Promotion of septation in irradiated Escherichia coli by a cytoplasmic membrane preparation

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.     

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.     

Structural and functional analysis of the respiratory burst in the phagocytosing macrophage

The interrelation between structural changes and oxygen consumption by the phagocyting macrophage was studied. The mean number of phagocyted particles was estimated by the method of stereological transformation. It is found that the uptake of yeast particles and CN- -nonsensitive oxygen consumption is related to the concentration of yeast cells in the incubation medium. A positive correlation was established between the oxygen consumption and the mean number of phagocyted particles. The results obtained may suggest that the "respiration burst" takes place in the contact area of the macrophage and the phagocyted material, and its extent probably depends on the surface of that contact area.     

Production of giant cells of Escherichia coli.

Giant cells, with volumes up to 500-fold those of normal cells, have been produced by both genetic and pharmacological means in Escherichia coli K-12. In the genetic approach, an envB or mon mutation (conferring rounded or irregular morphology) was combined with a lon mutation (block of septation after irradiation). UV irradiation and subsequent incubation for 2 to 5 h in a rich medium supplemented with 1% sodium chloride led t; production of polymorphic giant cells. In the pharmacological approach, incubation of several different strains of E. coli K-12 with the drug 6-amidinopenicillanic acid (FL1060) in the same rich medium gave rise to a homogeneous population of smoothly rounded giant cells.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

Significance of endogenous glutathione in realizing the oxygen effect in bone marrow cells

It has been established using hemopoietic bone marrow cells or EHrlich ascites carcinoma cells that endogenous glutathione affects the realization of only those lesions which appear in response to an increase in radioresistance, for example during anoxia. The involvement of glutathione in the realization of the oxygen effect is not limited to the time of irradiation. The effect of glutathione extends to the postirradiation period i. e., it is not determined only by the competition with oxygen for target radicals.     

Rapid postradiation recovery of yeasts: the relationship to gamma-induced reciprocal mitotic recombination and gene conversion

gamma-induced reciprocal mitotic recombination and gene conversion have been studied under conditions inhibiting "rapid" postirradiation recovery of diploid yeast Saccharomyces cerevisiae. It turned out that, if the first postirradiation cell division occurs at higher KCl concentrations ("rapid" postirradiation recovery is inhibited), the frequency of mitotic reciprocal recombination within the gene ADE2-centromere region decreases. Keeping of irradiated cells (in the G1 phase of the cell division cycle) in water at 28 degrees C prior to plating on the selective agar containing 1.5 M KCl leads to smaller frequency of gene conversion lys2-25/lys2-22----Lys+, as compared with that for the cells immediately plated on the selective agar. Correlation has been found between the coefficient of gene conversion frequency decrease, due to postirradiation keeping in water, and "rapid" recovery efficiency. Interpretation of the data is based on the hypothesis that recombination repair of DNA double-strand breaks induced by ionizing radiation is responsible for "rapid" postirradiation recovery.     

Plating efficiency as a function of time postirradiation: evidence for the delayed expression of lethal mutations

The plating efficiency (PE) of a gamma-irradiated (7 Gy) human cell hybrid line (HeLa X skin fibroblast, designated as CGL1) has been measured as a function of time postirradiation and compared to that of unirradiated cells at similar cell densities and under the same growth conditions. The results indicate that following irradiation, the PE of the irradiated cells initially increases but never returns to that of unirradiated cells during the experimental period that we have examined. Furthermore, after a period of 9 to 10 days (equivalent to at least 10 cell doublings) postirradiation and plating, the PE of the irradiated cells begins to decrease and continues to do so over the next 5 days. A decrease does not occur in unirradiated cells until much later (i.e., Day 15) corresponding to at least 5 additional cell doublings. The data are discussed in terms of a delayed expression of lethal mutations. The possible impact of these observations on the estimation of radiation-induced transformation frequencies is also considered.     

籼稻原生质体培养和植株再生

用籼稻IR52、IR8和IR45的幼花序和幼胚愈伤组织在LS培养基建立了稳定的悬浮培养物。悬浮系的建立经历三个阶段:褐变期,长根期,成熟期。建立了适合籼稻原生质体生长的Y8培养基,其植板率显著高于KPR和PCM培养基。悬浮细胞系间差异明显,只有部份系可以提供有分裂能力的原生质体或具看护活性。以上三个品种的原生质体均分裂良好,但只有IR52和IR8分化出苗,其中IR52分化率1.25%,得再生植株50余株,移至田间生长结实正常。     

THE EXPRESSION OF DIFFERENTIATION BY CHICK EMBRYO THYROID IN CELL CULTURE : II. Modification of Phenotype in Monolayer Culture by Different Media

Previous results with thyroid secretory cells in monolayer culture seem contradictory with respect to phenotypic stability of this cell type. On the one hand, in "minimal" medium the cells lose structural and functional specializations which can be returned only by three-dimensional growth in organ culture upon addition of fibroblasts derived from the thyroid capsule. On the other hand, in "rich" medium used for cloning, cytoarchitecture and function remain unaltered in either mass or clonal cultures. The apparent discrepancy has been resolved by plating cell suspensions in both media and changing to the alternate medium once the cells have become established. It has been shown that a number of characteristics, including hormone levels, are reversed each time such a change in medium is made. These modulations are discussed in terms of the normal variations in structure and function of the gland in vivo.     

Formation of reactive oxygen species in monocytes at adhesion to glass

Processes of oxygen activation in monocytes stimulated with adhesion to glass were studied by methods of luminol-dependent and lucigen-independent chemiluminescence. It was shown that monocyte chemiluminescence was caused by cell adhesion to glass surface. Generation of reactive oxygen species at monocyte adhesion to glass was dependent on calcium ion concentration in the medium. The increase in the level of cytosolic calcium, as the extracellular calcium concentration elevated, was accompanied by the activation of phospholipase A2, 5-lypoxygenase and cycloxygenases. Magnesium ions exerted no influence on oxygen activation by cells. Incubation of cells in glucose-free medium, or the addition of glycolysis blocker (2-deoxy-D-glucose) to cell suspension led to a decrease in chemiluminescence intensity. By means of inhibitory analysis, it has been established that processes of oxygen activation are related to arachidonic acid metabolism, and depend on the activity of phospholipase A2.     

Development under the control of insulin of lipogenic enzymes,lipoprotein lipase,isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture

In preadipose cellular fractions (I, II and III) isolated by density gradient centrifugation from the inguinal tissue of young rats, we followed the activity of fatty acid synthetase, ATP citrate lyase and lipoprotein lipase during differentiation in culture. 1.5 nM insulin when added at confluence markedly induced the activity of ATP citrate lyase and fatty acid synthetase in the cells derived from the lighter fractions (I and II). The magnitude of this response was 25–50-fold the initial value 15 days after plating. In the cells of the heaviest fraction (III) both enzymes exhibited low activity which was slightly stimulated by the presence of insulin, VLDL and heparin. In contrast, the activity of lipoprotein lipase appeared before confluence in cells from all three fractions and peaked at day 6 after plating. This early emergence was independent of the addition of insulin to the medium. However, insulin slightly enhanced the peak activity in post-confluent cells. The development of cAMP production in response to isoproterenol (100 μM) and to glucagon (0.3 μM) was determined in the cells of fraction II in the same culture conditions. The responsiveness to isoproterenol was present very early in these cells and rose rapidly during the exponential growth phase, reaching a peak value at day 8 after plating. In contrast, the development of glucagon sensitivity occurred only during late differentiation. The stimulatory effect of glucagon was enhanced when VLDL and heparin were added with insulin to the medium.     

Superoxide and hydrogen peroxide in oxygen damage.

Escherichia coli were damaged and killed by exposure to hyperbaric oxygen. Lethality was measured as the decrease in the number of colonies formed upon plating the exposed cells onto rich agar. Damage was assessed by plating onto both rich and minimal agar. Cells which gave rise to visible colonies on rich but not on minimal agar were considered to be damaged. That this differential colony count was largely due to reparable damage rather than to stable mutagenesis was shown by replica plating from the rich onto the minimal agar. Most of the cells which had been unable to grow when directly plated onto minimal agar regained this ability after growth upon rich agar. Repair of the damage imposed by exposure to oxygen was thus more readily accomplished on a nutritionally rich medium. The enzymes superoxide dismutase, catalase, and peroxidase appeared to protect against oxygen damage. It is thus likely that both O₂^? and H₂O₂ are important agents of oxygen toxicity. In accord with this conclusion were the observations that augmented intracellular levels of these enzymes correlated with increased resistance towards oxygen damage, whereas increased respiratory capacity correlated with increased sensitivity towards hyperbaric oxygen.     

Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: a possible mechanism for bystander-induced genomic instability?

Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy gamma rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.     

AN ANALYSIS OF THE EFFECT OF "CONDITIONED MEDIUM" UPON THE CELL CULTURE AT LOW DENSITY

A favorable effect of “conditioned medium” upon outgrowth of the cell culture with low density in vitro was analysed with the cells of chicken embryos. For preparing “conditioned medium”, cultures with a large number of cells were made with muscle, kidney, lung, liver and skin, while the biological activity of the medium was assayed by using the culture of a small number of the lung secondary cells. A use of “conditioned medium” was found to be necessary for encouraging the outgrowth of the cultured cells below a critical inoculum size. Of the various types of the media tested, the medium conditioned with muscle was most effective. “Conditioned medium” contained at least two different active factors, the first to enhance the plating efficiency of the inoculated cells to the surface of the culture dish, and the second to promote further outgrowth of the plated cells. “Conditioned medium” taken out of the mass culture at its exponentially growing phase had only the second factor, while that taken out of that at its stationary phase contained both factors. An activity of the first factor was not detected, when the mass culture was kept in such condition that the collagen synthesis was inhibited. The factor for enhancing the plating efficiency was eliminated from “conditioned medium” by preincubating the cells, before assaying the effect of the medium.     

Further studies on the radiation-induced expression of a tumor-specific antigen in human cell hybrids

The neoplastic transformation of human cell hybrids (HeLa x skin fibroblasts) is accompanied by the expression of a cell surface protein for which monoclonal antibodies have been raised. The gamma-radiation-induced neoplastic transformation of these cells has been studied where the expression of this cell surface protein, as detected by immunoperoxidase staining, has been used as an end point. The yield of foci of positively staining cells has been shown to increase with increasing time postirradiation at which the assay is done and decrease with increasing density of viable cells plated postirradiation. The time of plating postirradiation is also an important parameter with transformation frequencies increasing over the first 6 h of postirradiation holding at confluence, followed by a gradual decrease.     

Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.