~~Different cloning vectors (pC194, pBC16, PUB1 10, pBD10, pBD8, pAMP1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about lo3 c.f.u. per pg DNA) and allows different cloning vectors with molecular masses ranging from 1.8 to 17.7 MDa to be introduced into B. anthracis.

As the study of metaphase chromosomes with flow cytometry presupposes mixing all chromosomes from many cells before analysis, important information is lost. To overcome this limitation we have developed a novel cell‐oriented flow cytometric method for chromosome analysis. A flow cytometer supplied with a special device for disruption of metaphase chromosomes is the heart of the method. Cells with stained chromosomes are pressed into the disruption device and then converted in batches of chromosomes. Chromosome fluorescence and time intervals between fluorescence pulses are registered. There are large time intervals between neighboring batches because a sparse cell suspension is used and, in turn, there are small time intervals inside batches. Taking account of time intervals, it is possible to identify individual cell flow karyotypes. This highly productive method for individual cell analysis (100–200 cells/min) ensures detecting the changes in cell flow karyotypes, i.e., under‐ and overrepresentation of chromosomes, aberrations, and amplification of DNA. © 1996 Wiley‐Liss, Inc.

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Химиотерапия опухолей традиционно направлена на ингибирование пролиферации раковых клеток и активацию апоптоза. В данной работе на клетках карциномы (линия HeLaG63) и эндотелиоцитах (линия ECV304) человека проведено сравнительное исследование эффектов низких концентраций (1,5-10мМ) двух аналогов глюкозы: 2-DG и глюкозамина D. Оценка эффективности действия этих агентов проводилась по следующим параметрам: снижение жизнеспособности по МТТ-тесту, изменение проницаемости клеточной мембраны, изменение прогрессии по клеточному циклу, выраженность апоптотической гибели при культивировании клеток в питательной среде с различным содержанием глюкозы. Показано, что 48 часовая обработка 2-DG в исследованных концентрациях приводила к снижению доли клеток в G1 и S-фазах и аккумуляции их в G2\M фазах. Глюкозамин D, в отличие от 2-DG при тех же концентрациях блокировал эти же клетки в G1\S фазе клеточного цикла. При обработке клеток HeLaG63 глюкозамин D оказывал более токсичное действие, чем 2-DG. При сочетанном воздействии 2-DG и глюкозамина D регистрировали существенное увеличение суб- G1-популяции по сравнению с раздельным воздействием. Эффективность обработки увеличивалась при снижении концентрации глюкозы в питательной среде и/или с увеличением дозы агентов. Эндотелиоциты (ECV304) были менее чувствительны, как к действию глюкозамина D, так и 2-DG, и сочетанное воздействие даже при концентрациях 10мМ не отличалось от раздельного воздействия. Обработка клеток 10мМ 2-DG и глюкозамина D приводила к увеличению проницаемости клеточных мембран для флуоресцентного красителя пропидиум йодида, при этом наибольшую эффективность регистрировали для клеток HeLaG63. Chemotherapy for tumors has traditionally been aimed at inhibiting proliferation and activating apoptosis of cancer cells. Aim. To perform a comparative study of effects of two glucose analogues, 2-deoxy-D-glucose (2-DG) and glucosamine D, at low concentrations (1.5-10 mM) on carcinoma cells (HeLaG63 line) and endotheliocytes (ECV304 line). Methods. Efficacy of these agents was evaluated by decreased cell viability (MTT test), permeability of the cell membrane, changes in progression by the cell cycle, and apoptosis (cytometric method) of cells cultured in mediums with different glucose concentrations. Results. The 48-h 2-DG treatment of cells in the studied concentrations reduced the proportion of cells in G1 and S-phases and their accumulation in G2\M phases. The same concentrations of glucosamine D, as distinct from 2-D, blocked the same cells in the G1\S phase of the cell cycle. The same concentrations of glucosamine D were more toxic to carcinoma cells than 2-DG. A combination of 2-DG and glucosamine D significantly greater increased the sub-G1 population of HeLaG63 cells than either agent alone. The treatment effectiveness increased with a decrease in the glucose concentration in the medium and/or with an increase in the agent dose. Endotheliocytes (ECV304) were less sensitive to both glucosamine D and 2-DG, and the effect of their combination did not differ from the effect of either agent alone, even at concentrations of 10 mM. Treatment of cells with 10 mM 2-DG and glucosamine D increased the cell membrane permeability for the fluorescent dye, propidium iodide, with the greatest effect recorded for HeLaG63 cells. Conclusion. Therefore, the anticarcinogenic efficacy of glycolysis inhibitors can be enhanced, which would allow to considerably reduce their doses and avoid potential side effects induced by therapeutically effective drug concentrations.