Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.     

Response of Cultured Mammalian Cells to Diphtheria Toxin III. Inhibition of Protein Synthesis Studied at the Subcellular Level

Diphtheria toxin inhibited protein synthesis in intact KB cells. The action of the toxin upon the cell did not result in disaggregation of polyribosomes, or in impairment of their ability to function in protein synthesis. A reduction in single ribosomes and a concomitant increase in polyribosomes did result from the action of toxin. Nascent peptides were not cleaved from polyribosomes by the action of toxin, but treatment of fully intoxicated cells with puromycin resulted in cleavage of these peptides, and caused accelerated polyribosome breakdown. Our data indicated that the toxin must enter the cell to exert its effect. The component or components sensitive to toxin were localized in the 100,000 x g supernatant fraction of cytoplasmic extracts. When extracts from intoxicated cells were treated with nicotinamide, a significant proportion of their capacity to synthesize protein was restored. The specificity of this reaction suggested that nicotinamide adenine dinucleotide is involved in the action of toxin in the intact cell, and that one component inactivated by toxin is soluble transferase II.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

The effect of photodynamic treatment with thiopyronine on yeast nDNA and mtDNA

Summary In order to ascertain the main target of the photodynamic effect with the sensitizer thiopyronine (TP) and its interference with cellular DNA, the uptake of TP into the yeastSaccharomyces cerevisiae and the distribution of the dye within the cells were studied. After fractionation of the cellular components, about 1% of the TP was found to be bound to the nucleus in anaerobically grown yeast cells; in aerobically grown cells, about 7% could be detected at the mitochondria.After careful isolation of the DNA from the organelles, only 0.024% of the dye once taken up by the cells was detectable at nuclear DNA, whereas 0.652% was bound to mitochondrial DNA. A calculation of the number of TP-molecules bound per nucleotides revealed a ration of one molecule TP per about 6,000 nucleotides of nuclear DNA and one molecule TP per 53 nucleotides of mitochondrial DNA. In vitro, the maximal binding capacity was estimated to be about one molecule TP per nucleotide.The induction of single strand breaks in the DNA after photodynamic treatment in vivo and in vitro was investigated by comparing the sedimentation of nDNA and mtDNA through alkaline sucrose-gradients. No differences in the sedimentation profiles of nDNA after photodynamic treatment of the cells in vivo as compared to the untreated control could be observed. In contrast, the sedimentation coefficient of mtDNA was significally decreased after photodynamic treatment, indicating the induction of single strand breaks in vivo only in mtDNA and not in nDNA.     

Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland

1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

THE USE OF PHOSPHOLIPASE C IN THE STUDY OF RECEPTORS FOR NEUROMUSCULAR BLOCKING AGENTS

Abstract— Segments of diaphragmatic muscle and cerebral nerve-ending particles from the rat were treated exhaustively (180 min) with high concentrations of phospholipase C (300 μg/ml) (EC 3.1.4.3). This enzyme caused loss of approx. 50 per cent of muscle membrane P and 70 per cent of nerve-ending membrane P. At lower concentrations (3 μg/ml; 180 min), phospholipase C released 25 per cent muscle membrane P and 55 per cent of nerve-ending membrane P. At the lower concentration (3 μg/ml; 180 min), the enzyme caused a slow but progressive loss (90 per cent) of muscle twitch amplitude evoked by nerve stimulation (phrenic nerve-hemidiaphragm preparation). Loss of muscle twitch amplitude was notably slowed by removal of the enzyme. Phospholipase C-treated neuromuscular junctions developed no resistance to hemicholinium, botulinum toxin, or d-tubocurarine. Such preparations were unusually sensitive to the neuromuscular blocking action of di-isopropylfluorophosphate. The data do not encourage a belief that phospholipids which are substrates for phospholipase C are receptors for hemicholinium, botulinum toxin, d-tubocurarine or di-isopropylfluorophosphate.     

Polyribosomes, isolated from rat liver in a low ionic strength medium, capable of autonomic translation in a cell-free system without the addition of cellular fluid

Polyribosomes isolated from the liver in the presence of 10 mM KCl and purified by centrifugation through 2 M sucrose were shown to incorporate [3H]leucine both into aminoacyl-tRNA and polypeptides in a cell-free system without cell sap. The incorporation of [3H]leucine showed a linear increase within 80-100 min and was then levelled off. The system was sensitive to cycloheximide, puromycin and ethionine and needed ATP, GTP and unlabeled amino acids. The quantitation of tRNA in polyribosomes (the fraction which did not sediment with the subparticles after polyribosome dissociation) revealed more than two tRNA molecules per 80S monosome. It is likely that this tRNA excess as well as the earlier established presence of aminoacyl-tRNA synthetases and elongation factors promote the autonomic translation of polyribosomes.     

Altered ribosomes after inhibition of Escherichia coli by rifampicin

Addition of rifampicin to growing cells of Escherichia coli affected the ribosomes. The polyribosomes first decayed to 70S ribosomes. These later dissociated to particles distinct from ribosomal subunits. The altered ribosomes sedimented more slowly than the corresponding subunits and had lost some protein; their ribosomal RNA was intact, but they were more susceptible to degradation by ribonuclease than normal ribosomes. The addition of rifampicin to preparations of lysed cells caused no detectable changes in the ribosome fraction.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Photodynamic effects of Photofrin II on cell division in human NHIK 3025 cells

Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.     

Protoplast production from Candida utilis]

The protoplasts of Candida utilis 295 t were produced with the aid of the lytic enzyme from Helix pomatia. If the cell wall of C. utilis 295 t is not treated with SH-compounds (the best effect was found with L-cysteine), it is resistant to the action of the enzyme. The yield of the protoplasts was 100 per cent after 15 minutes of the incubation with the lytic enzyme if the cells were preliminarily treated with L-cysteine. Optimal conditions for the production of the protoplasts are described.     

Studies on the Function of Intracellular Ribonucleases : II. The Interaction of Ribonucleoprotein and Enzymes

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37°C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.     

In vitro evaluation of the cytotoxic and mutagenic potential of the 5-aminolevulinic acid hexylester-mediated photodynamic therapy

Photodynamic therapy (PDT) of tumors with 5-aminolevulinic acid hexylester (h-ALA) causes photo-oxidative reactions in treated tissues. In order to study cytotoxic and/or mutagenic effects, cells of the tumor cell line RPMI 2650 as well as fibroblasts of the cell line WS 1 were given photodynamic treatment in vitro. The cells were photosensitized with a 1mM h-ALA-medium solution for 5h and illuminated with different light doses (0.5, 1.0, 1.5 and 2.0 J/cm2) using red light (633+/-20 nm). PDT-induced cytotoxic effects were determined by measurement of the mitotic index (MI) and the nuclear division index (NDI). Chromosome aberrations (CA) and micronuclei (MN) were recorded to study mutagenicity. After treatment of the photosensitized RPMI 2650 cells with a light dose of 2.0 J/cm2, the MI was significantly decreased to 16.9 per thousand in comparison with that of the h-ALA control (33.8 per thousand ). In photosensitized WS 1 cells, light doses up to 2.0 J/cm2 showed no significant effect. The NDI of photosensitized RPMI 2650 cells was significantly decreased by light doses from 1.0 to 2.0 J/cm2, whereas no significant effect was seen in WS 1 cultures. Thus, h-ALA-PDT only induced desirable cytotoxic effects in tumor cells, but not in the fibroblasts. After application of light doses from 0.5 to 2.0 J/cm2, photosensitized RPMI 2650 cultures showed CA in 7.0-7.5% of the metaphases, which was not a significant increase (h-ALA control: 5.5%). In WS 1 cultures metaphases containing CA varied non-significantly from 5.0 to 7.5%. The MN rates were approximately the same in illuminated RPMI 2650 cultures and in the corresponding h-ALA control (4.4-4.9 per thousand ). The MN rates of the illuminated WS 1 cultures also varied non-significantly from 4.5 to 5.0 per thousand in comparison with the h-ALA control (5.5 per thousand ). In the mutagenicity tests the h-ALA-PDT had no significant effect, neither on the tumor cells nor on the fibroblasts. In addition to the cytogenetic analysis, spectral karyotyping (SKY) was used to characterize the cell lines and gain more detailed information on possibly PDT-induced CA. The SKY evaluation also showed no significant increase of the CA rate, but confirmed the result of the CA test. Thus, within the scope of the experiments performed, a mutagenic potential of the h-ALA-PDT can be excluded.     

Photodynamic effect of quinacrine on bacteria]

The acridine dye quinacrine (QA) was tested with regard to the photodynamic action on bacteria (Proteus mirabilis, Escherichia coli). The absorption maximum of the yellow dye QA ist in contrary to the photodynamically active dyes methylene blue (MB) and thiopyronine (TP) situated in the short wave region of the visible spectrum. Using for illumination a common light source--they have in general a weak emission in the short wave region--relatively high concentrations of QA are necessary for photodynamic action, and the difference between photodynamic inactivation and toxic effect is small. Using that light source XBO 500 with nearly equal emission in the range from 400 to 700 nm, a distinct photodynamic action of QA results. Comparing the photodynamic action of QA with those of MB and TP, QA has a low photodynamic effect, and the kinetics of inactivation of bacteria with QA is completely different from those obtained with the dyes MB and TP.     

Inhibitory action of erythromycin on protein biosynthesis by isolated polyribosomes

Consistent with the previous work by Pestka (Antimicrob. Agents Chemother.5, 255, 1974) on the binding of erythromycin to polyribosomes, we found that erythromycin does not inhibit protein synthesis catalyzed by polyribosomes. This is due to the presence of nascent peptidyl tRNA on the naturally occurring polyribosomes. In a soluble extract from E. coli pretreated to remove the ribosome releasing factor, polyribosomes without nascent polypeptides remain intact and can catalyze protein synthesis in the absence of initiation. In this system erythromycin effectively inhibited protein synthesis. The inhibition by erythromycin was caused by premature release of oligopeptidyl tRNA from polyribosomes.     

Thiopyronine and 8-methoxypsoralen sensitized photodynamic effect on DNA synthesis in yeast

Summary The synthesis of DNA in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine (TP) and visible light or with 8-methoxypsoralen (8-MOP) and UVA light.DNA synthesis was inhibited after photodynamic treatment with 8-MOP but not after photodynamic treatment with TP. This result is further evidence that the photodynamic effect with TP does not attack nuclear DNA in eucaryotic cells.