Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions.

V79 Chinese hamster cells were treated with hypertonic solutions of NaCl or KCl and irradiated rat various times before, during, or after exposure to the solution. In solutions of molarities between 0-2 and 0-5 M, the cellular radiosensitivity increases with the molarity of the bathing solution. At these molarities, the hypertonic solution need not be present during irradiation to sensitize cells. Furthermore, radiosensitivity of cells could be increased by exposing cells for longer times to the hypertonic solution before irradiation. At higher salt concentrations (at 1-5 to 1-8 M), significant radioprotection is observed. Survival curve data showed that this protection was characterized by an increase in DO and a decrease in n, while the survival curves of cells sensitized with 0-465 M NaCl or with lower concentrations exhibited mainly changes in DO. The 1-55 M NaCl solution must be present during radiation to give a protective effect. Prolonged exposure to the salt before irradiation reduced the amount of radioprotection afforded by the salt. The results are discussed in terms of the effects of ions on histones, cellular water structure and the cell-aging cycle.     

Rapid-mixing studies of radiosensitivity with thiol-depleted mammalian cells

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.     

Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.     

Effect of anisotonic NaCl treatment on cellular ultrastructure of V79 Chinese hamster cells. Part 1. Mitotic cells

The ultrastructural modifications produced by anisotonic NaCl treatment of Chinese hamster mitotic cells were observed at three NaCl concentrations which have been frequently used in radiosensitization studies: 0.05, 0.5 and 1.5 M. After exposure to 0.05 M NaCl, many well-spread chromosomes are visible. The chromatin fibres are well dispersed and membraneous material is associated with the chromosomes. After hypertonic treatment with 0.5 M NaCl, the chromosomes have a uniform, structureless appearance with some coalescing into larger anaphase-like masses. At 1.5 M NaCl, large scale cellular dehydration is apparent, and filamentous structures such as microfilaments are tightly constricted. The degree of chromosome staining is also reduced below the level of the cytoplasm. After both hypo- and hypertonic NaCl treatment the chromosomes appear swollen relative to untreated cells, but hypertonic treatment causes chromosome clumping and dissociates chromatin. Conformational changes in the chromatin may restrict the capacity for DNA repair and be related to cellular radiosensitivity.     

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.     

The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines.

Dimethylsulfoxide (DMSO) is a well-known solvent that is commonly used in the laboratory. We selected DMSO as the vehicle for an experiment designed to determine if several nonsteroidal anti-inflammatory agents inhibit the growth of Caov-3, OVCAR-3, and SK-OV-3 ovarian carcinoma cell lines. Using the tetrazolium conversion assay, however, we observed some variability in the number of cells present in each ovarian carcinoma cell line with varying concentrations of DMSO (10(-6)-10(-2) M) compared to medium alone. Similarly, when Caov-3, OVCAR-3, and SK-OV-3 cells were treated with 10(-4) M DMSO plus medium (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum) and plated on coverslips, the total number of cells present in 60 random fields increased significantly (P < 0.0001) for each ovarian carcinoma cell line treated with DMSO compared to medium alone. Ethanol did not demonstrate such prominent effects on cellular growth. Our observations are important to consider when selecting an appropriate solvent, especially for growth inhibition studies using Caov-3, OVCAR-3, and SK-OV-3 cell lines.     

Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors.

A sublethal dose of ethanol (5%, vol/vol), acid (HCl, pH 4.5 to 5.0), H2O2 (500 ppm), or NaCl (7%, wt/vol) was added to a Listeria monocytogenes culture at the exponential phase, and the cells were allowed to grow for 1 h. Exponential-phase cells also were heat shocked at 45 degrees C for 1 h. The stress-adapted cells were then subjected to the following factors at the indicated lethal levels--NaCl (25%, wt/vol), ethanol (17.5%, vol/vol), hydrogen peroxide (0.1%, wt/vol), acid (pH 3.5), and starvation on 0.1 M phosphate buffer at pH 7.0 (up to 300 h). Viable counts of the pathogen, after the treatment, were determined on Trypticase soy agar-yeast extract, and survivor plots were constructed. The area (h.log10 CFU/ml) between the control and treatment curves was calculated to represent the protective effect resulting from adaptation to the sublethal stress factor. Adaptation to pH 4.5 to 5.0 or 5% ethanol significantly (P < 0.05) increased the resistance of L. monocytogenes to lethal doses of acid, ethanol, and H2O2. Adaptation to ethanol significantly (P < 0.05) increased the resistance to 25% NaCl. When L. monocytogenes was adapted to 500 ppm of H2O2, 7% NaCl, or heat, resistance of the pathogen to 1% hydrogen peroxide increased significantly (P < 0.05). Heat shock significantly (P < 0.05) increased the resistance to ethanol and NaCl. Therefore, the occurrence of stress protection after adaptation of L. monocytogenes to environmental stresses depends on the type of stress encountered and the lethal factor applied. This "stress hardening" should be considered when current food processing technologies are modified or new ones are developed.     

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.     

The effects of various salt and sucrose solutions on the U.V.L. sensitivity of CHO cells.

The response of cultured CHO cells to U.V.L. irradiation during treatment with anisotonic solutions shows that treatment with hypotonic sucrose, NaCl or KCl solutions causes an increase in the cellular U.V.L. sensitivity, while exposure to hypertonic solutions causes a large decrease in U.V.L. sensitivity. Cells exposed to 1.8 M sucrose, NaCl or KCl solutions and given a U.V.L. dose of 252 erg/mm2 towards the end of the 20 min solution exposure time have survival levels which are respectively 228,26, and 23 times higher than the controls, i.e. cells irradiated in phosphate buffered saline. Cell volume data obtained using a Coulter counter, and nuclear area data of attached cells obtained using an optical microscope with a micrometer reticle, show that cell and nuclear size are related to U.V.L. sensitivity. That is, as cells shrink and the nuclear area decreases, the cells become more U.V.L.-resistant. During hypotonic treatment with 0.1 M NaCl, the cell volume, nuclear area and U.V.L. sensitivity increased in the first 2 to 4 min of exposure time, but at longer exposure times (greater than 3 to 4 min), cell volume, nuclear area and cellular U.V.L. sensitivity decreased. For 0.1 M KCl treatment the cells initially displayed a rapid increase in volume, nuclear area and U.V.L. sensitivity, but at the longer exposure times no decrease in cell and nuclear size were observed, and a slight increase in U.V.L. sensitivity occurred. Changes in U.V.L. sensitivity were related to changes in nuclear size and cell volume; however, calculations showed that during hypertonic treatment there is an ionic effect as well as an osmotic effect. That is, the cellular U.V.L. survival in equal hypertonic concentrations of NaCl or KCl was lower than in the same concentration of sucrose.     

Cell survival responses after exposure to modulated radiation fields

In the present study survival responses were determined in cells with differing radiosensitivity, specifically primary fibroblast (AG0-1522B), human breast cancer (MDA-MB-231), human prostate cancer (DU-145) and human glioma (T98G) cells, after exposure to modulated radiation fields delivered by shielding 50% of the tissue culture flask. A significant decrease (P < 0.05) in cell survival was observed in the shielded area, outside the primary treatment field (out-of-field), that was lower than predicted when compared to uniform exposures fitted to the linear-quadratic model. Cellular radiosensitivity was demonstrated to be an important factor in the level of response for both the in- and out-of-field regions. These responses were shown to be dependent on secretion-mediated intercellular communication, because inhibition of cellular secreted factors between the in- and out-of-field regions abrogated the response. Out-of-field cell survival was shown to increase after pretreatment of cells with agents known to inhibit factors involved in mediating radiation-induced bystander signaling (aminoguanidine, DMSO or cPTIO). These data illustrate a significant decrease in survival out-of-field, dependent upon intercellular communication, in several cell lines with varying radiosensitivity after exposure to a modulated radiation field. This study provides further evidence for the importance of intercellular signaling in modulated exposures, where dose gradients are present, and may inform the refinement of established radiobiological models to facilitate the optimization of advanced radiotherapy treatment plans.     

Effects of dimethyl sulfoxide, temperature, and sodium chloride on the activity of human matrix metalloproteinase 7 (matrilysin)

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

巯基物质在氧自由基损伤离体胃粘膜细胞中的作用

本文用离体胃粘膜细胞研究了细胞内流基物质在活性氧诱发细胞损伤中的作用。实验采用pronase-EDTA法分离大鼠胃粘膜细胞并进行短期孵育,以黄嘌呤氧化酶(XO)-黄嘌呤(X)系统产生氧自由基损伤细胞。实验结果表明,用XO-X损伤胃粘膜细胞时,细胞存活率显著降低,乳酸脱氨酶(LDH)漏出量增多,同时细胞内非蛋白质巯基(NPSH)和蛋白质巯基(PSH)含量均不同程度地下降;N-乙基顺丁烯二酰亚胺(NEM)在耗竭细胞内NPSH和PSH的同时,引起细胞死亡和LDH漏出增加,这一作用与NEM的作用时间和浓度是显著依从关系;在细胞孵育液中预先加入含-SH的化合物还原型谷胱甘肽(GSH)或半胱胺,可剂量依赖性地减轻XO-X引起的细胞损伤。上述结果提示,胃粘膜细胞内的巯基物质在自身防御机制中具有重要作用,氧自由基损伤胃粘膜细胞的机制之一可能与破坏细胞内巯基的稳态有夫。     

Effect of salinity of medium on cellular fatty acid composition of marine algaPorphyridium cruentum (Rhodophyceae)

Porphyridium cruentum Näg. (clone 161) was found to grow best in medium containing between 0.45 M and 0.8 M NaCl. From studies done on growing cultures, the palmitic acid content of the cells decreased with increasing NaCl concentration of the medium. Conversely, when the culture was transferred from a 0.8 M NaCl medium to 0.2 M NaCl, the amount of palmitic acid in thePorphyridium cells increased with time of incubation and it contributed up to 64.5% of the total fatty acid content. There appears to be a negative correlation between the cellular content of palmitic acid and the growth lag. The oleic acid content varied only marginally with increasing NaCl concentration. The poly-unsaturated acid content (linolenic and arachidonic acids) decreased initially and then increased with NaCl concentration up to and beyond ca. 0.8 M NaCl respectively. At 1.5 M NaCl, the poly-unsaturated fatty acids amounted to 78.2% of the total fatty acids in the cell. For stationary phaseP. cruentum cultures, a similar relationship existed between fatty acids and NaCl concentration. However, palmitic acid was accumulated up to three-fold more when compared to the exponential culture grown in low salinity. In addition stearic acid was also found in significant quantities.     

Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki-67 antibody.

This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki-67 antibody, or the proliferation-associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL-60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra- and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki-67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p120 was not significantly affected by 0.65-1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki-67.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of human tumor cells of differing radiosensitivity by WR-1065

We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of gamma radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently ( approximately 1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA-drug associations.     

Effect of salt solutions on radiosensitivity of mammalian cells. I. Specific ion effects.

The radiation isodose survival curve of cells subjected to a wide concentration range of sucrose solutions has two maxima separated by a minimum. Both cations and anions can alter the cellular radiosensitivity above and beyond the osmotic effect observed for cells treated with sucrose solutions. The basic shape of the isodose curve can also be modulated by changes in temperature and solution exposure times. Some of these alterations in radiosensitivity may be related to changes in the amount and structure of cellular water or macromolecular conformation or to the direct effect of the ions, expecially at high solute concentrations.     

Modification of cellular radiosensitivity by NO-synthase inhibitors

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect.We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.     

Evidence for the involvement of oxygen free radicals in the ethanol-induced late cellular injury in mouse myeloma cells.

In this study, we analysed the ethanol-induced long term cell injury on a general cell model (Sp2/0-Ag14 cell line). Cells were incubated in 1, 5, 10, 15 and 20% of ethanol (EtOH) for 5 min. After washing cell viability was tested by the Trypan Blue exclusion test in 5, 60 min, 4 and 24 h after EtOH exposure. Free radicals were monitored every 30 min by electron spin resonance (ESR) with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping technique. Scavenger compounds such as glutathione (GSH), dimethyl sulfoxide (DMSO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were applied for 24 h incubation after EtOH exposure. EtOH concentration dependently decreased the cell viability immediately after 5 min exposure, but with 4 and 24 h, a secondary cell destruction was found. Using ESR-spin trapping technique, an increased free radical activity could be detected. DMPO, DMSO and GSH significantly, but in different period protected the cells against free-radical induced cellular damage. EtOH produces an early (immediately after EtOH exposure) and a late (in about 4 h) cellular damage on Sp2/0-Ag14 cells. The oxygen free radicals can be detected in a short time after EtOH exposure, its biological effect manifested as a secondary cell destruction at 4 and 24 h. This phenomenon can be prevented by scavenger compounds.     

Competence in Escherichia coli cells. V. Interaction of the competence factor with the cells]

Some trends of the interaction of the competence factor with the cells are described on the model of phage lambda transfection. It is shown that this process depends on the composition of the recipients growth media, the temperature regime in the interaction processand on the concentration of the competence factor and the recipient cells. The preliminary treatment of the recipient by EDTA (0.0001 M), CaCl2 (less than 0.02 M), MgCl2 (less than 0.01 M), and NaCl (less than 0.01 M) resulted in the increaze of their sensitivity to subsequent treatment by the factor. Data are obtained demonstrating that after the treatment of cells by the competence factor their sensitivity to decreased CaCl2 concentration is increasing.