK cells adapted to growth in serum-free Hybris-2 medium].

Whether the MDCK cell line might adapt to grow in serum-free Hybris-2 medium and influenza viruses might be reproduced in the adapted cells was studied. Seventeen passages using the Hybris-2 medium yielded cells adapted to growth in this medium (MDCK-BS). The reproduction of influenza A (H1N1 and H3N2) and B viruses versus the cells cultured in Eagle's medium was studied. The laboratory strain of influenza A/Aichi/1/68 (H3N2) and the strain B/Ohio/01/05 of influenza B in equal titers were shown to be reproduced in both control cells on Eagle's medium and MDCK-BS cells adapted to growth in the Hybris-2 medium. The reproduction of the strains A/Brisbane/10/07 (H3N2) and A/Solomon Islands/3/06 (H1N1) was less active in the MDCK cells. Each strain of influenza viruses displayed varying infective activities. The developed serum-free Hybris-2 medium may be used for cultivation of monolayer continuous MDCK cells and for their reproduction of influenza A and B viruses.

Studying the proliferation of human peripheral blood T lymphocytes in serum-free medium.

We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 microg/ml, [corrected] respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3(+) and CD4(+) T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes.

Organ-sparing treatment of invasive transitional cell bladder carcinoma.

PURPOSE: To assess the effectiveness of two approaches of organ-sparing treatment in patients with invasive transitional cell bladder carcinoma. PATIENTS AND METHODS: During the period from June 1996 to June 2000, 33 patients with invasive transitional cell carcinoma (T2-4) of the bladder were treated. Patients were divided into two groups. Group A included 17 patients treated with CMV systemic chemotherapy (methotrexate 30 mg/m(2) and vinblastine 3 mg/m(2), day 1; and cisplatin 70 mg/ m(2), day 2) repeated every 3 weeks for 3 courses, combined with intravesical BCG. Complete responders (CR) received maintenance intravesical BCG, while partial responders (PR) were subjected to transurethral bladder resection (TURB) or partial bladder resection. Group B included 16 patients treated with maximal TURB followed by 2 CMV courses and radiotherapy. RESULTS: Group A patients have been followed-up for a period of 7 to 48 months (median 21.6 months). After completing the 2nd chemotherapy course, 2 patients refused further treatment and were excluded from the group. On completing chemoimmunotherapy 11 (73%) patients showed objective response (CR+PR) and preservation of the bladder was achieved. Four (27%) patients were treatment failures. Group B patients have been followed for a period of 9 to 47 months (median 27.5 months). On completing treatment 12 of 16 (75%) patients showed an objective response with preservation of the bladder. Treatment failure was diagnosed in 4 (25%) patients. The 2 groups differed significantly in terms of side-effects, which were more pronounced in Group B patients. Of the late complications in Group B a serious problem was the development of severe radiation-induced fibrosis leading to microcystis and a cystectomy was indispensable in one of the patients. CONCLUSION: The applied 2 approaches of combined organ-sparing treatment of invasive bladder carcinoma seem equally effective. Future randomized studies are needed to define reliable criteria for patient selection.

Treatment of an orchiectomized patient with hormone-refractory prostate cancer with LH-RH agonists.

This report confirms experimental data on direct inhibitory effects of LH-RH agonists in growth control of human prostate cancer cells. The patient had hormone-refractory prostatic carcinoma after hormonal therapy, including orchiectomy, and responded successfully to a 6-month treatment with goserelin acetate (3.6 mg depot s.c.). The treatment has led to a reduction of tumor mass and metastases as well as an improvement in the patient's paraclinical indicators and general status. The remission lasted 14 months. The treatment still continues.

[Effect of the gene for bacterial hemoglobin vhb on the effectiveness of the process of Escherichia coli-Streptomyces interspecies conjugation and production of antibiotics in streptomycetes]. / Vliianie gena bakterial'nogo gemoglobina vhb na éffektivnost' protsessa mezhrodovoi kon''iugatsii Escherichia coli-Streptomyces i produktsiiu antibiotikov u streptomitsetov.

The bacterial hemoglobin vhb gene was cloned from sliding bacterium Vitreoscilla sp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhb gene was transferred from Escherichia coli to some Streptomyces strains, producers of antibiotics, by the method of intergeneric conjugation using conjugative-integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhb gene on processes of conjugation and antibiotic production in a number of examined strains was shown.

Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation.

Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage phi C31 with the attachment site was transferred from E. coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces. The frequency of conjugant formation was 1 x 10(-3)-1 x 10(-5) depending on the strain. Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.

[Elements of regulatory systems of antibiotic biosynthesis in Streptomyces]. / Elementy sistem reguliatsii biosinteza antibiotikov u Streptomyces.

Studies of regulation of antibiotic synthesis involved both basic and applied aspects of biology of antibiotic producers. This paper is a survey of published experimental data and key hypotheses for the regulation of antibiotic synthesis in bacteria of the genus Streptomyces. Summarized data on transcriptional regulatory systems of gene clusters responsible for biosynthesis of some antibiotics in these microorganisms are presented. Results of our previous research of the regulation of antibiotic bialaphos production in the S. hygroscopicus strain are considered as well. On the basis of these results, a hypothetical scheme of the organization of lower levels of the regulatory system of the bialaphos biosynthetic cluster was constructed. It is assumed that an integrase-like product of the brpB gene cloned by the authors mediates transcriptional initiation of this gene cluster.

[Molecular-genetic study of the functional role of the regulatory gene brpB of the bialaphos producer Streptomyces hygroscopicus ATCC21705]. / Molekuliarno-geneticheskoe issledovanie funktsional'noi roli reguliatornogo gena brpB u produtsenta bialafosa Streptomyces hygroscopicus ATCC21705.

A chromosomal determinant of Streptomyces hygroscopicus, functioning as a positive regulator of the bialaphos-resistance gene bar in the model strain S. lividans, was conjugatively transferred into a bialaphos (BAP) producer through pVGTB24 plasmid. The plasmid is unable to replicate autonomously in S. hygroscopicus, but integrates into its chromosome via homology with the fragment containing this determinant. Integration of pVGTB24 was accompanied by a decrease in resistance of S. hygroscopicus exconjugant clones to the self-produced antibiotic and also by a decrease in their antibiotic productivity.

[Intergeneric Escherichia coli-Streptomyces conjugation as a means for the transfer of conjugative plasmids into producers of antibiotics chlortetracycline and bialaphos]. / Mezhrodovaia kon"iugatsiia Escherichia coli-Streptomyces kak sposob peredachi kon"iugativnykh plazmid v produtsenty antibiotikov khlortetratsiklina i bialafosa.

A system for introduction of plasmids into industrial producers of antibiotics chlortetracycline and bialaphos using intergeneric conjugation of Escherichia coli and Streptomyces was developed. Low level stability of inheritance of autonomously replicating DNA in recipient strains was shown. Site-specific integration of the conjugative-integrative vector pTO1 provided stable plasmid maintenance within the chromosomes of Streptomyces aureofaciens and S. hygroscopicus. Phenomenon of disturbance in differentiation and antibiotic production, resulting from pTO1 integration into S. hygroscopicus chromosome, was discovered.

[Chromosomal determinant of Streptomyces hygroscopicus with positive influence on resistance to bialaphos controlled by the bar gene]. / Khromosomnyi determinant Streptomyces hygroscopicus, pozitivno vliiaiushchii na uroven' ustoichivosti k bialafosu, kontroliruemyi genom bar.

Streptomyces hygroscopicus ATCC21705 is an organism producing bialaphos, a promising ecologically safe antibiotic of peptide nature. An unknown determinant was cloned within the 1.4-kb SsH fragment of chromosomal DNA of S. hygroscopicus. Its presence along with the bar gene in the hybrid plasmid PVGB21 increased expression of the phenotype resistance to bialaphos in the recipient strain of S. lividans TK 64. Plasmid PVG B22 carrying the isolated SsH fragment did not provide resistance to the antibiotic. Transcription from the cloned fragment was revealed by RNA-DNA hybridization. It was shown that the cloned determinant had a positive action on bar gene transcription correlating with a significant increase in the activity of N-acetyltransferase in the mycelium of the transformant pVG B21.

[Gravitational sensitivity of eukaryotic unicellular organisms]. / Gravitatsionnaia chuvstvitel'nost' éukarioticheskikh odnokletochnykh organizmov.

Unicellular organisms are viewed as the best suitable objects for studying environmental effects, including the field of gravity, on living beings at the cellular level. Investigations of unicellular free-living eukaryotic organisms in gravitational and space biology help resolve both theoretical problems and practical problems associated with the design and development of biological life support systems. This paper presents experimental data about the effect of hypergravity on the structure, function and behavior of unicellular organisms--Tetrahymena pyriformis and Euglena gracilis. They were centrifuged at a rate of 87, 108 or 142 rpm to produce acceleration of 2, 3 or 5 G. The following parameters were measured: time course variations in the culture growth, rate of biomass accumulation, cell shape and size. It was found that as the gravity level grew from 2 to 5 G the culture growth was first stimulated and then inhibited. Biomass accumulation as well as the cell size and volume of Tetrahymena pyriformis and Euglena gracilis also decreased. It was demonstrated that the functional activity of cells was inversely proportional to the gravity magnitude. Potential mechanisms of the effect of hypergravity on the cell structure and function are discussed.