[Effect of etoposide and amsacrine on mitotic progression of GM-130 and Hep-2 cell lines. The flow cytometry assay]. / Vliianie étopozida i amsakrina na prokhozhdenie mitoza kletkami linii GM-130 i Hep-2. Analiz metodom tsitometrii v potoke.

It has been shown that inhibitors of topoisomerase II (topo II) etoposide and amsacrine results in accumulation of GM-130 and Hep-2 cells with 4c DNA amount. The differential analysis based on flow cytometry (Zenin et al., 2001) and enabled us to discriminate cells with 4c DNA--G2, M, including metaphase and anaphase cells and cells in pseudo-G1. 1 microM etoposide evoked cell accumulation in G2 phase, while 40 microM etoposide blocked cell proliferation, which was confirmed by a complete absence of both mitotic cells and 4c DNA cell accumulation. GM-130 and Hep-2 cells that were first blocked and then washed from nocodazole, and after that treated with 50 microM etoposide or 20 microM amsacrine, were shown to enter pseudo-G1 with 4c DNA amount per cell. In the presence of nocodazole, 4 and 40 microM amsacrine evoked transition of all mitotic cells to pseudo-G1 within 1 h. 15 or 30 minutes pulse treatments of GM-130 cells with 40 microM amsacrine in the presence of nocodazole, followed by incubation in drug-free medium, resulted in the similar transition of cells to pseudo-G1.

[Optimization of conditions for isolating chromosomes in flow sorting]. / Optimizatsiia uslovii vydeleniia khromosom pri ikh sortirovke v potoke.

Factors for purity and efficiency of flow chromosome sorting were analysed on the base of quantitative analysis. The sorting rate and relative purity of individual chromosome fractions are determined both by the quality of initial chromosome suspension, instrument parameters and gate position on experimental histograms. The described procedure of analysing sorting efficiency and fraction purity allowed to formulate general tips for optimization of sorting conditions depending on a given strategy: maximization of quantity of obtained material, or achieving maximum purity for sorted fraction. The analysis is carried out on the bases of chromosome distribution parameters: their relative halfwidths and distances. These parameters can be obtained by the quantitative analysis programs for flow cytometry data. It is shown that the critical parameter for sorting purity is the level of contaminated objects in a zone of sorted chromosome signal registration. In addition, the fraction purity depends on the cover extent between different chromosome distributions. Created procedure allows to build up nomograms linking the sorting efficiency with fraction purity, depending on the position of sorting gates. These monograms permit to determine the position of sorting gates in relation to one or another strategy: 1) maximum rate for chromosome material obtaining, 2) maximum fraction purity, or 3) compromise between the two. The presented analysis allows to optimize the chromosome sorting process for subsequent genome investigations providing chromosome material with controlled characteristics.

[A novel method of chromosome distribution analysis in saccharose density gradient]. / Primenenie novogo metoda k analizu raspredeleniia khromosom v gradiente plotnosti sakharozy.

The human chromosomes distribution in a sucrose density gradient was studied using a new computer method of the quantitative analysis of flow karyotypes. The dual-parameter flow distributions of human chromosomes fluorescence intensities of the sucrose density gradient fractions were analyzed to obtain the quantity of each chromosome. The chromosomes were found to distribute over sucrose density gradient as follows: 1) fractions with low sucrose density mostly contain chromosomes 1-7, and their quantity is increased between 1.4- to 3.2-fold in comparison with the control unfractionated suspension; 2) medium density fractions are enriched with chromosomes 8-20 up to 2.4-fold; 3) fractions with a high sucrose density mostly contain small chromosomes 21-22 and fragments of broken chromosomes. So the new method of quantitative analysis of flow karyotypes allows one to determine the efficiency of enrichment and the maximally enriched fraction for any chosen chromosome. Maximally enriched fractions maximize the rate of preparative flow sorting of individual chromosomes for research or biotechnology purposes.

[A device for sample pretreatment during flow cytometry]. / Ustroistvo dlia predobrabotki obraztsa pri analize metodom tsitometrii v potoke.

A device for the preliminary treatment of samples immediately prior to flow cytofluorimetric analysis is described. The device is intended for several procedures: (a) mixing of batched sample volumes with the reagent and efficient stirring of the mixture; (b) disintegration of cell aggregates; and (c) disruption of cell membranes to release the cell contents (chromosomes, micronuclei, nuclei etc.). The pretreatment is useful for studying the kinetic parameters of fast cellular processes in the flow, a more correct analysis of the cell cycle and the study of karyotypes of single mitotic cells. The device was called a magnetic microstirrer.

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.

[Use of photo-anchoring of DNA probes for fluorescent in situ hybridization]. / Ispol'zovanie fotoprishivaemykh DNK-zondov dlia fluorestsentnoi gibridizatsii in situ.

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.

[Spectrofluorometric analysis of protein fractions of blood serum of health donors and patients with kidney diseases]. / Spektrofluorimetricheskii analiz belkovykh fraktsii syvorotki krovi zdorovykh donorov i bol'nykh s zabolevaniiami pochek.

A comparison was made of ultra-violet fluorescence characteristics of albumin enriched protein fractions of serum blood of 35 glomerulonephritis (GN) patients, 30 uremic haemodialysis patients, and 40 donors. It has been found that fluorescence spectra of the albumin enriched fractions of serum from GN patients are blue shifted as compared with those of donors' serum preparations. At the same time fluorescence spectra of the albumin enriched fractions of donors' serum are similar, while those of GN patients vary significantly. The ratio of fluorescence intensities at 320 and 365 nm (A = I320/I365), characterizing the spectrum position is 1.27 +/- 0.05, for protein preparations of donors, while that for GN patients varies within the limits of 1.3-2.1. Fluorescence spectra of protein fractions from blood serum of uremic patients are red shifted (A = 0.77-1.29) in comparison with those of donors' blood. Chromatographic investigations show that protein preparations of GN patients' blood contain monomers and dimers of albumin, that can be divided according to their molecular masses or hydrophobic properties of the surface. Joint chromatographic and spectral analysis allowed to distinguish up to six albumin enriched fractions. A protein fraction with blue fluorescence spectrum was obtained by gel-filtration and ion exchange chromatography. A higher concentration of this fraction determines a high value of parameter A, that is typical for protein preparations of GN patients' blood. Amino acid sequence shows that one component of this fraction is beta-haptoglobin.

[Two-parameter flow fluorescence analysis of human chromosomes. Quantitative processing]. / Dvukhparametricheskii fluprestsentnyi analiz khromosom cheloveka v potoke. Kolichestvennaia obrabotka.

A procedure for the quantitative analysis of the results of fluorimetric studies on human chromosomes in a flow is described. The procedure enables one to simultaneously follow two parameters by using two fluorochromes, one of which is selective to the GC-, and the other, to AT base pairs of DNA. Thus, it becomes possible to derive from the experimentally obtained distributions an information on the relative DNA content in individual chromosomes, differences in DNA content between homologous chromosomes, the relative number of chromosomes of each type in the sample tested, as well as on the percent ratio of AT and GC base pairs in a particular chromosome. In addition, the procedure enables one to assign, with a high degree of accuracy, the peaks on experimental distributions to the objects of analysis chromosomes. The analysis cutting off the extreme combines the method of components with the least square method, as applied to the peaks of the Gaussian shape. The first step involves the filtration of the starting experimental data from contaminating signals from both the parts of degraded chromosomes and stained cytoplasm fragments, and from chromosome aggregates. The procedure is realized as a package of programs for IBM-compatible PC/AT computers (486 and later versions), which permits one to perform a comprehensive analysis of two-parameter distributions of chromosome fluorescence signals to solve a number of problems, such as the identification of chromosome aberrations in cell lines tested, quantitative comparison of distinguishing features of homologous chromosomes and whole chromosome sets.

[The isolation and analysis of lymphoblastoid cell lines from patients with xeroderma pigmentosum and progeria]. / Poluchenie i analiz limfoblastoidnykh kletochnykh linii ot bol'nykh pigmentnoi kserodermoi i progeriei.

Lymphoblastoid cell lines from patients with xeroderma pigmentosum (2 forms) and progeria (unusual form) were established using transformation of peripheral blood lymphocytes by Epstein--Barr virus. The influence of different UV doses on cell vitality, proliferation and cell cycle progression was studied by means of flow cytometry. The cell vitality was determined after incubation of cells with etidium bromide and FDA. We used cytograms with two logarithmic signals (log green/log red) to discriminate the cell cycle status. Cell cultures were used with density of 500,000 cells per 1 ml, previously synchronized at G-phase by the incubation in a medium with low serum content. The effect of UV irradiation was followed during 72 h. Among four analysed cell lines only line XP2SP demonstrated enhanced UV sensitivity, expressed by decreasing of the amount of living cells after the UV dose of 2.5 J/m2 and higher. The cell cycle studies showed that cells were blocked in S-phase and simultaneously the amount of apoptotic cells with both reduced DNA content and ability to bind FDA was seen increased. Similar events were observed in the control line only after the dose of 20 J/m2 and higher.

[The use of lysolecithin for obtaining a chromosome suspension]. / Primenenie lizoletsitina dlia polucheniia suspenzii khromosom.

Lysolecithin treatment was used for obtaining isolated metaphase chromosomes from HeLa cells and lymphoblastoid cell lines BOLD and ROM. Chromosomes were stained with Hoechst 33258 and olivomycine. Chromosome quantity in suspension was determined by dual-laser sorter ATC-3000. It is shown that lysolecithin treatment of the cells allows to isolate chromosomes with higher concentration in suspension and less amount of debris in comparison with Triton X-100 treatment. A protocol for chromosome isolation using lysolecithin treatment is suggested.

[Quantitative processing of results of uniparametric fluorescent flow analysis of human chromosomes]. / Kolichestvennaia obrabotka rezul'tatov odnoparametricheskogo fluorestsentnogo analiza khromosom cheloveka v potoke.

The proposed procedure of computer analysis of the flow karyotype data, obtained in human chromosomes studies, is able to provide information about the basic parameters of the karyotypes: the positions of the peaks (corresponding to the relative size of chromosomes), peaks areas (relative number of chromosomes in the sample), coefficients of variation (CV) of the peaks--possible differences between homologous chromosomes. The analysis is based on the assumption that all chromosomal components of the experimental distributions are normal (Gaussians). The algorithm of the analysis uses a combination of two approaches: truncation method and least squares method. As the flow data are "contaminated" by background components, special tools for filtering off the contaminating signals were designed including the original integral Fourier filtering procedure. This analysis is realized in a program package utilizing IBM-compatible PCs. The user is able to get the desired parameters for most chromosomes of the karyotype under study from univariate flow data: differences between particular homologous chromosomes, presence of chromosome aberrations, extra chromosomes, etc., since structural aberrations and chromosome number variation lead to specific changes of the parameters of chromosome-related components.

[Uniparametric analysis of chromosomes from human fibroblasts using flow cytofluorometry]. / Odnoparamtericheskii analiz khromosom iz fibroblastov cheloveka metodom tsitofluorimetrii v potoke.

Metaphase chromosomes from human diploid fibroblasts were isolated by the polyamine method and stained with different fluorochromes: propidium iodide, chromomycine A2, bisbensimide H33258, and DAPI. The fluorescently stained chromosomes were analyzed using a cell sorter ATC-3000 ("Bruker-Spectrospine"), and the flow histograms were investigated to identify the peaks of individual chromosomes and to choose the chromosomes which can be isolated to a high purity. High resolution was obtained with H33258 or DAPI. It was demonstrated that the peaks of chromosomes 13, 17, 20 are well distinguished from the adjacent peaks, so that these chromosomes can be sorted with a high purity. The peaks formed by chromosomes 5, 6, 8, 14, 15 are slightly overlapped by the adjacent peaks, and the sorting on the basis of monovariate analysis cannot provide sufficient purity of the sorted fractions (< 0.5). Chromosomes 1-2, 3-4, 7-X, 9-12, 16-18, 19, 21-22 cannot be discriminated for successful sorting of an individual chromosome type. The data obtained provided an experimental basis for choosing the chromosomes to be sorted with high purity for the subsequent cloning of their DNA.

DNA content in Eurasian sturgeon species determined by flow cytometry.

The nuclear DNA content in 10 species of chondrostean fishes was measured by flow cytometry. The sterlet Acipenser ruthenus blood cells were used as an internal standard. The sterlet DNA content was calculated on the basis of comparison with the Xenopus laevis blood cells, 2C = 6.30 pg. In the tetraploid A. ruthenus and A. stellatus the DNA content comprises 3.74 pg/nucleus and is practically invariant; in Huso dauricus it is almost the same, 3.74-3.81 pg; and in A. nudiventris it is a little higher, 3.88-4.04 pg. In the oldest chondrostean, Pseudoscaphirhynchus kaufmanni, the nuclear DNA content is slightly lower, 2C = 3.46-3.48 pg, and in the American paddlefish Polyodon spathula it is lower still, 3.17 pg. In two octoploid sturgeons, A. baeri and A. gueldenstaedti, the DNA content is twice as high as that of the sterlet, 8.29-8.31 and 7.86-7.88 pg, respectively; a very similar amount, 8.24-8.42 pg, was determined in the hybrid Huso huso x A. ruthenus. In the Sakhalin sturgeon, A. medirostris (= A. mikadoi), the DNA content is two times higher than in the octoploids, 13.93-14.73 pg; therefore its ploidy may be 16n and the number of chromosomes could be 500.

Drop-delay measurement using enzyme-coated particles.

A simple technique is suggested for the measurement of drop delay for flow sorting. While the flow cytometer was set to sort a fixed number of particles, the drop-delay setting was changed step by step, and at each step the HRP-coupled particles were sorted into a well of an immunoassay strip. Then the HRP activity of the sorted samples was revealed by routine methods. The maximum level of the enzyme activity shows the proper drop-delay setting. Determination of the drop-delay setting takes only a few minutes. The technique is independent of the type of flow cytometer and does not require any additional equipment.

[Isolation of individual chromosomes and their use in studying the human and animal genome]. / Vydelenie individual'nykh khromosom i ikh ispol'zovanie dlia izucheniigenomov cheloveka i zhivotnykh.

This mini-review considers the main achievements in the field of human and animal chromosome separation: technical problems of flow-karyotyping and flow-sorting, the types of cell lines and chromosome source used, different protocols of chromosome suspension preparation, fluorochromes for chromosome staining, efficiency of chromosome sorting and purity of the fractions. The use of fractions for creating chromosome-specific human gene libraries and mapping cloned genes and DNA fragment are also considered.

[Effect of deproteinization on the in situ chromatin staining with 7-aminoactinomycin D]. / Vliianie deproteinizatsii na okrashivanie khromatina in situ 7-aminoaktinomitsinom D.

The possibility of use of 7-amino-actinomycin D (7aAMD)--fluorescent analog of actinomycin D--as a specific dye for DNA staining in the suspended cells was studied by means of laser flow-cytometry. The optimal conditions for staining were obtained: 7aAMD concentration 10(-5) M, pH 7, staining time 20 min, 37 degrees C, ionic strength 0.15 M Na+. In this case the fluorescent signal is proportional to the DNA amount and coefficient of variation is about 0.03. The influence of the stepwise extraction of the proteins from chromatin also was studied. In the course of the salt deproteinization the fluorescence intensity gradually rose thus showing the increase of the binding sides-number. The deproteinization of cells nuclei by 0.1 HCl increased the number of binding sites 2.5 times more. It was shown that the incubation of cells with RNAse at elevated ionic strength (0.3-0.7 M NaCl) leads to an additional increase of the cell fluorescence and produces no effect at low and normal ionic strength. The deproteinizing effect of RNAse and its possible mechanism is discussed.

[Fluorescence cytochemistry of nucleic acids. The current status and outlook: on the 35th anniversary of the method]. / Fluorestsentnaia tsitokhimiia nukleinovykh kislot. Sovremennoe sostoianie i perspektivy: k 35-letiiu metoda.

Most important fields of the fluorescence cytochemistry of nucleic acids are reviewed, special attention being paid to the following aspects of the problem: physicochemical basis of different methods of staining; new methods of cell analysis, including flow cytometry; functional studies, i.e. the use of fluorescence approaches to obtain information about the functional state of individual cells and cell aggregates.

[Kinetics of DNA-distaxin complex-formation]. / Kinetika kompleksoobrazovaniia DNK s distaktsinami.

Kinetics of DNA--distaxines complexes formation was studied spectrophotometrically by means of the stop--flow method. It has been found that increase of number of bulky pyrrolcarboxamide group in the row of distaxines: -0, -1, -2, - slows down the binding reactions and increases the number of their stages.

[Flow cytometry and cell sorting: current state and prospects for use in molecular biology]. / Protochnaia tsitometriia i sortirovka kletok: sovremennoe sostoianie i perspektivy ispol'zovaniia v molekuliarnoi biologii.

A survey of the specific features of flow cytometry, principals of instrumentation and main parameters of the modern cell sorting cytometers is given. Analytical capacities of flow cytometry as well as the main directions of its applications in cell biology, clinical diagnostics, immunology, biotechnology and molecular biology are considered. Also a possible future development of flow cytometry instrumentation and applications in molecular biology are briefly discussed.