Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm.
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ABSTRACT:
SUBMITTER: Mullock BM
PROVIDER: S-EPMC1172438 | biostudies-other | 1975 Oct
REPOSITORIES: biostudies-other
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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:Heterogeneity among the free small ribosomal subunits of rat liver was studied. Newly made free small subunits differ from the bulk of the free subunits in the following ways. (1) The newly made free small subunits sediment slightly faster than most free small subunits and form a band at slightly lower densities. (2) Mature free small subunits, prepared under conditions which minimize the amount of loosely bound protein, readily dimerize in solutions of low ionic strength. Little dimerization is found with newly formed subunits. This permits the separation of fractions enriched in newly formed subunits. (3) The fraction enriched in newly formed subunits contains a distinctive additional protein as compared the most of the free small subunits.
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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:A study has been made of the developmental changes that occur in the RNA and protein moieties of mRNA-protein particles isolated from newborn and adult rat forebrain free polyribosomes. mRNA-protein particles were isolated by oligo(dT)-cellulose chromatography from salt-washed polyribosomes dissociated by puromycin/0.5 M-KCl treatment as two fractions (E1 and E2) by using Tris/HCl/NaCl eluting buffers containing respectively 25 and 50% (v/v) formamide. Isopycnic centrifugation on CsCl gradients showed that the newborn-derived fractions E1 and E2 has buoyant densities of 1.48--1.50 and 1.41--1.43 g/cm3. Adult-derived E1 and E2 fractions had corresponding values of 1.47 and 1.42 g/cm3. The pooled mRNA-protein particles from the E1 and E2 fractions after deproteinization with proteinase K sedimented with a mean size of approx. 18 S on a sucrose gradient containing 85% formamide with little differences between mRNA molecules from newborn and adult. The mean lengths of the poly(A) segments were similar, being about 130 nucleotides long. Distinct changes were found in the protein composition of the mRNA-protein particles. Fractions E1 and E2 from the newborn contained two major proteins of mol.wts. 74 000 and 52 000 with differences in the relative proportions in each fraction. In contrast, adult fractions E1 and E2 contained predominantly the larger protein. However, the adult fraction E2 contained a more heterogeneous population of minor bands of proteins, including that of mol.wt. 52 000. The findings are discussed briefly in relation to other changes in the developing brain.
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Project description:5q-related Spinal muscular atrophy (SMA) is a hereditary multi-systemic disorder leading to progressive muscle atrophy and weakness caused by the degeneration of spinal motor neurons (MNs) in the ventral horn of the spinal cord. Three SMN-enhancing drugs for SMA treatment are available. However, even if these drugs are highly effective when administrated early, several patients do not benefit sufficiently or remain non-responders, e.g., adults suffering from late-onset SMA and starting their therapy at advanced disease stages characterized by long-standing irreversible loss of MNs. Therefore, it is important to identify additional molecular targets to expand therapeutic strategies for SMA treatment and establish prognostic biomarkers related to the treatment response. Using high-throughput nCounter NanoString technology, we analyzed serum samples of late-onset SMA type 2 and type 3 patients before and six months under nusinersen treatment. Four genes (AMIGO1, CA2, CCL5, TLR2) were significantly altered in their transcript counts in the serum of patients, where differential expression patterns were dependent on SMA subtype and treatment response, assessed with outcome scales. No changes in gene expression were observed six months after nusinersen treatment, compared to healthy controls. These alterations in the transcription of four genes in SMA patients qualified those genes as potential SMN-independent therapeutic targets to complement current SMN-enhancing therapies.
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Project description:1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg(2+) ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2-3 uridine residues, and there is evidence that these may be added to the 3'-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg(2+) ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5'-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5'-triphosphate. The optimum pH is in the range 7.0-8.5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [(32)P]UTP in the absence of ATP, GTP and CTP, 80-90% of (32)P was recovered in UMP-2' or -3' after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the (32)P was recovered in nucleotides other than UMP-2' or -3'. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.
| S-EPMC1186934 | biostudies-other
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:A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments.
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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:Messenger ribonucleic acid (mRNA) was found as the intermediary that transfers genetic information from DNA to ribosomes for protein synthesis in 1961. The emergency use authorization of the two covid-19 mRNA vaccines, BNT162b2 and mRNA-1273, is a significant achievement in the history of vaccine development. Because they are generated in a cell-free environment using the in vitro transcription (IVT) process, mRNA vaccines are risk-free. Moreover, chemical modifications to the mRNA molecule, such as cap structures and changed nucleosides, have proved critical in overcoming immunogenicity concerns, achieving sustained stability, and achieving effective, accurate protein production in vivo. Several vaccine delivery strategies (including protamine, lipid nanoparticles (LNPs), polymers, nanoemulsions, and cell-based administration) were also optimized to load and transport RNA into the cytosol. LNPs, which are composed of a cationic or a pH-dependent ionizable lipid layer, a polyethylene glycol (PEG) component, phospholipids, and cholesterol, are the most advanced systems for delivering mRNA vaccines. Moreover, modifications of the four components that make up the LNPs showed to increase vaccine effectiveness and reduce side effects. Furthermore, the introduction of biodegradable lipids improved LNP biocompatibility. Furthermore, mRNA-based therapies are expected to be effective treatments for a variety of refractory conditions, including infectious diseases, metabolic genetic diseases, cancer, cardiovascular and cerebrovascular diseases. Therefore, the present review aims to provide the scientific community with up-to-date information on mRNA vaccines and their delivery systems.
| S-EPMC10405914 | biostudies-literature