Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

Action spectra for photosynthetic adaptation in Scenedesmus obliquus

Photosynthetic adaptation of the unicellular green alga Scenedemus obliquus to different light conditions was investigated with respect to chlorophyll synthesis. Cultures were grown under white light (20 W · m^–2) from fluorescent lamps and were then transferred and subjected to the actual adaptation regime which consisted of a 24-h irradiation by different fluence rates and wavelengths. Fluence rate-response curves for chlorophyll synthesis were measured between 4 · 10^–2 and 1 · 10² W · m^–2. In white light from incandescent lamps, in blue and red light the fluence rate-response curves for chlorophyll (Chl) a and also for Chl b were bell-shaped. In red light the threshold was about the same as under blue light. The maximal amounts of Chl a and b were about twofold increased under blue light relative to the values obtained with red light. Action spectra for the stimulation of chlorophyll synthesis (Chl a + Chl b) as well as those for the separate chlorophylls showed two maxima near 450 and 500 nm. However, the action spectrum for Chl b synthesis demonstrated a considerably higher value in the 450-nm peak. Experiments with the photosynthesis inhibitor 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) indicated that photosynthetic energy supply supported the photostimulation of chlorophyll synthesis. The action spectra indicate the cooperation of two photoreceptors. The 460-nm peak is attributed to the typical blue-light receptor, being more active in Chl b formation. The peak at 500 nm may represent carotenoproteins acting as an accessory pigment system.Abbreviations PCV packed cell volume - Chl total amount of chlorophyll - Chl a, b chlorophyll a, b - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea This project was supported by the Deutsche Forschungsgemeinschaft. We thank Ms. K. Bölte for technical assistance.     

A comparison of the activities of aryl hydrocarbon monooxygenase in liver microsomes from mice of different strains during prolonged 3,4-benzo(a)pyrene administration.

The content and activity of the components of liver microsomal aryl hydrocarbon monooxygenase system change biphasically during long-term 3,4-benzo(a)pyrene administration of C57BL/6 mice as well as to (C57BL/6 X DBA/2)F1 hybrids. The first activity peak (4--14 days) is associated with the induction of aryl hydrocarbon monooxygenase by 3,4-benzo(a)pyrene; the second peak (70--84 days) is related to noninductive mechanism. In DBA/2 mice, the second peak is absent while the slight increase in aryl hydrocarbon monooxygenase activity observed on days 14--28 indicates the aberrant inductive capacity of 3,4-benzo(a)pyrene under its prolonged administration. It is suggested that the weak sensitivity to the blastogenesis caused by 3,4-benzo(a)pyrene observed in C57BL/6 mice and in (C57BL/6 X DBA/2)F1 hybrids is due to the high level of liver aryl hydrocarbon monooxygenase activity at the time of tumor appearance.     

Use of 8-hydroxyquinoline to enrich for temperature-sensitive mutants of Tetrahymena.

The effects of the chelating agent 8-hydroxyquinoline (Hq) on Tetrahymena thermophila were examined. Cell division was completely inhibited by 5 micrograms of Hq per ml. At this concentration deoxyribonucleic acid, ribonucleic acid, and protein syntheses were also completely and nonselectively inhibited. The inhibition was reversible after 6 h of Hq treatment. At concentrations above 20 micrograms/ml a 10,000-fold decrease in survival as seen after 2 h in the drug. The sensitivity of Tetrahymena to Hq was found to be dependent upon cell concentration, wild-type strain, medium, and length of time the culture is at 38 degrees C before Hq is added. Mutants of Tetrahymena that are unable to divide at the restrictive temperature, but which continue macromolecular synthesis, were found to be resistant to Hq treatment. Conditions were obtained in which more than a 1,000-fold difference in survival was seen between this class of mutant and the wild type. The effect of Hq on three other classes of temperature-sensitive mutants was examined, and the results are discussed.     

A comparison of the activities of aryl hydrocarbon monooxygenase in liver microsomes from mice of different strains during prolonged 3,4-benzo(a)pyrene administration

The content and activity of the components of liver microsomal aryl hydrocarbon monooxygenase system change biphasically during long-term 3,4-benzo-(a)pyrene administration to C57BL/6 mice as well as to (C57BL/6 × DBA/2)F1 hybrids. The first activity peak (4–14 days) is associated with the induction of aryl hydrocarbon monooxygenase by 3,4-benzo(a)pyrene; the second peak (70–84 days) is related to noninductive mechanism. In DBA/2 mice, the second peak is absent while the slight increase in aryl hydrocarbon monooxygenase activity observed on days 14–28 indicated the aberrant inductive capacity of 3,4-benzo(a)pyrene under its prolonged administration. It is suggested that the weak sensitivity to the blastogenesis caused by 3,4-benzo(a)pyrene observed in C57BL/6 mice and in (C57BL/6 × DBA/2)F1 hybrids is due to the high level of liver aryl hydrocarbon monooxygenase activity at the time of tumor appearance.     

Metabolic response of Chlorella emersonii to the herbicide sulfometuron methyl

Although it is clear that acetohydroxy acid synthase (AHAS; EC 4.1.3.18) is the target for sulfonylurea herbicides such as sulfometuron methyl (SMM), there is considerable uncertainty as to the mechanism(s) by which inhibition of AHAS inhibits or kills cells. We have further studied the mode of action of SMM, and its effects on metabolism and physiology in the unicellular green alga Chlorella emersonii var. emersonii. Addition of SMM to cells synchronized to a cycle of 16 h light-8 h dark showed that they were very sensitive to SMM toxicity in the first 16 h of the cell cycle, during which cell mass, protein and DNA increased. The increase in protein, DNA and chlorophyll was halted rapidly after SMM addition. Sulfometuron methyl prevented cell division even if added late in the light stages, when most of the protein and DNA were already synthesized, but did not affect cell division and autospore release if added after protein and DNA synthesis were complete. This suggests that SMM interferes with processes involved in preparation for division, beyond what would be expected if the cells were starved of the branched-chain amino acids needed as precursors for synthesis of proteins in general. The accumulation of -ketobutyrate (KB) in the cells in response to addition of SMM, and its possible role in the growth inhibition, was also investigated (in continually illuminated cultures). Intracellular KB accumulated rapidly within 30 min of SMM addition, but declined nearly to basal levels in several hours. This paralleled the decrease and subsequent recovery of extractable AHAS activity. Despite this, growth of the algal culture did not recover. We suggest that metabolites formed by misincorporation of KB in place of -ketoisovalerate (e.g., in the ketopantoate hydroxymethyl transferase reaction) might be responsible for the persistence of growth inhibition. We note that an important difference between the effect of SMM and that observed with externally added KB is that the ratio between intracellular KB and -ketoisovalerate is expected to be high in the first case, but not necessarily in the second.Abbreviations AHAS acetohydroxy acid synthase - BCAA branched-chain amino acids - IM imidazolinone - KB -keto-butyrate - SMM sulfometuron methyl - SU sulfonyl urea This research was supported in part by grant 338/92 from the Israel Science Foundation. It was also supported by the Lily and Sidney Oelbaum Chair in Applied Biochemistry, of which D.M.C. is the incumbent.     

Studies on the dependence of chlorophyll synthesis on protein synthesis in Euglena gracilis,together with a nomogram for determination of chlorophyll concentration

Summary Experiments have been carried out to determine the basis for the dependence of chloroplast pigment synthesis on protein synthesis in dark-grown cells of Euglena gracilis greening in the light. The complete inhibition of chlorophyll synthesis brought about by actidione (10 g/ml) when added half way through the greening process was not relieved, even to the slightest extent, when 0.01 M -aminolaevulinic acid (ALA) was also present. The much smaller inhibition of chlorophyll synthesis brought about by chloramphenicol (2 mg/ml) was also relieved little, if at all, by the addition of ALA. It is concluded that the inhibition of chlorophyll synthesis by actidione can not be solely or primarily due to lack of ALA resulting from the decay of possibly labile enzymes of ALA synthesis, but could be due to inhibition of synthesis of the thylakoid structural protein. The results obtained with chloramphenicol are difficult to interpret because of the possibility that the drug, at high concentration, directly inhibits processes other than protein synthesis.Chlorophyll and carotenoid synthesis by E. gracilis were both markedly stimulated by the addition of ALA. It is suggested that the rate of chlorophyll synthesis in the greening cells is limited by the rate of formation of ALA. The stimulation of formation of carotenoids as well as chlorophyll may indicate that the cells have a mechanism for ensuring that the rate of carotenoid synthesis does not fall below a certain proportion of the rate of chlorophyll synthesis.A nomogram has been devised from which the concentrations of chlorophylls a and b, and total chlorophyll can be read off once the absorbances of an 80% acetone extract at 663 and 645 m have been determined.     

Development of Oxidative Stress in the Cells of Chlorella stigmatophoraand Their Bleaching by the Glycolate Pathway Inhibition under Salinization Conditions

The changes in chlorophyll and proline contents, the rate of photosynthetic oxygen evolution, the activity of superoxide dismutase (SOD), and NADPH concentration in the cells of Chlorella stigmatophoraButcher were followed under the conditions when the glycolate pathway was inhibited by 10 mM -hydroxy-2-pyridinemethane sulfonate + 10 mM isonicotinoyl hydrazine or with 425 mM NaCl. The oxidative stress exerted by the inhibition of the glycolate pathway developed in three phases. At the first phase (15 to 60 min), the photosynthetic rate slightly increased, chlorophyll and proline contents and SOD activity declined, whereas NADPH pool increased considerably. At the second phase (1 to 7 h), in addition to a short-period chlorophyll accumulation, proline synthesis enhanced together with SOD activity, whereas NADPH pool decreased. Degradation processes dominated the third phase (24 to 72 h): cells absorbed rather than evolved oxygen, and the contents of proline, chlorophyll, and NADPH as well as the SOD activity decreased dramatically. The authors conclude that the inhibition of photorespiration in chlorella cells disrupted the functions of the electron transport chain (ETC); as a result, the oxygen stress developed, and cells became bleached. The accumulation of free proline in the cells under salinization provides a mechanism subduing ETC overreduction, and photorespiration is a component of the cell antioxidant system.     

Inhibition of stage-specific DNA synthesis in rat spermatogenic cells by polycyclic aromatic hydrocarbons

Changes in the rate of DNA synthesis in spermatogenic cells after treatment of segments of rat seminiferous tubule at defined stages of epithelial cycle with benzo[a]pyrene (BP) or 7,12-methylbenz[a]anthracene (DMBA) were studied. The incorporation of labeled thymidine into DNA was used as a measure of the rate of DNA synthesis. Very little or no inhibition of DNA synthesis at stages V and VIII of the cycle was observed at BP and DMBA concentrations lower than 100 microM. In contrast, in the presence of added mitochondria and/or microsomes from whole rat testis, 20 microM BP or DMBA inhibited DNA synthesis 5% and 80%, respectively. This inhibition of DNA synthesis was prevented by inhibitors of the cytochrome P-450 system and by free radical scavengers. These results suggest that polycyclic aromatic hydrocarbons (PAH) require metabolic activation in order to inhibit DNA replication in seminiferous tubules. The first step of this biotransformation is cytochrome P-450-dependent and occurs in Leydig cells. However, the metabolites produced in this step may be further metabolized to reactive metabolites by peroxidative pathways in the seminiferous tubules; these latter products may affect DNA replication.     

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts

High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon pH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by pH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.Abbreviations 9-AA_max = Maximum yield of 9-aminoacridine fluorescence - DCMU = 3(3,4-dichlorophenyl)-1,1-dimethylurea; F_max ± Maximum yield of chlorophyll fluorescence - hr = hour - PAR = Photosynthetically Active Radiation - Q_A = Primary stable electron acceptor within photosystem II - qE = High energy state quenching of chlorophyll fluorescence - qI = quenching of chlorophyll fluorescence related to photoinhibition - qP = Quenching of chlorophyll fluorescence by oxidised plastoquinone - qQ = photochemical quenching of chlorophyll fluorescence - qR = (F_max—maximum level of chlorophyll fluorescence induced by the addition of saturating DCMU) - qT = Quenching of chlorophyll fluorescence attributable to state transitions     

Metabolic products of microorganisms. 192. The anthraquinones of the Aspergillus glaucus group. II. Biological activity

From the mycelia of Aspergillus cristatus the following anthraquionic pigments were isolated: catenarin, emodin, erythroglaucin, rubrocristin, physcion, physcion-9-anthrone, questin, viocristin, and isoviocristin. The latter two do not belong to the 9, 10-anthraquinone series but to the 1,4-anthraquinones, and so far they have not been reported among naturally occurring quinones.Emodin, catenarin, viocristin, and isoviocristin snowed antibacterial activity with minimal inhibitory concentrations ranging from 1–10 g/ml. In Bacillus brevis catenarin and emodin inhibited the incorporation of uracil and leucine preferentially. At higher concentrations the incorporation of thymidine into the trichloroacetic acid-precipitable fraction of cells was also affected. In the presence of viocristin or isoviocristin all three macromolecular syntheses came to a halt. Rubrocristin, erythroglaucin, and physcion showed no significant inhibitory effects.In Ehrlich ascites carcinoma cells catenarin, emodin, and viocristin inhibited the incorporation of uridine and thymidine. The incorporation of leucine was hardly affected.In vitro, inhibition of DNA-dependent RNA polymerase from Escherichia coli by catenarin and to a lesser extent by emodin was observed, whereas rubrocristin (catenarin-8-methyl ether), physcion, and erythroglaucin were not active.Abbreviations MIC minimal inhibitory concentration - TCA trichloroacetic acid - ECA Ehrlich ascites carcinoma Metabolic Products of Microorganisms. 191. W. Keller-Schierlein und B. Joos; Über das 4-Oxohomotyrosin, ein Abbauprodukt des Echinocandins. Helv. Chim. Acta (in press)     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

Inhibition of carotenoid biosynthesis by the herbicide SAN 9789 and its consequences for the action of phytochrome on plastogenesis

Treatment of the mustard (Sinapis alba L.) seedling with the herbicide SAN 9789 inhibits synthesis of colored carotenoids and interferes with the formation of plastid membrane lipids without affecting growth and morphogenesis significantly. In farred light, which is hardly absorbed by chlorophyll, development of plastid ultrastructure, synthesis of ribulosebisphosphate carboxylase and synthesis of chlorophyll are not affected by SAN 9789. It is concluded that normal phytochrome actions on plastid structural development, protein and chlorophyll syntheses are not affected by the absence of carotenoids provided that there is no significant light absorption in chlorophyll. The findings show that the inhibition of synthesis of one set of plastid membrane components (the carotenoids) does not stop synthesis of other components such as chlorophyll and does not halt membrane assembly. Supplementary experiments with the closely related compound SAN 9785, which affects the amount and composition of plastid lipids but not carotenoid and chlorophyll syntheses, suggest that the effect of the herbicide SAN 9789 is due exclusively to its inhibition of synthesis of colored carotenoids. In the presence of SAN 9789 white or red light at high fluence rate causes photodestruction of chlorophyll and ribulosebisphosphate carboxylase and photodecomposition of thylakoids. These effects are interpreted as resulting exclusively from the self-photooxidation and photosensitizing action of chlorophyll once the protection by carotenoids of chlorophyll against self- and sensitized photooxidation is lost.Abbreviations Carboxylase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - Chl chlorophyll a plus chlorophyll b - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - SAN 9789 -chloro-5-(methylamino)-2-(, , -trifluoro-m-tolyl)-3 (2H) pyridazinone - SAN 9785 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazione. SAN 9789 is sold commercially under the trade name Norflurazon - fr far red - wl white light     

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 M Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 M Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 M Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.     

Indications about the mechanism of action and toxicity of netilmicin on the basis of the antiplastidial activity inEuglena gracilis

Summary The effects of the new antibiotic netilmicin (NT) were studied onEuglena gracilis green cells. It was found that, in the presence of the drug, chlorophyll synthesis was strongly inhibited and plastid structure dramatically altered as revealed by fluorescence and electron microscopic observations. Importantly, NT at low concentrations (10–20 g/ml) and for short periods of time (36–72 hours) induced a marked bleaching effect that was permanent and accompanied by the persistence in the colorless cells of poorly differentiated plastids. Other cell components were not influenced as shown by their appearance and also by the kinetics of growth and the cell viability. On the basis of the results and of the literature on the bleaching agents it is suggested that NT is an untoxic antibiotic which specifically inhibits protein synthesis at prokaryotic level.This work was supported by a grant from Italian Research Council (CNR), Contract N. 82.02016.04.     

Effect of 7,8-benzolfavone on the formation of benzo(alpha)pyrene-DNA-bound products in hamster embryo cells.

The hydrocarbon-deoxyribonucleoside products present in enzyme digests of DNA from hamster embryo cultures that had been treated with[3H]-benzo[alpha]pyrene (BP) were isolated by chromatography on Sephadex LH20 columns. The products isolated from cells treated with 7,8-benzoflavone (7,8-BF) for 18 h prior to the addition of [3H] BP were indistinguishable from the products isolated from untreated cultures, but the amounts of these products decreased with increasing concentrations of 7,8-BF. The amount of BP metabolized was also decreased in 7,8-BF-treated cultures. The decrease in the amounts of hydrocarbon-deoxyribonucleoside products per mg DNA was logarithmic with respect to the decrease in BP metabolism. The findings are consistent with the hypothesis that 7,8-BF inhibits both an initial and a later metabolic step involved in the conversion of BP to a reactive species that binds to cellular DNA.     

Relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii in heterotrophic culture

The relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii grown heterotrophically on acetate was investigated. At low acetate concentrations (<0.4 g l^–1), where no inhibition of cell growth was observed, the cell growth yield and specific growth rate could be represented by the Pirt model, 1/Y=1/Y _g +m/ with a constant value of maintenance energy coefficient m. However, at high acetate concentrations (>0.4 g l^–1), inhibition of cell growth occurred, in which m became variable and dependent on the acetate concentration. A simple mathematical model was proposed to predict the actual maintenance energy coefficient m in the inhibited cultures and experimentally validated.Author for correspondence     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.