Physiology of rat-liver polysomes. The stability of messenger ribonucleic acid and ribosomes.
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ABSTRACT:
SUBMITTER: Wilson SH
PROVIDER: S-EPMC1270442 | biostudies-other | 1967 May
REPOSITORIES: biostudies-other
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Project description:Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.
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Project description:A 60-year-old Japanese woman was hospitalized for cardiogenic shock 24 days after receiving the second dose of the coronavirus disease 2019 BNT162b2 vaccine. Impella CP left ventricular assist device implantation and venoarterial peripheral extracorporeal membranous oxygenation were immediately initiated along with inotropic support and steroid pulse therapy, as an endomyocardial biopsy specimen showed myocarditis. Three weeks later, her cardiac function had recovered, and she was discharged. An immune response associated with the presence of spike protein in cardiac myocytes may be related to myocarditis in the present case because of positive immunostaining for severe acute respiratory syndrome coronavirus 2 spike protein and C4d in the myocardium.
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Project description:Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing [3H]glycine, [3H]lysine and [3H]serine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.
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Project description:Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.
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Project description:1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.
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Project description:Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.
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Project description:ObjectiveTo determine whether the COVID-19 mRNA vaccines can negatively impact the semen parameters of young healthy men in the long-term.DesignWe conducted semen analyses on 12 men before, 3 and 9 months after achieving fully vaccinated status. Individuals who admitted a history of infertility or previous azoospermia were excluded from study participation.SubjectsHealthy male volunteers between the ages of 18-50 years old were recruited between September 2021 - March 2022.Main outcome measuresSemen analyses were performed and evaluated volume, sperm concentration, total motility, and total motile sperm count (TMSC). The primary outcome was median change in the TMSC at baseline, 3 months, and at least 9 months following vaccination.ResultsA total of 12 men volunteered in our study (median age 26 [25 - 30] years). Subjects provided follow-up semen samples at a median of 10 months following the second vaccine dose. There were no significant changes in any semen parameters between baseline, 3 months, and 10 months following vaccination. Baseline samples demonstrated median sperm concentrations and TMSC of 29.5 million/cc [9.3 - 49] and 31 million [4-51.3], respectively. At 9-month follow-up, sperm concentration and TMSC were 43 [20.5 - 63.5] (P=.351) and 37.5 [8.5 - 117.8] (P=.519), respectively. Of note, there were no significant changes in semen volume nor total motility (%) for participants at follow-up.ConclusionCOVID-19 mRNA vaccines and the booster dose does not appear to negatively impact the semen parameters of healthy males up to 10 months following vaccination.
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Project description:1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.
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