ATP and Protein Synthesis Assays to Evaluate the Toxicities of Preservatives in Vitro

1991 ◽  
Vol 19 (1) ◽  
pp. 60-67
Author(s):  
Corrado L. Galli ◽  
Barbara Viviani ◽  
Giampiero Casartelli ◽  
Marina Marinovich
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Cellular Protein ◽  
Cell Protein ◽  
Leucine Incorporation ◽  
Maximal Effect ◽  
Atp Level ◽  
Cellular Protein Content

Cellular protein content, protein synthesis, ATP level and lactate dehydrogenase (LDH) activity, measured in a murine epidermal cell line (HEL/30), were used as the endpoints for determining the cytotoxicities of 17 antimicrobial chemicals. The relative toxicities of the test compounds were quantified by the determination of the concentrations inducing a 50% inhibition of [3H]-leucine incorporation into proteins (IC50), causing a 50% reduction of ATP level or final cell protein content or producing the maximal effect on LDH leakage (EC50) after 2 hours of treatment. The results indicate a good correlation between both the reduction of ATP level and inhibition of protein synthesis and the minimal inhibitory concentration (MIC) on different microorganisms, suggesting that ATP and protein synthesis assays could be useful as prescreening methods for testing the cytotoxicities of preservatives.

scholarly journals Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

1971 ◽  
Vol 124 (2) ◽  
pp. 385-392 ◽  
Author(s):  
R. W. Wannemacher ◽  
C. F. Wannemacher ◽  
M. B. Yatvin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Free Amino Acids ◽  
Free Amino ◽  
Cellular Protein ◽  
Deficient Diet ◽  
Cellular Protein Content ◽  
Regulate Protein

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.

The Effect of Dilution Rate upon Protein Content and Cellular Amino Acid Profiles in Chemostat Cultures of Saccharomyces Cerevisiae CABI 039916

2010 ◽  
Vol 6 (4) ◽  
Author(s):  
Beverley Finn ◽  
Linda M Harvey ◽  
Brian McNeil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Content ◽  
Dilution Rate ◽  
Specific Growth ◽  
Cellular Protein ◽  
Cell Protein ◽  
Protein Pool ◽  
Cellular Protein Content ◽  
Amino Acid Profiles

In the present study we examined the use of a chemostat system to investigate the impact of changes in the specific growth rate of Saccharomyces cerevisiae CABI 039916 on cellular amino acid profiles and total protein content. This experimental system allowed the unambiguous examination of the link between changes in dilution rate and the culture response, which would have been difficult in batch or fed-batch cultures. Alteration of the specific growth rate (via manipulation of the dilution rate) within a carbon and energy-limited chemostat has a significant impact on the physiology of Saccharomyces cerevisiae. Low dilution rates (<0.1h-1) led to predominantly respiratory metabolism and the maximisation of cellular protein content within the cell (58%), by contrast high dilution rates (>0.2h-1) led to respirofermentative metabolism, where the cellular protein content was minimal (~40%). The content of nearly all amino acids in the yeast protein pool fell significantly as dilution rate increased in parallel with the decline in cell protein content. By contrast, the concentration of two related key food/feed amino acids in the cell protein pool—glutamic acid and arginine could be increased within the cellular protein by 5% (increasing the dilution rate from 0.05h-1 to 0.25h-1) and 1.5% respectively (decreasing the dilution rate from 0.05h-1 to 0.2h-1). Despite previous studies showing that metabolic change was associated with major changes in free amino acid levels, the present study indicates that the total cellular yeast protein amino acid composition is largely invariant despite profound metabolic changes, with one or two key exceptions.

Long-term Cytotoxicity Testing of the First Twenty MEIC Chemicals by the Determination of the Protein Content in Human Embryonic Lung Cells

1992 ◽  
Vol 20 (2) ◽  
pp. 285-289
Author(s):  
Paul J. Dierickx ◽  
Björn Ekwall
Keyword(s):  
Protein Content ◽  
Good Alternative ◽  
In Vitro Cytotoxicity ◽  
Cell Protein ◽  
Cytotoxicity Test ◽  
Hep G2 ◽  
Human Embryonic Lung

Within the framework of the MEIC (the multicentre evaluation of in vitro cytotoxicity) programme, the long-term cytotoxicity of the first twenty MEIC chemicals was investigated on MRC-5 cells. After treatment of the cells with each chemical for six weeks, the PI50 value was determined. The PI50 is the concentration (mM) of test compound required to induce a 50% reduction in total cell protein content. The results were compared with the acute PI50 values previously measured after treatment for 24 hours with the same compounds in Hep G2 cells. A good correlation between these two sets of data (r2 = 0.94) was observed, with the exception of digoxin. Together with other preliminary MEIC validation results, this study indicates that a good alternative cytotoxicity test may be found for the prediction of human long-term toxicity.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽  
1980 ◽  
Vol 28 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Luanne M. Deal ◽  
J. T. Reeves ◽  
B. A. Larkins ◽  
F. D. Hess
Keyword(s):  
Protein Synthesis ◽  
Sand Culture ◽  
Leucine Incorporation ◽  
Insoluble Protein ◽  
In Vitro System ◽  
A Cell ◽  
Protein Incorporation ◽  
Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination

1983 ◽  
Vol 3 (7) ◽  
pp. 1212-1221 ◽  
Author(s):  
A Babich ◽  
L T Feldman ◽  
J R Nevins ◽  
J E Darnell ◽  
C Weinberger
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Cellular Protein ◽  
Host Protein ◽  
Cell Protein ◽  
Viral Mrna ◽  
Initiation Of Translation ◽  
Viral Mutant ◽  
Cellular Mrnas ◽  
Cellular Protein Synthesis

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.

Effects of oestradiol, the oestrous cycle and pregnancy on weight, metabolism and cytosol receptors in the uterus of the brush-tail possum (Trichosurus vulpecula)

1986 ◽  
Vol 108 (2) ◽  
pp. 201-210 ◽  
Author(s):  
J. D. Curlewis ◽  
G. M. Stone
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Corpus Luteum ◽  
Metabolic Activity ◽  
Weight Ratio ◽  
Oestrous Cycle ◽  
Uterine Weight ◽  
In Vitro Protein Synthesis ◽  
Vitro Protein

ABSTRACT Uterine weight, RNA, DNA, protein content, in-vitro rate of protein synthesis, cytosol oestrogen and progesterone receptors were examined after administration of oestradiol to ovariectomized animals and on days 0, 5, 9 and 13 of the non-pregnant cycle and day 13 of pregnancy. In ovariectomized animals, oestradiol increased uterine weight, RNA: DNA and protein: DNA ratios and the concentration of cytosol receptors for oestradiol and progesterone. During the oestrous cycle there was a linear increase in uterine weight and a significant effect of the corpus luteum on the weight of the ipsilateral uterus. Changes in RNA, DNA and protein content between days 0 and 5 were not observed, but between days 5 and 13 RNA: DNA and protein: DNA ratios increased and the DNA: tissue weight ratio decreased. Thus, cellular hypertrophy and/or increased metabolic activity rather than hyperplasia occur over this period, which is coincident with the known rise in plasma progesterone levels. The rate of in-vitro protein synthesis (per unit tissue protein) during the non-pregnant cycle was greatest at day 0. These changes in uterine metabolic activity were associated with alterations in cytosol receptor concentrations for both steroids. Cytosol progesterone receptor concentrations were highest at day 0 after which they declined to a minimum at day 13. Cytosol oestradiol receptor concentrations, however, rose between days 0 and 5 and then declined. Although lutectomy on day 8 of the cycle does not interfere with the development of a histologically normal luteal phase, high peripheral progesterone levels which occur after day 8 in intact animals are associated with major increases in uterine metabolic activity. The unilateral effect of the corpus luteum on uterine weight was associated with a decrease in DNA: g tissue ratio and an increase in rate of in-vitro protein synthesis indicating hypertrophy and/or extracellular accumulation of secreted material as well as enhanced metabolic activity. There was a significant effect of pregnancy on uterine weight at day 13 and this was associated with an increase in DNA content of both uteri. There was a unilateral effect of pregnancy on RNA: DNA ratio and in-vitro rate of protein synthesis, but not on uterine weight. J. Endocr. (1986) 108, 201–210

scholarly journals ISG56/IFIT1 is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis

2013 ◽  
Vol 94 (1) ◽  
pp. 59-68 ◽  
Author(s):  
J. Andrejeva ◽  
H. Norsted ◽  
M. Habjan ◽  
V. Thiel ◽  
S. Goodbourn ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Virus Type ◽  
Cellular Protein ◽  
Parainfluenza Virus ◽  
Hydroxyl Group ◽  
Virus Protein ◽  
Viral Mrnas ◽  
Tetratricopeptide Repeats ◽  
Virus Transcription

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2′-O-methyltransferase activity, an enzyme that methylates the 2′-hydroxyl group of ribose sugars in the 5′-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2′-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Activation of Protein Synthesis in Aging Potato Tuber Slices

1971 ◽  
Vol 26 (10) ◽  
pp. 1064-1067 ◽  
Author(s):  
Günter Kahl
Keyword(s):  
Protein Synthesis ◽  
Potato Tuber ◽  
Actinomycin D ◽  
Leucine Incorporation ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Fresh Potato ◽  
Polyuridylic Acid ◽  
Storage Organs

Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.

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