Thymineless Death and Ultraviolet Sensitivity in Micrococcus radiodurans

Thymine-requiring mutants of Micrococcus radiodurans have been isolated by selection on solid medium containing trimethoprim. Strains requiring either high concentrations of thymine (50 μg/ml) or low concentrations (2 μg/ml) for normal growth were obtained. The Thy⁻ mutant requiring low thymine concentrations has been characterized. It was shown to retain the high ultraviolet light (UV) resistance typical of wild-type M. radiodurans, but it was not resistant to thymineless death. Preliminary exposure of the cells to thymineless conditions resulted in enhanced UV sensitivity, and this interaction occurred under conditions where “unbalanced growth” was inhibited by the addition of chloramphenicol. Upon addition of thymine to deprived cells, UV resistance was gradually restored, and this recovery took place in the absence of protein synthesis. A model is proposed to account for the similarity of thymineless death in bacteria whose deoxyribonucleic acid repair efficiencies differ widely.     

Response of murine erythroleukemia cells to cis-and trans-diamminedichloro-platinum (II)

Summary Cis-diamminedichloroplatinum II (cis-DDP), an antitumor drug and the inactive trans-isomer were studied to evaluate their effects on cell multiplication, DNA synthesis, and surface morphology of the murine erythroleukemia cells (clone 6A11A). Short-term treatment of cells (1h) with 5 and 10μg/ml of cis-DDP resulted in a significant inhibition of cell multiplication. Continuous treatment with cis-DDP (up to 144 h) significantly arrested cell growth at 1,5, and 10μg/ml. The cells exposed to 10 μg/ml trans-DDP exhibited a slight decrease in cell multiplication; however, the 25-μg/ml treatments showed a modest inhibition of cell growth. Continuous treatment with cis-DDP resulted in a concentrationdependent decrease in DNA synthesis, although low-dose treatment (0.05 and 0.1 μg/ml), with a few exceptions, had no relative inhibitory effect. Likewise, trans-DDP treatments decreased tritiated thymidine incorporation; however, this inhibitory effect was not as drastic as with corresponding concentrations of cis-DDP. Scanning electron microscope studies revealed the formation of many giant cells and blebs at all short-term treatment concentrations of cis-DDP past the 48 h interval. Continuous treatment of cis-DDP at 1 μg/ml concentration produced giant cells with minute holes, whereas the 5 and 10 μg/ml exposure resulted in the formation of blebs and large holes and reduction of microvilli past the 48-h treatment period. At higher concentrations the continuous treatment of cis-DDP completely destroyed the cells. The surface morphology of trans-isomer treated cells, in most instances, resembled the corresponding untreated control cells.     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Concentration-dependent differntial effects of cortisol on synthesis of α-lactalbumin and of casein in cultured mouse mammary gland explants: Importance of prolactin concentration

Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10⁻⁹ M to 2.8×10⁻⁷ M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10⁻⁸ M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10⁻⁶ M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Effect of dikegulac on growth and alkaloid production inCatharanthus roseus (L.) G. Don. (pink flowered)

Application of sodium-dikegulac reduced plant height with associated increase in branch and leaf number and root biomass inC. roseus (L.) G. DON. Chlorophyll content reduced significantly after first month of 100 and 250 μg/ml DK application. However, such reduction was replaced by significant rise after forth month in 250 μg/ml DK application and fifth month in 100 μg/ml DK application followed by appreciable decline only in 250 μg/ml DK treatment but 100 μg/ml DK maintained higher level till harvest. Total sugar content was significantly high during forth and fifth month stage of growth after DK application. Amino acid content was higher during third to fifth month in 100 μg/ml DK treatment and during third to forth month in 250 μg/ml DK treatment. Tryptophan, on the other hand showed higher content at the fifth month stage of growth after application of DK in both the concentrations. Leaf and root dry weight as well as total alkaloid content were highest in 100 μg/ml DK application. DK, therefore, appears to be a potential chemical for increasing biomass and alkaloid content inC. roseus.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Different effects of 5-fluorouracil on Methanosarcina barkeri and on Methanobacterium thermoautotrophicum

Abstract Growth of Methanosarcina barkeri (strain Fusaro) was found to be inhibited by 5-fluorouracil (FU) only at relatively high concentrations (>50 μg / ml ). Inhibition could not be relieved by uracil. Therefore, FU probably did not exert its effect via inhibition of DNA synthesis as is the case in other organisms. Control experiments with Methanobacterium thermoautotrophicum (strain Marburg) on the other hand revealed that the effect of FU on this archaebacterium is probably exerted at the level of nucleic acid synthesis. The M. thermoautotrophicum cultures rapidly acquired resistance towards the pyramidine analog.     

The influence of thymine and 5-bromouracil on the sensitivity ofEscherichia coli to alkylation,UV and X-irradiation

The influence is followed of an alkylating agent (triethylene-melamine) UV and X-irradiation on the survival ofEscherichia coli 15 T∋ells grown in a minimal medium containing enzymatic hydrolysate of caseine. Thymine-less death of a considerable number of cells was observed in a culture grown in this medium. A conclusive difference in the sensitivity to the lethal agents used was found between a culture grown in a thymine-less medium and bacteria grown in a medium containing excess (20 μg/ml) of thymine. The culture grown with a sufficient thymine concentration was more sensitive to alkylation and X-rays, whereas bacteria surviving conditions of thymine-less death were more resistant to the above agents. However, such cells were more sensitive to UV-irradiation. The differences found are discussed from the point of view of DNA concentrations found in the individual cultures.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.     

The effects of folic acid and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of folic acid on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by l-NAME in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180–200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40–120 cmH₂O). Coronary flow and markers of oxidative stress: nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were calculated. The experiments were performed during control conditions and in presence of folic acid (100 μM) alone or folic acid (100 μM) plus l-NAME (30 μM). Control values of coronary flow varied in range from 4.37 ± 0.10 ml/min/g wt at 40 cmH₂O to 12.05 ± 0.42 ml/min/g wt at 120 cmH₂O. Nitrite outflow varied from 1.68 ± 0.17 nmol/min/g wt at 40 cmH₂O to 3.56 ± 0.17 nmol/min/g wt at 120 cmH₂O and was parallel with coronary perfusion pressure-coronary flow curve. Folic acid significantly increased coronary flow (40–120 cmH₂O, 5.63 ± 0.10 ml/min/g wt and 15.2 ± 0.42 ml/min/g wt, respectively) and was accompanied by significant increase in nitrite outflow (2.28 ± 0.29 nmol/min/g wt at 40 cmH₂O to 6.66 ± 0.50 nmol/min/g wt at 120 cmH₂O). In addition, folic acid significantly decreased superoxide anion production especially at upper coronary perfusion pressure values (60% at 120 cmH₂O) and increased index of lipid peroxidation (37.16% at 120 cmH₂O), respectively. Folic acid plus l-NAME did not change control values of coronary flow significantly. However, folic acid plus l-NAME increased nitrite outflow especially at upper coronary perfusion pressure values (43.05% at 120 cmH₂O) and did not change significantly superoxide anion production or index of lipid peroxidation versus control values, respectively. The results clearly showed that on isolated rat hearts at gradually increased constant perfusion pressure, folic acid increased coronary flow, increased nitrite outflow, decreased superoxide anion production, and increased index of lipid peroxidation. These effects were reversed or blocked by l-NAME thus demonstrating mediation or at least participation of NO in the mechanism of the folic acid-induced effects.     

In vitro antifungal activity of the berberine and its synergism with fluconazole

Berberine with and without fluconazole was tested by an agar disk diffusion assay in which clinical isolates of Candida albicans were applied onto yeast extract-peptone-dextrose agar plate. Berberine, which had no intrinsic antifungal activity at the concentration tested, exerted a powerful antifungal activity in combination of fluzonazole. Combinations of berberine and fluconazole were also tested by the checkerboard assay to determine whether they had favorable or unfavorable antifungal interactions. The MIC of fluconazole was 1.9 μg/ml when the drug was tested alone and decreased to 0.48 μg/ml in the presence of berberine concentrations of 1.9 μg/ml. However, berberine at concentrations of >1.9 μg/ml combined with a fluconazole supra-MIC (i.e., >1.9 μg/ml) eliminated the residual turbidity in the incubation wells. This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of a trailing effect, which is the result of a residual growth at fluconazole concentrations greater than the MIC.     

Maternal and neonatal scalp hair concentrations of zinc, copper, cadmium, and lead

Postpartum scalp hair samples from 82 term-pregnancy mother/neonate pairs were analyzed for their concentration of zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb), using inductively coupled plasma-mass spectrometry. Maternal and neonatal Zn concentrations had geometric means (and 99% confidence intervals) of 122.5 μg/g (117.9–131.5 μg/g) and 146.9 μg (141.5–156.7 μg/g) respectively. Corresponding Cu values were 18.4 μg/g (17.6–23.8 μg/g) and 6.7 μg/g (6.3–7.6 μg/g). Those of Cd were 0.49 μg/g (0.47–0.69 μg/g) in the mothers and 0.57 μg/g (0.55–0.86 μg/g) in the neonates. For Pb, they were 7.95 μg/g (7.60–9.32 μg/g) and 4.56 μg/g (4.39–5.56 μg/g). Cigaret smoking, despite its relatively low prevalence (19.5%), was associated with lower Zn and higher Cd and Pb concentrations and in lower Zn/Cd and Zn/Pb molar concentration ratios. Smoking also altered interelemental relationships, particularly those of Zn with Cd and Pb and those between Cd and Pb. Smoking frequency appeared to show negative dose-response effects on maternal and neonatal Zn concentrations, Zn/Pb molar concentration ratios, and birth weight. Mothers with a history of oral contraceptive (OC) usage had significantly higher Cu concentrations and lower Zn/Cu molar concentration ratios than nonusers, with the highest Cu concentrations and lowest Zn/Cu values being associated with third-generation OCs. No similar effects were elicited in the respective neonatal Cu concentrations. Neither alcohol consumption nor prenatal supplementation with iron and/or folic acid had discernible effects on the maternal or neonatal elemental concentrations. The data from this study suggest that in a given population of term-pregnancy mothers and neonates, significant interindividual variations in hair trace element concentrations can occur, irrespective of commonality of general environment, and that lifestyle factors, including cigaret smoking and OC usage history, can be significant contributory factors to such variations. The data are discussed in relation to the effects of smoking-associated exposure to Cd and Pb exposure on Zn availability for placental transfer, as well as on the quantitative maternal Zn supply levels to the fetus resulting from the known tendency of smokers to have lower dietary intakes of Zn. The higher Cu concentrations in OC users are discussed in relation to altered Cu metabolism, characterized by increased synthesis of the Cu-binding protein, ceruloplasmin, as an acute-phase antioxidant response to altered lipid profile and increased lipid oxidation.     

TAN-1057A: A Translational Inhibitor with a Specific Inhibitory Effect on 50S Ribosomal Subunit Formation

The inhibitory activities of a novel antibiotic compound have been investigated. A synthetic version of the natural product TAN-1057A was examined for its effects on translation and ribosomal subunit formation. The antibiotic at 6 μg/ml reduced the growth rate of wild-type Staphylococcus aureus cells by 50%. The IC₅₀ for inhibition of protein synthesis in these cells was 4.5 μg/ml. Pulse and chase labeling kinetics showed a strong inhibitory effect on 50S ribosomal subunit formation as well. The IC₅₀ for this process was 9 μg/ml, indicating an equivalent inhibitory effect of the antibiotic on translation and 50S synthesis. The post-antibiotic effect of the drug was investigated. Protein synthesis resumed rapidly after removal of the drug from cells, but full recovery of the normal 50S subunit complement in treated cells required 1.5 h. The dual inhibitory effects of this compound are compared with other antimicrobial agents having similar effects on cell growth. Received: 27 December 2000 / Accepted: 22 March 2001     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Concentration dependence of the effect ofBacillus intermedius ribonuclease on the yeastSaccharomyces cerevisiae

Bacillus intermedius RNase added at a low concentration (0.001 μg/ml) stimulated yeast growth, while a high RNase concentration (1500 μg/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.     

Effect of some inhibitors on kanamycin formation byStreptomyces kanamyceticus

The formation of kanamyein is markedly inhibited by mercuric chloride, sodium iodoacetate, 2,4-dinitrophenol, sodium arsenite and sodium azide particularly when these are added at the start of fermentation. Less inhibition of kanamyein synthesis is observed in case of sodium 5,5-diethylbarbiturate, malonic acid, sodium arsenate and sodium fluoride. Inhibition of kanamycin synthesis is associated with growth inhibition in case of 2,4-dinitrophenol, sodium arsenite and sodium azide. Bacitracin and D-cycloserine have a stimulatory effect on kanamycin synthesis with slight inhibition of cellular growth. This stimulation might be due to accumulation of cell wall intermediates — aminosugar and sugar — which are shunted to the pathway of kanamycin synthesis. Penicillin lowers kanamycin synthesis by 65 percent as compared with 19 percent reduction of cellular growth. Chloramphenicol has a stimulatory effect at lower concentration (20 μg/ml), when it is added at 24 h of fermentation. At higher concentration (60 (μg/ml) chloramphenicol shows marked inhibition of both cellular growth and antibiotic biosynthesis.