Analysis of the kinetic growth patterns of cultured cells. III. The effect of inoculation density, of a geroprotector-antioxidant and of stationary-phase aging

With the help of a previously devised model of cultured cell growth kinetics it was shown that the "plateau" level on the growth curve of human embryo diploid fibroblasts increased, if the cells were grown with Epigid geroprotector-antioxidant (equal results with two drug concentrations: 10(-5) and 10(-7) M, resp.). It was also found that the "plateau" level on the growth curve of cultured "3 days aged" Chinese hamster cells (subcultivated to a fresh medium 3 days after a 1:6 subcultivation) lays higher than the "plateau" level of "14 days aged" cells but lower than that of "7 day aged" cells. Furthermore it was shown that an increase in inoculation density of Chinese hamster cells increased proportionately the rate of cell population growth but had a little effect on the "plateau" level of the growth curve. The data obtained are discussed in terms of some "proliferative" theories of cellular ageing.     

Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata.

Thirty-three wild type strains of Rhodopseudomonas capsulata were examined for ability to engage in genetic recombination through mediation by "gene transfer agent" (GTA) particles. The genetic exchange assays were based on capacity of strains to produce or receive GTA required for restoration of photosynthetic growth competence to a non-photosynthetic "white" mutant or for acquisition of resistance to rifampicin. A majority of the strains could either produce or receive GTA, and it was demonstrated that the agent is species specific. Possible relations between GTA and bacteriophages or bacteriocins were investigated. Sixteen types of virulent phages active on Rps. capsulata were isolated and their host ranges determined. Tests for transduction by the phages gave uniformly negative results. The viruses showed strict species specificity, but there was no apparent correlation between capacity of the Rps. capsulata strains to donate or receive GTA and susceptibility to the phages. A comparable survey disclosed that most of the bacterial strains were sensitive to or capable of producing bacteriocins; the latter also appear to be unrelated to GTA activity. The collection of bacterial strains was also screened for detection of lysogenic properties. None of the isolates is a "true" lysogen, but phages were detected in cultures of two strains, which may be "phage carriers" or pseudolysogens.     

Protein release in recombinant Escherichia coli using bacteriocin release protein.

The effect of induction level of the bacteriocin release protein (BRP) on cell growth characteristics, protein expression, and protein release in a recombinant strain of Escherichia coli RR1 was investigated. Mitomycin C, the inducing agent, when added to the growth medium in moderate amounts (up to 200 ng/mL) was observed to enhance the release of periplasmic proteins from the cell to the fermentation broth substantially. The percentages of release of the proteins alpha-amylase and beta-lactamase were increased by factors of about 7 and 3, respectively, upon induction of the BRP. The percentage of alpha-amylase released into the broth increased from only about 5% to almost 50% with the aid of BRP. The cell growth curve and low extracellular activity of the cytoplasmic protein beta-galactosidase were indicative that cell lysis did not occur in an appreciable amount at a low induction level, with a mitomycin C concentration of less than 300 ng/mL.     

Growth factor production by Creutzfeldt-Jakob disease cell lines.

Creutzfeldt-Jakob disease (CJD), a progressive dementia of humans, is caused by an infectious agent that is closely related to the scrapie agent of sheep. Although the molecular nature of these "unconventional" agents is still a matter of speculation and controversy, even less is known concerning the mechanism(s) of their effects on the central nervous system. To gain insight into the cellular effects of these agents, we have examined a series of cell lines derived directly from CJD-infected hamster brain or produced from nontransformed rodent lines by exposure to CJD infectious fractions in vitro. These cell lines appear transformed by a variety of criteria and secrete growth factors into the culture medium. All CJD lines produce a factor that is like alpha-transforming growth factor (alpha-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glial fibrillary acidic protein in normal astrocytic cells in vitro. This effect is mimicked by purified alpha-TGF and platelet-derived growth factors. Further study of CJD-induced growth factor production may elucidate fundamental properties of these unconventional agents.     

Special traits of morphogenesis of some forms of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> L.: II. Ontogenetic variability of vegetative organs

The growth of the main stem of the investigated varieties of Lupinus angustifolius L. may be characterized by a gooseneck curve which is negatively related to the average daily air temperature and positively related to the amount of precipitation. The size of leaf sections and of the stem between nodes varies regularly depending on their location on the shoot and on growth conditions.     

Studies on antitumor effects and mechanism of action of l-hexylcarbamoyl-5-fluorouracil by DNA/BrdU double staining using flow cytometry.

An investigation was carried out to elucidate the mechanism of action of the oral antitumor agent 1-hexylcarbamoyl-5-fluorouracil (HCFU) by determining its effects on the growth and cell cycle of epipharyngeal carcinoma cells (KB cell) by DNA/BrdU double staining using flow cytometry (FCM). As a result, it was found that HCFU stimulates KB cells in the S phase to proliferate for the first 3 days of treatment in a low concentration (8 micrograms/ml) and caused cell accumulation in the later G2M phase. On the other hand, when administered in the concentration (20 micrograms/ml) that produces a 50 per cent cell kill, as determined from the cell growth curve, HCFU appeared to exhibit a cytocidal effect by blocking cells in S and G2M for the first 3 days after exposure. It was revealed by FCM for the first time that HCFU operates by a similar mechanism to that of 5-FU. This method seems to be of significance to therapeutic schemes that take into consideration the mechanism of action of antitumor drugs.     

Synergistic effect of glycine and bacteriocin release protein in the release of periplasmic protein in recombinantE. coli

Summary The synergistic effect of using mitomycin C-induced bacteriocin release protein (BRP) and glycine on cell growth, protein expression and release in a recombinant strain RR1 ofE. coli was investigated. An optimal combination of 50 ng/ml mitomycin C and 0.5% glycine concentration enhanced the release of periplasmic proteins from the cell into the fermentation broth without significantly affecting protein productivity. Under this optimal condition, the percentage of -amylase released into the broth increased from 7–13% to as much as 78%. The cell growth curve and low extracellular activity of the cytoplasmic protein -galactosidase show that there is no appreciable cell lysis.     

胶体金对 K526 细胞影响的研究

本文论述了有关人类白血病细胞系K562细胞的体外培养的特点。以及胶体金纳米颗粒独特的物理性质,并应用胶体金治疗肿瘤的原理和意义,成功的在体外条件下培养出生长状态稳定的K562细胞,最后通过观察、记录体外生长的状况,绘制了细胞生长曲线,并计算出细胞的倍增时间。利用柠檬酸三钠来还原氟金酸的方法制备了胶体金,通过紫外／可见分光光度计和电子透射显微镜检测了胶体金的吸收光谱以及它的颗粒直径、形状,进行了胶体金颗粒对K562细胞的影响研究。竞验结果表明,加入胶体金和没加胶体金的细胞生长状况基本相同。     

秃杉引种幼龄生长规律初步研究

 引种研究表明,秃杉幼苗生长缓慢,苗期生长为双峰倒“W”形,春秋两季速生,盛夏趋于停滞。幼树造林初期生长缓慢,3年后加快;5—10年速生,生长量已接近或超过原产地。季节生长：春末夏初(5—6月)速生,6月出现峰值,盛夏遇高温减缓,生长呈单峰倒“V”形。树高昼夜生长：夜间>白天;阴雨天>阴天>晴天。引种地气温,相对湿度,净日照时数是影响幼龄秃杉生长的重要气象因子。秃杉幼树年生长与观察年树龄的关系可用数学模型y= a+bt表示;季节生长与观察月份的关系可用数学模型y=a+blnt表示。     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

Growth effects of 2-thiouracil and possibility of selective inhibition of floral differentiation inChenopodium rubrum L.

The effect of 2-thiouracil on vegetative growth and floral differentiation was investigated inChenopodium rubrum plants grown in water cultures. Between the low concentrations of the agent, stimulating vegetative growth and floral differentiation, and those inhibiting both these processes, a narrow concentration range was found (1.10^?5 m to 2.10^?5 m), where growth was inhibited selectively. At a concentration of 1.10^?4 m a selective inhibition of development was found when 2-thiouracil was applied at the beginning of photoperiodic induction. Inhibition of development was strong regardless of whether 2-thiouracil was applied before, during or closely after 4 days of photoperiodic induction; the degree of growth inhibition, however, changed in dependence on photoperiodic induction. The strongest relative inhibition of development, calculated as a ratio between development and growth, was observed always at the beginning of photoperiodic induction. Investigation of plant growth as well as the anatomical and autoradiographic study after the application of 2-thiouracil indicate that the inhibition becomes evident at the end of 4 days of application by an overall growth inhibition and a decrease of mitotic activity. Reversal by uracil was possible after simultaneous application of 2-thiouracil. The nature of the selective inhibition is discussed and two possible interpretations of the data obtained are analyzed: a) different response of growth processes in apices and young vegetative organs respectively with regard to different participation of cell division and elongation, b) specific inhibition of floral differentiation.     

ATP synthesis in plasma membrane enriched particles by the action of insulin and related growth factors.

Early biosynthesis of short-life ATP was observed in plasma membranes of target cells stimulated by insulin or other polypeptide growth factors in the presence of all components of aerobic phosphorylation and cytochrome c. The effect is always mediated by the binding of insulin or growth factors to specific receptors. Erythrocyte plasma membranes are a convenient model to study the phenomenon. Insulin-stimulated synthesis of the plasma membrane "signal" ATP in an amount of 1-10 nM is potentized by ionophores carbonyl cyanide p-trifluorometoxyphenylhydrazone and monensin and inhibited by amiloride and ouabain. It is supposed that the plasma membrane "signal" ATP readily generated in response to a growth or mitogenic factor is an "amplifier" or "coupling agent" in the transduction of a signal to growth, proliferation, and mitogenesis. Biosynthesis of the plasma membrane "signal" ATP seems to be associated with partial reversion of Na+, K+ -ATPase with the participation of the plasma membrane redox chain as a proton generator.     

Studies on the substrate specificity and inducibility of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system in Bacillus megaterium A.T.C.C. 13368 was investigated with regard to its appearance in the cell with respect to the growth curve of the organism, with regard to its inducibility by a number of agents (among them some of the classical inducers of the mammalian liver microsomal cytochrome P-450 system) and with regard to its capacity to convert non-steroidal substances into oxygenated compounds. The enzyme was found to reach a maximum concentration in the cell during the stationary phase of the growth curve. Of all the agents tested as inducers, none showed any capacity to induce cytochrome P-450meg. Finally, of the substances tested as substrates only aniline (p-hydroxylation) was metabolized by the microbial enzyme system. This conversion might be related to the general oxygenase activity of haemoproteins. It is concluded that the substrate specificity of the B. megaterium hydroxylase system is narrow.     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

"Phoenix phenomenon" in the growth of Clostridium perfringens.

The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III). The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied. This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar). When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon. The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed. The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited. This indicated that phases I and II were an injury-recovery process.     

Development of EDTA Media for the Rapid Growth of Chlamydomonas mundana

SYNOPSIS. An empirical method is used to determine mixtures of the chelating agent, EDTA (ethylenediamine tetraacetate), and trace metals which allow maximum growth rates of Chlamydomonas mundana under optimum environmental conditions. In this method which resembles an immunological titration one trace metal (Ca, Fe, Mn or Zn), in increasing concentrations, is used to "saturate" a standard amount of EDTA. Other required trace metals are added in a range of minimum concentrations. Extent of growth with each mixture is evaluated after 24 hours of incubation. Mixtures allowing greatest growth are then used to evaluate growth rate. Media with manganese as the EDTA-saturating trace metal allow most rapid growth rates equivalent to 11.0 doublings per day. Magnesium as the EDTA-saturating metal does not allow growth, suggesting that Mg does not displace other required trace metals from EDTA-complex. Since the mixtures allowing maximum growth rate are "underchelated" it appears that the free metals rather than the metal-chelate complexes are utilized by the alga in rapid growth.     

The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assessed by the colony-forming assay

The radiosensitivity of human lymphocytes was investigated by the method of colony formation in the absence of interleukin-2 (IL2) and feeder cells, both of which enhance growth of T-cell colonies. The shape of the survival curve and the radiosensitivity was shown to depend upon the ability of lymphocytes to produce IL2: the survival curve for lymphocytes that were the most competent producers of IL2 is the closest to linearity; the lymphocytes that were poor producers show biphasic survival curves. The radiosensitivity of the lymphocytes from the first group is less than that of the latter, when the comparison is based on the first part of the biphasic survival curve. This is more easily seen when cultures are irradiated 24 h after stimulation by phytohemagglutinin (the time of the peak IL2 production) than when cultures are irradiated 2 h before stimulation. This study demonstrates that growth conditions influence the response of lymphocytes to irradiation and that optimal growth conditions result in a linear survival curve.     

Candida albicans--adriamycin interactions: ultrastructural and spectrofluorometric study of whole yeasts and spheroplasts

The occurrence of candidiasis in cancer patients who undergo chemotherapy requires the interrelation of Candida albicans and the antimitotic drug Adriamycin (ADM) which is well known as an intercalating agent. The whole yeasts were not affected by 2 h of contact with the drug at 10(-4) M neither for their growth curve nor for their ultrastructure, despite the presence of free ADM on their surface. Spheroplasts displayed a delay in their growth and exhibited altered nucleoli with segregation of their granular and fibrillar components. The modified emission spectrum of ADM, determined by spectrofluorometry, corresponded neither to the free ADM nor to the DNA-bound drug, but it could be related to a metabolite of the drug. The cell wall appeared to be one of the main sites for ADM resistance of Candida albicans in vitro.     

Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S.

The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells. Stationary-phase induction of bolA is dependent on sigma S. The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter.     

Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents

An envelope of tails test was used to show that the delayed rectifier K+ current (IK) of guinea pig ventricular myocytes results from the activation of two outward K+ currents. One current was specifically blocked by the benzenesulfonamide antiarrhythmic agent, E-4031 (IC50 = 397 nM). The drug-sensitive current, "IKr" exhibits prominent rectification and activates very rapidly relative to the slowly activating drug-insensitive current, "IKs." IKs was characterized by a delayed onset of activation that occurs over a voltage range typical of the classically described cardiac IK. Fully activated IKs, measured as tail current after 7.5-s test pulses, was 11.4 times larger than the fully activated IKr. IKr was also blocked by d-sotalol (100 microM), a less potent benzenesulfonamide Class III antiarrhythmic agent. The activation curve of IKr had a steep slope (+7.5 mV) and a negative half-point (-21.5 mV) relative to the activation curve of IKs (slope = +12.7 mV, half-point = +15.7 mV). The reversal potential (Erev) of IKr (-93 mV) was similar to EK (-94 mV for [K+]o = 4 mM), whereas Erev of IKs was -77 mV. The time constants for activation and deactivation of IKr made up a bell-shaped function of membrane potential, peaking between -30 and -40 mV (170 ms). The slope conductance of the linear portion of the fully activated IKr-V relation was 22.5 S/F. Inward rectification of this relation occurred at potentials greater than -50 mV, resulting in a voltage-dependent decrease in peak IKr at test potentials greater than 0 mV. Peak IKr at 0 mV averaged 0.8 pA/pF (n = 21). Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (225 ms) at membrane potentials (-20 to +20 mV) typical of the plateau phase of cardiac action potentials.