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Results for “Ribosome Profiling”

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PubMed2003

Differential expression of ribosomal proteins in human normal and neoplastic colorectum.

Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithilia except immature mucosal cells, whereas their expression levels were significantly enhanced in colorectal cancer cells. In addition, L7 ribosomal protein was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.

Colon↗

PubMed2004

characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 +/- 7.9 per sample and 13.4 +/- 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

Bacteria↗

PubMed1983

Biochemical characterization of three mycobacterial ribosomal fractions.

The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity.

Animals↗

PubMed1986

Identification of 30-kDa fat body protein of Sarcophaga peregrina larvae selectively phosphorylated in the presence of 20-hydroxyecdysone as ribosomal protein S6.

Previously, we showed that 20-hydroxyecdysone induces selective phosphorylation of a fat body protein of Sarcophaga peregrina with a molecular mass of about 30,000 (30-kDa protein) (Itoh, K., Ueno, K., & Natori, S. (1985) Biochem. J. 227, 683-688). This paper describes the identification of this 30-kDa protein. From the electrophoretic profile of 40S ribosomal proteins on two-dimensional polyacrylamide gel electrophoresis, the 30-kDa protein was identified as S6.

Adipose Tissue↗

PubMed1995

Synaptic connectivity of local circuit neurons in laminae III and IV of hamster spinal cord.

The present study was undertaken to examine the morphological bases of local synaptic interactions between dorsal horn interneurons. Seven interneurons responding to innocuous mechanical stimuli were intracellularly recorded in lamina III/IV of an isolated preparation of hamster spinal cord with partially intact innervation from an excised patch of hairy skin. Axonal arborizations were stained with horseradish peroxidase (HRP) and examined with an electron microscope. Five cells had extensive synaptic terminations (375-1,785 boutons/axon) with localized distributions (rostrocaudal distance, 425-1,251 microns) overlapping the dendritic trees. Two cells gave rise to deep stem axons that bifurcated into rostrocaudal daughter branches with collaterals ventral to the parent cell bodies (79-661 boutons/axon). Axons of local interneurons were thinly myelinated and formed terminal and en passant enlargements (mean [+/- S.D.] diameter = 0.88 +/- 0.24 microns, n = 157) containing clear, round vesicles 20-60 nm in diameter. Collateral branches of deep axon cells produced round, vesicle-containing boutons comparable in diameter (0.93 +/- 0.22 microns, n = 31) to local axon cells. Both types of interneurons formed asymmetric synaptic contacts with dendritic profiles, but not with cell bodies or axon terminals. Postsynaptic profiles contained sparse ribosomes and had a mean diameter of 1.0 +/- 0.5 microns (n = 49), significantly smaller than a population of identified proximal dendrites (2.3 +/- 0.9 microns, n = 47). HRP-labeled boutons were rarely (5/45 or 11%) in synaptic contact with more than one profile. We conclude that lamina III/IV interneurons make axodendritic synapses predominantly with distal dendrites. Thus, terminations of deep dorsal horn interneurons appear to have a postsynaptic distribution overlapping with axodendritic contacts formed by several functional classes of cutaneous sensory fibers signaling innocuous mechanical stimuli. Such overlap suggests that local spinal networks selectively and strongly influence afferent signals at initial stages of somatosensory integration.

Adaptation, Physiological↗

PubMed2024

Ribosomal DNA copy number variation associates with hematological profiles and renal function in the UK Biobank.

The phenotypic impact of genetic variation of repetitive features in the human genome is currently understudied. One such feature is the multi-copy 47S ribosomal DNA (rDNA) that codes for rRNA components of the ribosome. Here, we present an analysis of rDNA copy number (CN) variation in the UK Biobank (UKB). From the first release of UKB whole-genome sequencing (WGS) data, a discovery analysis in White British individuals reveals that rDNA CN associates with altered counts of specific blood cell subtypes, such as neutrophils, and with the estimated glomerular filtration rate, a marker of kidney function. Similar trends are observed in other ancestries. A range of analyses argue against reverse causality or common confounder effects, and all core results replicate in the second UKB WGS release. Our work demonstrates that rDNA CN is a genetic influence on trait variance in humans.

Humans↗

PubMed1997

Quantitative ultrastructural evaluation of satellite cells in soleus muscle from rats kept in hypokinesia.

The aim of the present study is a morphometric, ultrastructural evaluation of satellite cells (SC) derived from the soleus muscle (SOL) of rats exposed to conditions of hypokinesia for a period of 7 or 21 days. Qualitative and quantitative analysis of 320 electron micrographs of SC from each group was carried out. After 7 and 21 days of immobilization, profiles of the SC, in contrast to the control group, had a lower mean surface area, had a cylindrical shape, and exhibited more folded membrane. Analysis of electron micrographs of SC showed that after immobilization, a lower number of SC contained profiles of mitochondria, rough endoplasmic reticulum (RER), and Golgi. Volume fractions of RER were twofold lower after 7 days of hypokinesia and fivefold lower after 21 days compared with the control group. The SC of SOL of rats subjected to hypokinesia differed from the control group by a markedly decreased number of ribosomes and RER profiles. After 21 days of immobilization the ultrastructural characteristics of SC were typical for cells in an inactive state showing various degrees of degeneration. The results of our study presented here permit the conclusion that 21 days of hypokinesia induce a depression of SC activity, whereas subtle changes in SC appear only after 7 days of immobilization.

Animals↗

PubMed2003

New Klebsiella oxytoca beta-lactamase genes bla(OXY-3) and bla(OXY-4) and a third genetic group of K oxytoca based on bla(OXY-3).

The two genetic groups (oxy-1 and oxy-2) previously identified in the Klebsiella oxytoca taxon are recognizable by four independent molecular markers: (i). ERIC-1R profiles, (ii). 16S ribosomal DNA (rDNA) signature sequences, (iii). singular nucleotides in a defined fragment of the rpoB gene, and (iv) the type of the strain's bla(OXY) gene (i.e., bla(OXY-1) or bla(OXY-2)). K. oxytoca strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and bla(OXY) gene sequence. With regard to the gene identity percentages between the bla(OXY-1) and bla(OXY-2) gene groups (86.8% +/- 0.4%) and within a bla(OXY) gene group (>99%), it was concluded that the bla(OXY) gene of strain SG271 was representative of a new bla(OXY) gene group (bla(OXY-3)), since the mean identity percentages between it and the two bla(OXY) gene groups were 85.5% +/- 0.2% and 84.4% +/- 0.4%, respectively. Since the corresponding percentages were 95.0% +/- 0.4% and 86.2% +/- 0.3% for strain SG266, it was impossible to classify its bla(OXY) gene, which was therefore named bla(OXY-4). The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the rpoB sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new K. oxytoca genetic group (oxy-3), whereas strain SG266 could not be classified.

Anti-Bacterial Agents↗

PubMed1975

Interaction of the 5'-ends of 28S RNA in dimerization of hamster ribosomes.

Free ribosomes extracted from hamster cells and 28S RNA purified from these ribosomes are known to form dimers. We find that spleen phosphodiesterase inhibits ribosomal dimer formation, but only when a free 5'-hydroxyl end group, produced by the action of alkaline phosphatase, is present. Hence, formation of dimer ribosomes probably involves interaction at or near the phosphorylated 5'-ends of 28S RNA. Dimer RNA molecules show a modal length, when measured on electrom micrographs, of 2.1 mum, which is about double the length of 28S RNA. Electron micrographs of 115S dimer ribosomes often show profiles consistent with our interpretation that in dimers the 28S RNA chains are loosely linked by their 5'-ends.

Animals↗

PubMed1984

Sertoli cell development of pig testis in the fetal and neonatal period.

The Sertoli cells of pig fetuses from 35 days postcoitum until 1 mo after birth have been investigated by light and electron microscopy in decapitated animals and their control littermates, as well as in untreated animals. Until 52 days postcoitum, Sertoli cells change in form during the formation of sex cords but from then onwards they are rather uniform. They primarily display an elongated nonindented nucleus with one or more prominent nucleoli, a well-developed Golgi apparatus, and in the basal compartment below or beside the nucleus, a large lipid droplet. There are large quantities of rough endoplasmic reticulum (RER) from 52 days postcoitum onwards, often with complex whirl forms and a parallel arrangement of profiles with relatively few ribosomes. After birth their numbers seem to be somewhat less, and by 1 mo after birth the RER profiles are often shorter and almost free of ribosomes. Clustered ribosomes are found in large quantities throughout the period under investigation. Especially in the early fetal period, the endoplasmic reticulum (ER) profiles show prominently filled cisternae. Mitochondria are mostly long and slender, or small and ovoid. Most have lamellar cristae, but mixed lamellar-tubular cristae can also be seen. Between decapitated, control and untreated animals no obvious ultrastructural differences could be observed. The peritubular cell sheath surrounding the sex cords did not show signs of differentiation into a layer of myoid cells.

Animals↗

PubMed1999

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants.

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Animals↗

PubMedDate not supplied

Protein synthesis and the components of protein synthetic machinery during cellular aging.

The slowing down of protein synthesis is a change widely observed during the aging of organisms. It has also been claimed that a decline in the rate of protein synthesis occurs during cellular aging. However, the evidence in favour of this view is not clear-cut, and reliable estimates of rates of protein synthesis during cellular aging have yet to be made. Studies on various components of the protein synthetic machinery during cellular aging have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Detailed studies on the structure and function of ribosomes, tRNA isoacceptor profiles, activities of aminoacyl-tRNA synthetases, levels and activities of initiation factors, rates of protein elongation, and the accuracy of protein synthesis will be needed before the molecular mechanisms of the regulation of protein synthesis during cellular aging can be understood.

Amino Acids↗

PubMed1990

Polyadenylated RNAs as error sources in ribosomal RNA turnover analyses.

An approach to ribosomal RNA turnover studies in which cytoplasmic RNA was extracted and subsequently fractionated to isolate ribosomal RNA is reported. The presumption that the pool of 28S and 18S RNAs represented ribosomal RNA, exclusively, proved false and led to erroneous results of ribosomal RNA turnover. Polyadenylated RNAs exhibited a heterogeneous size distribution and, although constituting only 3% (w/w) of the cytoplasmic RNA extract, accounted for fully 10% of radioactivity of the presumptive ribosomal RNA pool. Profiles from the radioactivity data suggested that the discrepant results were due to these polyadenylated RNAs. An additional analytical procedure, an oligo (dT) cellulose column chromatography of the RNA extract prior to the sucrose density gradient fractionation step, performed as described in this paper, proved an effective remedy for this error.

Animals↗

PubMed1978

Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts.

Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg(2+). Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases.

Journal Article↗

PubMed1988

Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts.

Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.

Adenosine Triphosphatases↗

PubMed1975

The influence of oestradiol benzoate and progesterone on the secretory activity of the epithelium of the ampulla tubae in ovariectomized ewes: an electron microscopical study.

The influence of oestradiol benzoate (ODB) and progesterone on the ultrastructural organization of secretory and ciliated cells of the ovine oviduct was studied in ovariectomized animals. In untreated control animals, the absence of any influence of ovarian steroids resulted in a low cuboidal epithelium with a quiescent ultrastructural appearance. A loss of cilia has not been clearly established. The first sign of an oestrogenic influence, such as an increasing number of ribosomes and RER profiles can be observed 24 hours after a single injection of 75 mcg ODB, but immature granules do not appear in the cells until the third day after treatment. In normal cycling ewes, however, immature granules and release can be observed on D-O (first day of heat) and on D-1, i.e., 1-2 days after the peak level of oestradiol-17 beta. Some possible explanations for this difference in time are discussed. On the fourth and fifth day after ODB treatment, a similar ultrastructural organization of the secretory cells has developed as on D-2 and D-3 of the oestrous cycle. A combined treatment of ODB and progesterone resulted in a distinctly lesser secretory activity than with ODB only. An extended ODB treatment, consisting of three daily injections of 8.25 mcg ODB during 5-9 successive days, did not prevent release, The probability that a declining oestrogen level serves as the trigger for the onset thereof, must be excluded. It is concluded, that progesterone is probably not involved in creating the optimal environment in the ampullae tubae, which seems to be essential to a satisfactory completion of processes like survival of the gametes, fertilization and early embryonic development. Progesterone might be able to shorten the response of the epithelium to oestrogen, so that the optimal intratubal environmental conditions are attained at the proper time after ovulation.

Animals↗

PubMed1984

Fine structure of dividing astroglia and oligodendroglia during myelin formation in the developing mouse spinal cord.

To study the morphology and cellular relationships of dividing glial cells during myelin formation, were perfused newborn and 5-day mouse pups and embedded slices of cervical, thoracic, and lumbar cord for light and electron microscopic study. In semithin epon sections stained with toluidine blue, all levels of spinal cord at both ages contained mitotic glia in gray columns and funiculi. In electron micrographs of funiculi, dividing astroglia containing bundles of glial filaments, many glycogen granules, and had large processes extending into the surrounding neuropil. Cytoplasmic organelles of many immature interphase oligodendroglia and mitotic oligodendroblasts were similar and included microtubules, clusters of free ribosomes, and scattered profiles of granular endoplasmic reticulum. Unlike astroglia, dividing oligodendroblasts lacked large processes and in metaphase they were ellipsoids and had smooth plasma membranes. When these cells were studied in alternating serial thin and semithin sections over 10-15 micrometers distances, we did not identify connections between myelin sheaths and mitotic oligodendroblasts. Our findings indicate that oligodendroglia in developing white matter multiply before developing large processes. Our data also suggest that oligodendroglia do not divide while forming myelin.

Animals↗

PubMed1987

Ultrastructure of ependymal cells in primary cultures of cerebral cortex.

Ultrastructural features of ependymal cells growing in primary cultures of dissociated cerebral cortical tissue were investigated using electron microscopy. The ependyma exhibited a specific orientation in tissue culture such that the cell surface corresponding to the apical surface in situ was directed toward the culture medium. The membrane on the apical surface of cultured ependyma was characterized by the presence of cilia and microvilli. The lateral margins were marked by a variety of junctions: zonula occludens, zonula adherens, and membrane interdigitations. The cell cytoplasm contained pleomorphic mitochondria, Golgi profiles, clusters of ribosomes, rough endoplasmic reticulum, and particularly profuse scattered microfilaments and microtubules. These features support the identity of the cells as ependymal in the cultures and establish them as a relatively accurate reflection of ependyma in situ.

Animals↗