[Effects of myostatin and other growth factors on cultured human cells]. / Vliianie miostatina i nekotorykh rostovykh faktorov na kul'tiviruemye kletki cheloveka.

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.

Comparison of cytotoxicity of aminoglycoside antibiotics using a panel cellular biotest system.

The cytotoxicity of four aminoglycoside antibiotics was studied by estimation of the dose-effect relationship using a panel cellular biotest system including cell cultures for test objects. The cultures represented 4 differentiation types: normal human fibroblasts and myoblasts, human or Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells. It was found that three widely used antibiotics gentamicin, kanamycin, and neomycin exhibit similar, but not identical cytotoxicity parameters and differ distinctly from geneticin. Hence, the proposed panel biotest system helps to quantitatively evaluate and differentiate the effects of bioactive substances with similar chemical structure.

[Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation]. / Ekspressiia gena distrofina u bol'nykh miodistrofiei Diushenna posle transplantatsii mioblastov.

Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used.

Cell culture test system for express analysis of cytotoxic and growth-stimulating effects of bioactive compounds.

Cultures of human and mammalian cells presenting 4 types of differentiation (normal human fibroblasts and myoblasts, human and Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells) were used in a panel biotest system. This system allowed to evaluate the cytotoxic and stimulatory effect of bioactive compounds by determining the dose-effect relationships and some quantitative parameters including LD(50). Examination of some biolactive compounds of different nature (sangviritrin, escin, deltostim, cycloheximide, dexamethasone) confirmed high efficacy of this biotest system.

[Human myoblast culture as muscle stem cells in medical and biological studies]. / Kul'tiviruemye mioblasty cheloveka kak stvolovye kletki myshechnoi tkani v medikobiologicheskikh issledovaniiakh

The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

Use of cultured human fibroblasts for rapid evaluation of cytotoxic effects of bioactive substances.

A test system for detecting cytotoxic effects of bioactive substances based on human fibroblast culture is proposed. The effects of acrylamide, streptomycin, cycloheximide, sodium dodecyl sulfate, sanguiritrine, and ethanol were evaluated by organic stain binding. Typical dose-effect relationships were detected for all substances except cycloheximide. The proposed test system can be used for screening of bioactive substances in preclinical trials.

[Study of protein products of gene expression in cells with altered chromosome sets for genetic mapping]. / Izuchenie belkovykh produktov gennoi ékspressii v kletkakh s izmenennym naborom khromosom dlia geneticheskogo kartirovaniia.

Two-dimensional electrophoresis was used for analyzing proteins in hybrid cells that contained single human chromosomes (chromosome 5, chromosome 21, or chromosomes 5 and 21) against the background of the mouse genome. By comparing the protein patterns of hybrid and parent cells (about 1000 protein fractions for each kind of cell), five fractions among proteins of hybrid cells were supposedly identified as human proteins. The genes of two of them are probably located on chromosome 5, and those of other three, on chromosome 21. Moreover, analysis of proteins in fibroblasts of patients with the cri-du-chat syndrome (5p-) revealed a decrease in the content of two proteins, as compared with those in preparations of diploid fibroblasts. This fact was regarded as evidence that two corresponding genes are located on the short arm of chromosome 5. Methodological problems associated with the use of protein pattern analysis in cells with altered chromosome sets for the purposes of genetic mapping are discussed.

[Characterization of cloned alpha-satellite sequence from the extrachromosomal DNA of HeLa cell culture]. / Kharakteristika klonirovannykh al'fa-satellitnykh posledovatel'nostei iz ékstrakhromosomnykh DNK kul'tur kletok HeLa.

We have obtained the alphoid DNA clones, pK1 and pK2, from the extrachromosomal DNA of Hela cells treated by cycloheximide (30 micrograms/ml). Nucleotide sequences of the clones were aligned. The sides of the pK1 and pK2 are 390 and 184 bp, respectively. The marked RELP for the clones was not observed. The results of in situ hybridization have shown an approximately equal distribution of Ag-grains over major part of human chromosomes, with a slight preference for chromosomes 1, 5 and 19 (the 1-st group of alpha-satellite DNA). Therefore, the obtained alphoid sequences seem to be rather conservative and non-chromosome-specific. We suppose that increase of the alphoid DNA content in the fraction of the extrachromosomal DNA under the cycloheximide treatment is a result of the sporadic statistical processes rather then consequence of the specific excision.

[Effects of cycloheximide on protein, RNA and DNA synthesis in cultures of CHO cells and human diploid fibroblasts]. / Vliianie tsiklogeksimida na sintez belka, RNK i DNK v kul'turakh kletok CHO i diploidnykh fibroblastov cheloveka.

In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go--an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long-term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels.

[Increase in the quantity of highly repetitive sequences in extrachromosomal circular DNAs under inhibition of translation by cycloheximide]. / Uvelichenie soderzhaniia vysokopovtoriaiushchikhsia posledovatel'nostei v ékstrakhromosomnykh kol'tsevykh DNK pri tormozhenii transliatsii tsiklogeksimidom.

Small polydispersed circular DNA(spcDNA) was isolated from cultivated HeLa cells. Cells were treated by cycloheximide in concentrations of 1 and 50 micrograms/ml. Gradient fractions were dot blotted to nitrocellulose filters and were hybridized with different repetitive DNAs. The pool of repetitive DNA sequences in fraction of spcDNA increased for cycloheximide treated cells. The content of Alu sequences increased by 1.5-2.5 times, "classical" satellite DNA--by 5-7 times, alpha-satellite--by 3-5 times.

[The role of the cytoskeletal elements during the return to the cell center of their nuclei displaced by centrifugation]. / Rol' élementov tsitoskeleta v protsesse vozvrashcheniia v tsentr kletok ikh iader, smeshchennykh tsentrifugirovaniem.

Living cells of the monolayer cultures of embryonal pig kidney epithelium (PKE-cells) and of embryonal bovine tracheal cells (FBT-cells) were ultracentrifuged at 20,000g. The centrifugal force was directed parallel to the surface of the culture slides. Just after centrifugation, the cellular nuclei were displaced to the centrifugal parts of cells. Centrifuged slides with cells were returned to the normal culture conditions, and 22 h later the nuclei were seen to restore their central position in the cells. The motion of the nuclei to the cell center was rather chaotic both in direction and speed. The speed of this motion never exceeded several microns per hour. After nocodasole treatment (0.1-10 mkg/ml) of the cells or in a hypotonic medium, the distance of nuclear dislocation during centrifugation was longer, and the nuclei returned to the cell centers faster than in the control ones. After cytochalasin B treatment (2 mkg/ml), the nuclei moved to the cell centers somewhat more slowly than they did in the control cells. Thus, the establishment of the central position of nuclei in the cells takes place in the absence of microtubules or intermediate filaments. Probably, the central position of nuclei depends mainly on the action part of the cytoskeleton.