[ENTRY OF FACULTATIVE PATHOGEN SERRATIA GRIMESII INTO HELA CELLS. ELECTRON MICROSCOPIC ANALYSIS].

Facultative pathogens Serratia grimesii are able to invade eukaryotic cells where they have been found in vacuoles and free in the cytoplasm (Efremova et al., 2001; Bozhokina et al., 2011). However, efficiency of this invasion is low, and the mechanisms of the invasion related to the initial steps of the process are not known. In the present study, we have increased the invasion efficiency by incubation of HeLa cells with N-acetylcysteine (NAC) preceding the infection. In the NAC-pretreated cells, two modes of S. grimesii to enter HeLa cells were observed. In the most cases, the penetration of S. grimesii into the cell was consistent with the "zipper mechanism", involving specific interaction of bacterial invasin with a host cell surface receptor. However, in some cases, bacteria were trapped by membrane ruffling probably produced by injected bacterial proteins that trigger the bacterial uptake process, as described in the "trigger mechanism". Further elucidation of bacterial and cellular factors involved in the bacteria-host cell interaction should clarify whether two different mechanisms or a predominant one operate during S. grimesii invasion.

[Assembly of actin filaments induced by sequestration of membrane cholesterol in transformed cells].

Cholesterol is one of the major lipid components of plasma membrane and it plays an important role in various signaling processes in mammalian cells. Our study focused on the role of membrane cholesterol in organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by weakly developed actin network and reduced stress fibers--human embryonic kidney HEK293 cells, epidermoid larynx carcinoma HEp2 cells and mouse fibroblasts 3T3-SV40. Using F-actin labeling with rhodamine-phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin, and polyene macrolide antibiotic filipin. In cells treated with methyl-beta-cyclodextrin or filipin, similar processes of actin cytoskeleton reorganization involving filament assembly were revealed. In carcinoma HEp2 cells and fibroblasts 3T3-SV40, cholesterol-sequestering reagents induced intensive stress fiber formation and enhanced cell spreading which corresponded to reversion of transformed phenotype. The rearrangements of cytoskeleton are likely initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.

[The influence of Mycoplasma salivarium in the absence and presence of L-arginine on karyotypic variability in cell line of the Indian muntjak skin fibroblasts under long-term cultivation].

The influence of Mycoplasma salivarium on the numerical and structural karyotypic variability has been investigated in the "markerless" cell line of the Indian muntjak skin fibroblasts (line M) during long-term cultivation in the absence and presence of L-arginine. Cultivation of the mycoplasmal contaminated cells for 15 and 30 days did not change the character of cell distribution for the chromosome number. In the contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number was changed. These changes involved bimodal distribution for the chromosome number due to a significant decrease in the frequency of the cells with the modal number of chromosomes with main structural variant of karyotype (SVK)--2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with submodal number of chromosomes with main SVK--2 + 2 + 1 + 1. Besides, a significant increase in the frequency of the cells with lower chromosome number was observed in 60 days compared to that in 75 days of cultivation. Cultivation of the contaminated and control cells in the medium with increased concentration of L-arginine during 60 days did not change the numerical parameters relative to the control. Cultivation of the contaminated cells for 60 days followed by addition of L-arginine for 15 days restored the numerical parameters the numerical parameters to the control level. In the contaminated cells the frequency of chromosomal aberrations significantly increased for 30, 60 and 75 days cultivation relative to the control variant. In 30 days, the small but significant increase took place due to increase in the frequency of chromosomal aberrations of all the types. In 60 and 75 days, a greater increase took place due to a significant increase in the frequency of chromosomal and chromatid breaks. Moreover, in 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as one of the ways for adaptation of the "markerless" cell lines to condition of cultivation and the role of L-arginine in the restoration of normal karyotypic structure of cell population of line M under mycoplasmal contamination are discussed.

Probing for actinase activity of protealysin.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

Decreased sensitivity of transformed 3T3-SV40 cells treated with N-acetylcysteine to bacterial invasion.

Long-term treatment of transformed 3T3-SV40 mouse fibroblasts with antioxidant N-acetylcysteine decreased cell level of ROS and increased the concentration of reduced glutathione. Removal of N-acetylcysteine from the medium led to the appearance of well-expressed stress fibrils, virtually absent in control cells. In contrast to control cells, these cells were not invaded by apathogenic Escherichia coli A2 strain producing ECP32 protease specifically cleaving actin. Antioxidant N-acetylcysteine can cause partial reversion of transformed phenotype at the expense of a shift of cell redox balance in favor of reduced glutathione.

[Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts]. / Perestroiki aktinovogo tsitoskeleta v kletkakh 3T3 i 3T3-SV40 v prisutstvii antioksidantov.

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.

Invasive characteristics of apathogenic Shigella flexneri 5a2c mutant obtained under the effect of furazolidone.

Apathogenic Shigella flexneri 5a2c mutant treated with furazolidone can infect eucaryotic cells. These bacteria contain no virulence genes responsible for Sh. flexneri invasion, which seems to be the cause of their apathogenicity. The capacity of bacteria to penetrate into eucaryotic cells correlates with the appearance of ECP 32 protease specifically cleaving actin.

[Effect of agents changing the intracellular level of reactive oxygen species on the cell cycle phase distribution in 3T3 and 3T3SV40 cell lines]. / Vliianie agentov, izmeniaiushchikh vnutrikletochnyi uroven' aktivnykh form kisloroda, na kharakter raspredeleniia kletok linii 3T3 i 3T3SV40 po fazam kletochnogo tsikla.

A comparison of cell cycle phase distribution of 3T3 cells and their transformants 3T3SV40 treated with different substances changing the intracellular level of reactive oxygen species (ROS) has been made. In this study the following glutathione synthesis modulating agents were tested: two precursors of intracellular glutathione, antioxidant N-acetyl-L-cysteine (NAC) (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and inhibitor of glutathione synthesis, DL-buthionine-S, R-sulfoximine (BSO). It has been shown that both NAC (10-20 mM) and OTZ (20 mM) decreased the intracellular level of ROS in both cell lines. OTZ was more potent than NAC. However, only NAC caused changes in cell cycle progression of both cell types in dose-dependent manner. These changes differed in 3T3 and 3T3SV40 cells. Flow cytometric analysis of cell cycle phase distribution indicated that NAC (20 mM) blocked cell cycle in the G1 phase. The G1--arrest was completely reversible after removal of NAC from the medium. NAC (10-20 mM) caused a decrease in S and G2/M phases of transformants 3T3SV40. Moreover, a part of the population died apoptoticaly. Different mechanisms of NAC effect on normal and transformed cells are discussed. It is suggested that there is no strong correlation between cell cycle progression and intracellular level of ROS.

[The effect of mycoplasmal contamination of human embryonic lung cell line MRC-5 on the karyotypic variability]. / Vliianie mikoplazmennoi kontaminatsii kletochnoi linii legkogo émbriona cheloveka MRC-5 na kariotipicheskuiu izmenchivost'.

Karyotypic variability has been investigated for nonimmortalized human embryonic lung cell line MRC-5, cultivated with Acholeplasma laidlawii strain PG-8 for 15-45 days. The character of cell distribution for chromosome number did not change during this time. In all investigated variants the number of polyploid cells increased considerably with the lengthening of the term after decryoconservation. The number of chromosomal aberrations in 15-45 days contaminated cells increased significantly as compared to the control at the expense of dicentrics (telomeric associations). The number of dicentrics had a tendency to increase with the lengthening of the term of contamination. Thus, in 45 days the number of dicentrics increased twice as much as that in 15 days. The increase of polyploids may be due presumably to the specific character of karyotypic variability in nonimmortalized cell lines with the long-term cultivation. Our present and previous results made it possible to suppose that the formation of dicentrics (telomeric associations) in nonimmortalized "markerless" cell line, following the long-term mycoplasmal contamination, may prove additionally the role played by dicentrics in cell adaptation to in vitro conditions whatever the degree of transformation may be--nonimmortalized line or immortalized nontumorogenic or high tumorogenic lines.

[Invasion of Escherichia coli A2 induces reorganization of actin microfilaments in Hep-2 cells]. / Reorganizatsiia aktinovykh mikrofilamentov v kletkakh HEP-2 v rezul' tate invazii bakterii Escherichia coli A2.

Bacteria of spontaneously isolated non-pathogenic strain E. coli A2 have been previously shown to produce a new proteinase, referred to as protease ECP 32, which specifically cleaves actin (Khaitlina et al., 1988; Matveyev et al., 1996). Similar proteinase activity was found in revertants of Shigella flexneri L-forms. In this work immunofluorescence and electron microscopy were used to address a question of whether E. coli A2 can invade epithelial cells similarly as it has been demonstrated for Sh. flexneri. Infection of Hep-2 cells with E. coli A2 resulted in bacterial invasion of the cells followed by cytoskeleton reorganization. On one end of intracellular bacteria bundles of actin filaments resembling a comet-like tail were observed. Bacteria of referent strain CCM 5172, not producing protease ECP 32, were not taken up by the cells. These data suggest that protease ECP 32 may be involved in the process of bacterial invasion and cytoskeleton reorganization.

[Effect of mycoplasma contamination of the human uterine leiomyosarcoma cell line SK-UT-1B on karyotype structure]. / Vliianie mikoplazmennoi kontaminatsii kletochnoi linii leiomiosarkomy cheloveka SK-UT-1B na kariotipicheskuiu struktury.

The karyotypic variability has been investigated for human uterine leiomyosarcoma cell line SK-UT-1B, cultivated for 30-90 days after contamination with Acholeplasma laidlawii, strain PG-8. The character of cell distribution for chromosome number gradually changes in contaminated cells, comparatively to the control, with the lengthening of the term of contamination. So, in 30 days the analysed distributions do not differ in the experimental and in the control variants, the modal number of chromosomes being equal to 46. In 60 days the frequency of cells with modal number of chromosomes have a tendency to decrease, and the range of variability in the number of chromosomes tend to increase. In 90 days, the frequency of cells with modal number of chromosomes decreases significantly, and the range of variability on the number of chromosomes increases significantly. The number of chromosomal aberrations gradually increases in contaminated cells, as compared to the control, with the lengthening of the term of contamination. So, in 30 days the number of chromosomal aberrations does not increase, only the number of dicentrics (telomeric associations) has the tendency to increase. In 60 days, the number of chromosomal aberrations, mainly dicentrics, increases significantly. In 90 days, the number of chromosomal aberrations increases significantly, including both dicentrics and chromatid breaks. The possible reasons of the observed character of karyotypic variability is discussed. Our previous results make it possible to suppose that the increase in the number of dicentrics in "markerless" line SK-UT-1B with long term contamination may be an additional evidence on the role of dicentrics in cell adaptation to in vitro conditions in such lines.

[The dependence of the frequency of stable and unstable chromosome aberrations on the dose of the irradiation of human lymphocytes in vitro]. / Zavisimost' chastoty stabil'nykh i nestabil'nykh aberratsii khromosom ot dozy oblucheniia limfotsitov cheloveka in vitro.

Two methods (FISH and Giemsa) were used to estimate the frequency of stable and unstable chromosome aberrations in human lymphocytes exposed to gamma-irradiation in vitro in the dose range of 0.1 to 1.5 Gy. The DNA-probes specific to the chromosomes 1, 4, and 12 were used in combination with a pancentromeric probe. It was revealed that the dose-effect dependences for translocations (FISH) and dicentrics (FISH and Giemsa) were similar and could be adequately described by a linear-quadratic function.

[The effect of the Mycoplasma contamination of 2 kidney cell sublines from the kangaroo rat on their karyotypic structure]. / Vliianie mikoplazmennoi kontaminatsii dvukh sublinii kletok pochki kengurovoi krysy na kariotipicheskuiu strukturu.

The karyotypic variability has been investigated for two cell sublines of Rat kangaroo kidney cultured for 40-160 days after contamination with Acholeplasma laidlawii, strain PG-8. The contaminated cultures did not differ from non-contaminated ones in cell distribution for chromosome number. The majority of cells of subline NBL-3-11 with modal number of chromosomes displayed the main structural variant of the karyotype (SVK)--2+2+2+2+2+1; in subline NBL-3-17 the main SVK being 3+3+3+3+3+2. A comparison of intact cultures of these sublines in cell distribution for chromosome number show just the opposite direction of aneuploidy processes: cell heterogeneity for chromosome number decreased in NBL-3-11 and increased in NBL-3-17. The quantity of chromosomal aberrations, primarily chromosomal breaks, increases within 40-160 days of cultivation of contaminated cells of subline NBL-3-11. The number of chromosomal aberrations, mainly at the expense of dicentrics due to telomeric associations, increases after 40 days of cultivation of subline NBL-3-17 contaminated cells. During a long-term cultivation (110 days) of subline NBL-3-17 intact cells, there is an increase in the number of chromosomal aberrations, mainly dicentrics, whereas the extent of chromosomal breaks appears much less. The present results and other additional experimental data make it possible to suppose that the increase in chromosomal instability seen in subline NBL-3-17 at a long-term cultivation may be characteristic of this culture, in distinction to subline NBL-3-11. The most frequent breaks were seen in chromosomes 1, 2 and X of intact and contaminated cells in both the sublines. Chromosomes 1, 2 and 4 are mainly involved in dicentric formations by q (long) arms. The role of dicentrics in cell adaptation to in vitro conditions is discussed.

[The effect of mycoplasmal contamination of cultures of skin fibroblasts from the Indian muntjac and of the subsequent decontamination of the cultures using ciprofloxacin on the karyotypic structure of the cell line]. / Vliianie kontaminatsii mikoplazmoi kul'tur fibroblastov kozhi indiiskogo muntzhaka i posleduiushchei dekontaminatsii kul'tur s pomoshch'iu tsiprofloksatsina na kariotipicheskuiu strukturu kletochnoi linii.

The karyotypic variability of Indian muntjac skin fibroblast cell line, cultured for 95-168 days after contamination with Acholeplasma laidlawii strain PG-8, has been investigated. The contaminated cultures differ from noncontaminated ones in cell distribution for chromosome number. The noncontaminated cultures have modal number of chromosomes equal to 7 with the main structure variant of the karyotype (SVK) 2+2+1+1+1. In the contaminated cultures the cell number with 7 chromosomes and the main SVK 2+2+1+1+1 decreased, whereas the cell number with 6 chromosomes increased along with the main SVK 2+2+1+1 resulting from the loss of chromosome Y1. The treatment of cells with ciprofloxacin for mycoplasma decontamination did not restore the normal cell distribution for chromosome number. The frequency of chromosomal aberrations, mainly dicentrics, due to telomeric associations, increased after 95-168 days of cultivation of contaminated cells. Chromosomes 1 and 2 and their combination are mainly involved in dicentric formations. The treatment of contaminated cells with ciprofloxacin restores the initial frequency of chromosomal aberrations. Chromosomes with altered structures have not been demonstrated. It has been shown that cells became mycoplasma-free after 15 days of treatment with ciprofloxacin. The role of aneuploidy and dicentrics in cell adaptation to culture conditions is discussed.

[A cytogenetic study of the Chinese hamster lung cell line V-79 infected with Mycoplasma arginini and decontaminated using ciprofloxacin]. / Tsitogeneticheskoe issledovanie linii kletok legkogo kitaiskogo khomiachka V-79, infitsirovannoi mikoplazmoi Mycoplasma arginini i dekontaminirovannoi s pomoshch'iu tsiprofloksatsina.

Karyotypic variability has been studied in a line of the Chinese hamster cells artificially contaminated with Mycoplasma arginini. The contaminated cultures differed from mycoplasma-free cells in cell distribution for chromosome number. The frequency of cells with modal chromosome number 21 decreased, while that of cells with 20 chromosomes increased. Decontamination of cell culture with ciprofloxacin (10 mg/ml) and a subsequent cultivation of cell in the antibiotic-free medium did not restore the original cell distribution for chromosome number. In-53--131 days after infection, the increased frequency of chromosomal aberrations was registered. Eradication of Mycoplasma by ciprofloxacin and a long-term cultivation in antibiotic-free medium restored the frequency of chromosomal aberrations to the control level corresponding to mycoplasma-free cultures. In contaminated cultures no cyto- or genotoxic effect of ciprofloxacin was observed. These data, together with the previous ones, enable the authors to recommend ciprofloxacin to make cell cultures free from mycoplasmas with minimal risk to change the original properties of cell lines.

[Diagnosis of mycoplasma infections using directed amplification]. / Diagnostika mikoplazmennykh zarazhenii s pomoshch'iu napravlennoi amplifikatsii.

Test systems for rapid detection of mycoplasmas in biological samples have been elaborated on the base of the polymerase chain reaction (PCR). Amplification of the conservative rDNA sequences was used for testing of cell cultures for mycoplasmal contamination. Mycoplasma pneumoniae detection system has been developed based on amplification of the species-specific DNA sequences. Inversions of some repeated sequences in the Mycoplasma pneumoniae genome make it possible to run the PCR with a single primer. The revealed spacer length polymorphism for 16S-23S rDNA operons can be used in the mycoplasmas identification.

[The effect of Mycoplasma contamination and decontamination with ciprofloxacin on the karyotypic structure of the Chinese hamster V-79 lung cell line]. / Vliianie mikoplazmennoi kontaminatsii i dekontaminatsii s pomoshch'iu tsiprofloksatsina na kariotipicheskuiu strukturu kletochnoi linii legkogo kitaiskogo khomiachka V-79.

Karyotypic variability was investigated for the Chinese hamster lung cell line V-79, infected (contaminated) with a mycoplasma Acholeplasma laidlawii A. The mycoplasmal contamination did not affect cell distribution for the chromosome number. However, 30-70 days following cell culture contamination the increase in chromosomal aberrations was observed in the contaminated cell line primarily at the expense of chromosomal breaks. The following cyprofloxacin treatment of the culture resulted in mycoplasmal elimination and decrease in the frequency of chromosomal aberrations up to the control level. The analysis of G-banded chromosomes showed no significant differences in karyotypes of originally non-infected cells and cells after decontamination. The karyotypic variability, induced by mycoplasmal infection in V-79 cell line, differed from that in the muntjac skin fibroblast cell line, the latter being described elsewhere. A predominant type of chromosomal variability in V-79 cell line are chromosomal breaks, whereas in the muntjac fibroblast cell line dicentrics (telomeric fusions) were primarily observed, possible explanations of these differences being discussed. This may be presumably due to differences in karyotypic structure of these two cell lines, and to their different adaptation to culture conditions.

[The effect of Mycoplasma contamination on the karyotypic structure of a skin fibroblast cell line from the Indian muntjac]. / Vliianie mikoplazmennoi kontaminatsii na kariotipicheskuiu strukturu kletochnoi linii fibroblastov kozhi indiiskogo muntzhaka.

The karyotypic variability has been studied in a low chromosomal cell line of the Indian muntjak skin fibroblasts contaminated with Mycoplasma arginini R-16 and Acholeplasma laidlawii A. The mycoplasmal contamination exerted influence on the cell distribution for the chromosome number. The frequency of the modal class cells with 7 chromosomes, having the main structure variant of the karyotype (MSVK) 2 + 2 + 1 + 1 + 1, was seen to decrease, while the frequency of the submodal class cells with 6 chromosomes, having MSVK 2 + 2 + 1 + 1 because of the loss of Y1 chromosome from MSVK 2 + 2 + 1 + 1, to increase. The frequency of chromosomal aberrations, predominantly that of dicentrics, was higher in the contaminated variants. Dicentrics were mainly produced by chromosomes 1 and 2. They were formed by a nonrandom combination of chromosomes and their arms. Possible correlations are discussed between aneuploidy and dicentric chromosome formation in low chromosomal cell lines, and cell adaptations to culture conditions.