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Results for “Ribosome Profiling”

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PubMed2004

Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities.

Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.

Bacteria↗

PubMed2005

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination.

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum.

Animals↗

PubMed1970

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation.

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.

Amino Acids↗

PubMed2005

Diamond Blackfan anemia: A paradigm for a ribosome-based disease.

Diamond Blackfan anemia is characterized by a severe hypoplastic anemia and a heterogeneous collection of other clinical features. Approximately 25% of Diamond Blackfan anemia cases are associated with mutations in the gene encoding ribosomal protein S19. The hypothesis presented here ties together molecular and clinical features of the disease, and establishes a conceptual framework for understanding many of the unusual characteristics of a growing number of diseases linked to factors involved in ribosome synthesis. The hypothesis states that ribosomal proteins are expressed in amounts that differ relative to one another in a tissue-specific manner, and that haploinsufficiency for a particular protein may make that protein limiting for ribosome assembly in some tissues, while other tissues remain unaffected. Further, polymorphisms in factors controlling the expression of a particular ribosomal protein gene may alter its expression and expand or contract the number of tissues affected from individual to individual. Support for the hypothesis comes from the observation that promoters in ribosomal protein genes exhibit little conservation and transcription profiling indicates that the absolute amounts of mRNAs for individual ribosomal proteins can vary dramatically relative to one another. Balanced expression of ribosomal proteins is achieved post-translationally, where excess proteins not assembled into ribosomal subunits are often rapidly degraded. The number of ribosomes per cell is therefore determined by the factors that limit assembly. In principle, any essential ribosomal protein could become limiting for assembly if its level of expression falls below a critical threshold. Whether an inactivating mutation in ribosomal protein gene would affect protein synthetic capacity of a tissue would depend on the ratio of the ribosomal protein relative to other ribosomal proteins in that tissue. If the ratio were high, the tissue may not be affected as the level of functional protein may not fall to a point where it becomes limiting for subunit assembly. In contrast, if the ratio were low, an inactivating mutation could make the protein limiting for subunit assembly resulting in a clinical phenotype. Polymorphisms in the myriad of cis- and trans-acting factors, which govern the expression of ribosomal proteins in response to developmental and physiological signals, could act to increase or decrease ribosomal protein expression and thereby impact the profile and severity of clinical phenotypes. Therefore, these factors represent targets for the development of new therapies to treat Diamond Blackfan anemia and other ribosome based diseases.

Anemia, Diamond-Blackfan↗

PubMed1999

Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8. Generation of a mutant lacking ribosomal protein S17.

S17 is a primary rRNA-binding protein which has been implicated in ribosome assembly and translational fidelity. We describe the generation and biochemical characterization of an S17 minus ribosomal mutant, a ribosomal protein-lacking mutant obtained in Thermus thermophilus HB8. The S17 mutant was obtained by insertional inactivation of the target gene with the kanamycin adenyl transferase (kat) gene, making use of a Thermus-Escherichia shuttle vector and the natural ability of Thermus to transform. In the final construct used to transform Thermus cells, the S17 coding region was replaced with the kat gene cloned in-frame with the first three amino acids of S17. Hence, in vivo transcription of the kat gene was under the control of the ribosomal operon promoter. As in Escherichia coli, the Thermus S17 mutant exhibited a temperature-sensitive phenotype. Two-dimensional PAGE, Western blot, and ELISA confirmed the absence of S17 from the mutant ribosomes. Sucrose-gradient profiles of mutant cells showed a clear separation and normal proportions of 50S and 30S subunits and a normal ratio between them. In addition, the S17 mutant showed the presence of a 20S peak representing assembly-defective particles. The successful re-incorporation of protein S17 into the mutant ribosomes was demonstrated when reconstitution with isolated S17 was performed at 60 degrees C.

Anti-Bacterial Agents↗

PubMed1993

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.

Adenosine Triphosphatases↗

PubMed1987

Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves.

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q(B) polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.

Journal Article↗

PubMedDate not supplied

Population differentiation of the malaria vector Anopheles aquasalis using mitochondrial DNA.

Populations of the malaria vector Anopheles aquasalis from Venezuela, Trinidad, and Brazil were analyzed using restriction enzyme digestion of mitochondrial DNA (mtDNA). The five enzymes surveyed yielded 12 mtDNA haplotypes. Three restriction endonuclease profiles (Bcl I, Eco RI, and Hind III) differentiated the Venezuelan and Trinidadian A. aquasalis from two Brazilian populations. Estimates of mtDNA sequence divergence between the Venezuelan/Trinidadian populations and each Brazilian population (0.023-0.032), as well as divergence between the two Brazilian populations (0.030), were within the range of interspecific distances calculated for members of anopheline species complexes. Ribosomal DNA (rDNA) restriction profiles of the A. aquasalis populations examined were identical.

Animals↗

PubMed1983

Intermittent high altitude hypoxia - induced structural changes in the pulmonary myocardium in young mice.

Seven-day-old mice, strain H, were exposed to intermittent high altitude hypoxia (IHA) in a barochamber (7,000 m, 4 h/day, 5 days a week); a total number of exposures was 10. It has been shown that the layer of cardiac musculature in the adventitia of the pulmonary veins, the so-called pulmonary myocardium, reacts to IHA hypoxia by enlargement even sooner than the right ventricular myocardium. The average thickness of the layer of pulmonary myocardium was significantly greater in animals exposed to IHA hypoxia as compared with the controls. Furthermore, IHA hypoxia induces the extension of the pulmonary myocardium to the periphery of the pulmonary venous bed. Ultrastructural investigation of the pulmonary myocardium in hypoxic animals revealed the presence of unoriented myofilaments in the peripheral myofibril-free sarcoplasma, increase in the number of ribosomes and appearance of profiles of granular endoplasmic reticulum. Our data provide quantitative support for the hypothesis that it is not only the contraction of pulmonary arteries, but also venoconstriction which contribute to the hypoxic pressure response in mice.

Altitude↗

PubMed2003

Shifts in diversity and microscale distribution of the adapted bacterial phenotypes due to Hg(II) spiking in soil.

In a previous experiment [Ranjard et al. (2000) FEMS Microbiol Ecol 31:107-115], the spatial heterogeneity of a mercury impact on soil bacterial community was revealed by an increase of mercury-resistant (HgR) bacterial numbers in the outer fraction and the sand fractions when compared to those in the silt fractions. The objectives of the present study were (i) to investigate whether mercury exposure affects the diversity and the distribution within the various fractions of the HgR populations and (ii) to evaluate the contribution of the HgR populations to the overall community adaptation. A total of 236 strains isolated before (104 isolates) and 30 days (132 isolates) after spiking were characterized by an amplified ribosomal DNA restriction analysis (ARDRA) and grouped into 30 different genotypes. Whereas almost the same numbers of different genotypes were observed at both sampling times when considering all microenvironments, important changes in their evenness were observed. At the microscale level, we also noticed a heterogeneous distribution of the genotypes. Partial 16S rDNA sequences of each genotype were determined to permit phylogenetic affiliation. Whereas Pseudomonas-like species were dominant in all microenvironments at T = 0, genotypes not detected before spiking such as genotypes closely related to Xanthomonas, Pseudoaminobacter, and Sphingomonas-like species became dominant at T = 30. Similarly, several genotypes close to Bacillus, Streptomyces, and Rhodococcus species were only detected in the sand fractions at T = 0 and could not be detected in these fractions at T = 30. Probes defined within the intergenic spacer between the rrs and rrl genes were designed for the dominant genotypes and were hybridized toward the RISA (Ribosomal Intergenic Spacer Analysis) profiles derived from the T = 0 and T = 30 bacterial communities associated to the unfractionated soil. Results showed that the culturable dominant HgR genotypes partially contributed to the adaptation of the whole bacterial community.

Adaptation, Biological↗

PubMed2003

Spatial analysis of archaeal community structure in grassland soil.

The complex structure of soil and the heterogeneity of resources available to microorganisms have implications for sampling regimens when the structure and diversity of microbial communities are analyzed. To assess the heterogeneity in community structure, archaeal communities, which typically contain sequences belonging to the nonthermophilic Crenarchaeota, were examined at two contrasting spatial scales by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis followed by unweighted pair group method with arithmetic mean analysis of 16S rRNA- and ribosomal DNA-derived profiles. A macroscale analysis was carried out with soil cores taken at 2-m intervals along triplicate 8-m transects from both managed (improved) and natural (unimproved) grassland rhizosphere soils. A microscale analysis was carried out with a single soil core by assessing the effects of both sample size (10, 1, and 0.1 g) and distance between samples. The much reduced complexity of archaeal profiles compared to the complexity typical of the bacterial community facilitated visual comparison of profiles based on band presence and revealed different levels of heterogeneity between sets of samples. At the macroscale level, heterogeneity over the transect could not be related to grassland type. Substantial heterogeneity was observed across both improved and unimproved transects, except for one improved transect that exhibited substantial homogeneity, so that profiles for a single core were largely representative of the entire transect. At the smaller scale, the heterogeneity of the archaeal community structure varied with sample size within a single 8- by 8-cm core. The archaeal DGGE profiles for replicate 10-g soil samples were similar, while those for 1-g samples and 0.1-g samples showed greater heterogeneity. In addition, there was no relationship between the archaeal profiles and the distance between 1- or 0.1-g samples, although relationships between community structure and distance of separation may occur at a smaller scale. Our findings demonstrate the care required when workers attempt to obtain a representative picture of microbial community structure in the soil environment.

Crenarchaeota↗

PubMed2005

Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos.

This study was carried out to optimize conditions for using real time RT-PCR as an efficient and precise quantitative method for estimating the transcript levels of genes expressed in samples containing miniscule amounts of RNA, such as single mammalian oocytes and embryos. First, using mouse eggs and blastocysts, we tested three kinds of RNA isolation or collection methods: TRIZOL reagent, oligo-dT conjugated beads, or three freeze/thaw cycles with the reverse transcription buffer. There were no significant differences among three groups in mRNA quantity as assayed by real time RT-PCR analysis. Second, we compared the efficacy of real time analysis between TaqMan fluorescent probes and the SYBR-green dye system. The two systems presented similar real time RT-PCR profiles for the 16s ribosomal protein gene from oocytes to blastocysts. Third, RNA from mouse embryos at defined stages of preimplantation development were isolated and the levels of transcripts encoded by several housekeeping genes (GAPDH, beta-actin, ribosomal protein L7, 16s ribosomal protein, histone H2A.Z) were quantitatively analyzed by real time RT-PCR. The histone H2A.Z and 16s ribosomal protein slightly increased from the egg to blastocyst stages by approximately 10- and 30-fold, respectively. However, other transcripts increased more than 300-fold as a function of developmental stage from eggs to blastocysts. Our results suggest that the simple freezing/thawing method for RNA collection, the economic SYBR-green dye system, and histone H2A.Z gene as an internal control should be useful for the real time RT-PCR analysis of single mouse eggs and preimplantation embryos.

Animals↗

PubMed2005

Molecular assessment of inoculated and indigenous bacteria in biofilms from a pilot-scale perchlorate-reducing bioreactor.

Bioremediation of perchlorate-contaminated groundwater can occur via bacterial reduction of perchlorate to chloride. Although perchlorate reduction has been demonstrated in bacterial pure cultures, little is known about the efficacy of using perchlorate-reducing bacteria as inoculants for bioremediation in the field. A pilot-scale, fixed-bed bioreactor containing plastic support medium was used to treat perchlorate-contaminated groundwater at a site in Southern California. The bioreactor was inoculated with a field-grown suspension of the perchlorate-respiring bacterium Dechlorosoma sp. strain KJ and fed groundwater containing indigenous bacteria and a carbon source amendment. Because the reactor was flushed weekly to remove accumulated biomass, only bacteria capable of growing in biofilms in the reactor were expected to survive. After 26 days of operation, perchlorate was not detected in bioreactor effluent. Perchlorate remained undetected by ion chromatography (detection limit 4 mug L(-1)) during 6 months of operation, after which the reactor was drained. Plastic medium was subsampled from top, middle, and bottom locations of the reactor for shipment on blue ice and storage at -80 degrees C prior to analysis. Microbial community DNA was extracted from successive washes of thawed biofilm material for PCR-based community profiling by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences characteristic of strain KJ were recovered from any RISA bands. The most intense bands yielded DNA sequences with high similarities to Dechloromonas spp., a closely related but different genus of perchlorate-respiring bacteria. Additional sequences from RISA profiles indicated presence of representatives of the low G+C gram-positive bacteria and the Cytophaga-Flavobacterium-Bacteroides group. Confocal scanning laser microscopy and fluorescence in situ hybridization (FISH) were also used to examine biofilms using genus-specific 16S ribosomal RNA probes. FISH was more sensitive than RISA profiling in detecting possible survivors from the initial inoculum. FISH revealed that bacteria hybridizing to Dechlorosoma probes constituted <1% of all cells in the biofilms examined, except in the deepest portions where they represented 3-5%. Numbers of bacteria hybridizing to Dechloromonas probes decreased as biofilm depth increased, and they were most abundant at the biofilm surface (23% of all cells). These spatial distribution differences suggested persistence of low numbers of the inoculated strain Dechlorosoma sp. KJ in parts of the biofilm nearest to the plastic medium, concomitant with active colonization or growth by indigenous Dechloromonas spp. in the biofilm exterior. This study demonstrated the feasibility of post hoc analysis of frozen biofilms following completion of field remediation studies.

Bacteria↗

PubMed2006

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.

Bacteria↗

PubMed1997

Enhancement of resolution of low molecular weight RNA profiles by staircase electrophoresis.

Stable low molecular weight (LMW) RNA comprises molecules used in the taxonomy of microorganisms and in studies on the microbial diversity of populations. However, the use of electrophoretic techniques has been hampered due to the low resolution obtained with techniques used for the separation of this kind of molecule. In this work we develop an electrophoretic method (staircase electrophoresis) that increases the resolution of the technique. This improvement in the resolution adequately resolves the three zones that integrate the profiles of LMW RNA: ribosomic 5S RNA (5S rRNA), class 2 transfer RNA (tRNA), and class 1 transfer RNA, allowing the technique to be applied to taxonomic studies (diagnostic, the identification of the individuals), phylogenetic studies and studies on naturally occurring microbial populations.

Electrophoresis↗

PubMed1983

Cytological study of the cellular changes after transection of the proximal radix of the rat trigeminal ganglion.

Cytological changes following transection of the proximal root of the trigeminal ganglion in adult rats were assessed by light and electron microscopy. Radices were transected about 3-5 mm from the ganglia and animals were killed from 1 to 60 days after the operation. Light microscopically, it was found that all Nissl granules became uniformly stained and evenly distributed throughout the cytoplasm within 3 days. Three types of cell alteration involving Nissl granules occurred within 3 to 12 days after the operation: 1) chromatolysis, 2) dark staining of the cytoplasm accompanied by an increase of Nissl granules, and 3) faint staining of the cytoplasm accompanied by dispersion of Nissl granules. Electron microscopically, the chromatolysis pattern was characterized by peripheral concentration of the granular endoplasmic reticulum (gER) and ribosomes in the cytoplasm. Neurons of the dark-staining type showed an increased number of polysomal complexes throughout the cytoplasm, whereas those of the faint-staining type had diffusely dispersed cisternae of the gER which were shortened and bore reduced numbers of attached ribosomes. Perinuclear localization of profiles of Golgi complexes disappeared temporarily 1-3 days after the operation, but the normal perinuclear pattern appeared to return after 1 week. Enzyme histochemistry of acid phosphatase activity revealed an increase in the number of very fine reaction products in the cytoplasm up to 14 days following the operation. Cells recovered the normal pattern of Nissl staining by 48 days. Myelin figures, which are rarely observed in normal ganglia, were still observed in dense lysosomal bodies after 30 days. Nuclear size in affected neurons steadily increased up to about 2 weeks postoperation but returned to normal by 48 days.

Animals↗

PubMed1995

Hutchinson-Gilford progeria fibroblasts exhibit metabolically normal uridine uptake and RNA synthetic rates.

The accelerated aging disease Hutchinson-Gilford progeria syndrome displays altered messenger RNA levels in cultured fibroblasts, yet little is known of effects on transcription by RNA polymerases other than RNA polymerase II. Total RNA metabolism was examined by incubation of Hutchinson-Gilford progeria fibroblasts with [5-3H]uridine in asynchronous culture. Uptake of radiolabel was quantitative and was incorporated preferentially (99 +/- 0.3%) into newly synthesized RNA. Progeria and control cultures showed comparable rates of uptake of radiolabel, time courses of RNA synthesis, and relative intensity profiles of newly synthesized ribosomal RNA. These characteristics held over a greater than ten-fold range of cell densities (5 x 10(3) to 8 x 10(4) cells/cm2). Progeria and control fibroblasts thus have comparable metabolic capacities for uridine uptake and net RNA production, emphasizing the relative specificity of transcriptional changes previously identified in progeria cells.

Biological Transport↗

PubMed1989

Morphological studies on cerebral cortical lesions induced by transient ischemia in Mongolian gerbil--diffuse and peripheral pallor of the neuronal perikarya.

Unilateral transient cerebral ischemia was produced in Mongolian gerbils by clipping the left common carotid artery for 1 h. About 60% of the gerbils with neurological symptoms had post-ischemic seizures. The majority of those that had seizures died within a few days, and sections of their cerebral cortices contained many dark and shrunken neurons. However, the gerbils that did not have seizures survived without any severe complications. In the cerebral cortex of the latter, the neurons with diffuse or peripheral pallor of the perikarya were seen along with a small number of dark and shrunken neurons. Diffuse pallor occurred within a few hours following ischemia in layers III, V, and VI, and disappeared 1 or 2 days after recirculation. Electron microscopically, these neurons showed dispersion of ribosomes, simple and elongated profiles of rough endoplasmic reticulum (r-ER), clustered vacuoles, and mild to moderate mitochondrial swelling. Occasional net-like tubulomembranous structures, probably derived from r-ER, were observed. On the other hand, peripheral pallor became apparent after 5 days following ischemia, usually involving layer II first and gradually extending to the deeper layers. Concomitantly, the amount of neuropil decreased and the dendrites exhibited tortuosity and irregularity in layer II. Electron microscopically, these neurons showed marked swelling of peripheral perikarya and polyribosomes and organelles were located peripherally to the nuclei. In addition, numerous degenerated axon terminals and distended dendrites were observed around the neurons. These observations indicate that diffuse pallor represents damage directly induced by ischemia and subsequent recirculation, while peripheral pallor is the delayed and remote effect of ischemia, probably due to degeneration of neuronal processes.

Animals↗