Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Dependence of mammalian DNA synthesis on DNA supercoiling. III. Characterization of the inhibition of replicative and repair-type DNA synthesis by novobiocin and nalidixic acid

Novobiocin and nalidixic acid, inhibitors of the bacterial enzyme DNA gyrase, inhibit DNA, RNA and protein synthesis in several human and rodent cell lines. The sensitivity of DNA synthesis (both replicative and repair) to inhibition by novobiocin and nalidixic acid is greater than that of protein synthesis. Novobiocin inhibits RNA synthesis about half as effectively as it does DNA synthesis, whereas nalidixic acid inhibits both equally well. Replicative DNA synthesis, as measured by incorporation of [3H]thymidine, is blocked by novobiocin in a number of cell strains; the inhibition is reversible with respect to both DNA synthesis and cell killing, and continues for as long as 20--30 h if the cells are kept in novobiocin-containing growth medium. Both novobiocin and nalidixic acid inhibit repair DNA synthesis (measured by BND-cellulose chromatography) induced by ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine (but not that induced by methyl methanesulfonate) at lower concentration (as low as 5 micrograms/ml) than those required to inhibit replicative DNA synthesis (50 micrograms/ml or greater). Neither novobiocin nor nalidixic acid alone induces DNA repair synthesis. Incubation of ultraviolet-irradiated cells with 10--100 micrograms/ml novobiocin results in little, if any, further reduction of colony-forming ability (beyond that caused by the ultraviolet irradiation). Novobiocin at sufficiently low concentrations (200 micrograms/ml) apparently generates a quiescent state (in terms of cellular DNA metabolism) from which recovery is possible. Under more drastic conditions of time in contact with cells and concentration, however, novobiocin itself induces mammalian cell killing.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

Estradiol-17 beta inhibition of lutropin and dibutyryl cyclic AMP induced steroidogenesis in intact bovine luteal cells. Absence of effect on induced protein phosphorylations

Estradiol-17 beta is known to inhibit in a dose dependent manner the lutropin-induced stimulation of progesterone synthesis in luteal cells without affecting the intracellular cyclic AMP increase produced by the hormone. The hypothesis that this inhibitory action could involve an inhibition of the cyclic AMP dependent phosphorylation of cytosolic proteins was investigated by using incubations of selected small bovine luteal cells. Doses of 10 and 100 micrograms/ml of estradiol-17 beta inhibited respectively 60 and 90% of the progesterone synthesis induced by lutropin as well as by dibutyryl cyclic AMP in small bovine luteal cells. At the concentrations of 10 and 100 micrograms/ml, estradiol-17 beta was unable to affect the cyclic AMP dependent protein kinase activation induced by lutropin. At the concentration of 10 micrograms/ml the steroid was without effect on the lutropin or dibutyryl cyclic AMP induced protein phosphorylations. However 100 micrograms/ml of estradiol-17 beta seemed to produce a slight inhibition of the induced protein phosphorylations.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Effect of tunicamycin on glycosylation of a 50 kDa protein and thermotolerance development.

We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a heat treatment at 45.5 degrees C administered 12 hr later. The thermotolerance ratio at 10(-3) isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2-2 micrograms/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 micrograms/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 micrograms/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance development by treatment with a combination of 2 micrograms/ml tunicamycin and 10 micrograms/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis-independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro.

The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 micrograms of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 micrograms of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-beta may be present in this acid/ethanol extract of skeletal muscle.     

黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.     

Lack of complete cooperativity of ribosome assembly in vitro and its possible relevance to in vivo ribosome assembly and the regulation of ribosomal gene expression.

Earlier studies have shown that the reconstitution of Escherichia coli 50S as well as 30S ribosomal subunits from component rRNA and ribosomal protein (r-protein) molecules in vitro is not completely cooperative and binding of more than one r-protein to a single 16S rRNA (or 23S rRNA) molecule is required to initiate a successful 30S (or 50S) ribosome assembly reaction. We first confirmed this conclusion by carrying out 30S subunit reconstitution in the presence of a constant amount of 16S rRNA together with various amounts of total 30S r-proteins (TP30) and by analyzing the physical state of reconstituted particles rather than by assaying protein synthesizing activity of the particles as was done in the earlier studies. As expected, under conditions of excess rRNA, the efficiency of 30S subunit reconstitution per unit amount of TP30 decreased greatly with the decrease in the ratio of TP30 to rRNA, indicating the lack of complete cooperativity in the assembly reaction. We then asked the question whether the cooperativity of ribosome assembly is complete in vivo. We treated exponentially growing E coli cells with low concentrations of chloramphenicol which is known to inhibit protein synthesis without inhibiting rRNA synthesis, creating conditions of excess synthesis of rRNA relative to r-proteins. Several concentrations of chloramphenicol (ranging from 0.4 to 4.0 micrograms/ml) were used so that inhibition of protein synthesis ranged from 40 to 95%. Under these conditions, we examined the synthesis of RNA, ribosomal proteins and 50S ribosomal subunits as well as the synthesis of total protein. We found that the synthesis of 50S subunits was not inhibited as much as the synthesis of total protein at lower concentrations of chloramphenicol, but the degree of inhibition of 50S subunit synthesis increased sharply with increasing concentrations of chloramphenicol and was in fact greater than the degree of inhibition of total protein synthesis at chloramphenicol concentrations of 2 micrograms/ml or higher. The inhibition of 50S subunit synthesis was significantly greater than the inhibition of r-protein synthesis at all chloramphenicol concentrations examined. These data are consistent with the hypothesis that the cooperativity of ribosome assembly in vivo is also not complete as is the case for in vitro ribosome reconstitution, but are difficult, if not impossible, to explain on the basis of the complete cooperativity model.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of cholesterol in the glucocorticoid-mediated inhibition of cell cycle progression in human acute lymphoblastic leukemia cells

Two glucocorticoid receptor-containing clones of human acute lymphoblastic leukemia, one (CEM-C7) sensitive and one (CEM-C1) resistant to dexamethasone (dex) were studied in an effort to identify the time course of the biochemical changes responsible for dex-induced growth inhibition of CEM-C7 cells. Cells were synchronized by treatment with 0.25 mM (C7) or 0.50 mM (C1) thymidine for 12 h followed by 0.025 micrograms/ml (C7) or 0.050 micrograms/ml (C1) colcemid for 12 h, then released either in the presence or absence of 1 microM dex. The inhibition of cellular proliferation which occurs at 48 h after release in the dex-treated CEM-C7 cells was preceded by an inhibition of acetate incorporation into cholesterol, first evident at 24 h, inhibition of protein synthesis at 30 h, and the development of a cell cycle block in G1 at 36 h. No inhibition of any of these parameters was seen in the resistant CEM-C1 cells. Thus the inhibition of cholesterol synthesis in the sensitive cells may be one of the earliest parameters affected by glucocorticoids.     

Effect of cycloheximide on nuclear maturation of pig and mouse oocytes

Culture of mouse oocytes in medium with 1 or 100 micrograms cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 micrograms cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 micrograms/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture. The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Influence of Macromolecular Biosynthesis on Cellular Autolysis in Streptococcus faecalis

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.