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Results for “Ribosome Profiling”

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PubMed2002

New targets for antivirals: the ribosomal A-site and the factors that interact with it.

Many viruses use programmed -1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins. Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation. Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited -1 frameshifting and the ability of yeast cells to propagate the L-A and M(1) dsRNA viruses (J. D. Dinman, M. J. Ruiz-Echevarria, K. Czaplinski, and S. W. Peltz, 1997, Proc. Natl. Acad. Sci. USA 94, 6606-6611). Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R. E. Schwartz, J. Liesch, O. Hensens, L. Zitano, S. Honeycutt, G. Garrity, R. A. Fromtling, J. Onishi, and R. Monaghan, 1988. J. Antibiot. (Tokyo) 41, 1774--1779), also inhibits -1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin. Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome. Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed -1 ribosomal frameshifting efficiencies and killer virus phenotypes. Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor. These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds.

Anisomycin↗

PubMed1979

Stromal sarcoma of breast: a light and electron microscopic study.

A case of stromal sarcoma of the breast was studied by light and electron microscopy. The tumor comprised of uniform, plump spindle cells with neither epithelial nor "metaplastic" mesenchymal elements. Ultrastructurally most of the tumor cells appeared immature containing abundant free ribosomes, variable amounts of microfilaments and profiles of rough endoplasmic reticulum, with scant intercellular collagen and elastic fibers. The cells often showed prominent nesting as well as a variety of cell junctions, including desmosomes. We believe this tumor is a poorly differentiated, precursor mesenchymal sarcoma which belongs in the category of "stromal sarcoma of breast," a term coined by Berg et al. in 1962. To our knowledge, this represents the first published electron microscopic study on this tumor. Despite a high mitotic figure count in the tumor, the patient has survived a simple mastectomy with no evidence of metastasis 30 months later.

Breast Neoplasms↗

PubMed2003

Standard and Swedish variant types of the hybrid alder Phytophthora attacking alder in Hungary.

A new Phytophthora disease of common alder (Alnus glutinosa) similar to that previously reported in several countries in Europe has been observed in Hungary. Based on these earlier studies, the alder Phytophthora was considered likely to be a hybrid between P cambivora and a P fragariae-like species: across Europe a range of new alder Phytophthora is spreading that comprise a range of heteroploid hybrids including a 'standard' hybrid type and several other hybrid types termed 'variants'. Phenotypic and molecular features of the pathogen in Hungary were characterised and compared with isolates from elsewhere. The morphologies of five isolates from one region (Hévíz) resembled the common, 'standard' type, whereas the three isolates from another region (Hanság) exhibited traits similar to those of one of the 'variant' types, ie the Swedish 'variant'. Molecular markers of these two groups of Hungarian isolates also represented a good fit to those of the standard type and the Swedish variant, respectively. Isozyme patterns and profiles of restriction fragments of the entire internal transcribed spacer (ITS) region or mitochondrial DNAs and of RAPD-PCR products did not differ within a group, but distinct polymorphisms were exhibited between the two groups of isolates. Southern analysis of random amplified polymorphic DNA (RAPD) revealed the homologous nature of co-migrating bands of P cambivora and the isolates of alder Phytophthora. Furthermore, restriction fragment profiles of the ITS region of ribosomal DNAs and the mtDNAs were consistent with reported biparental origin of alder Phytophthora. The hybrid status of these continuously evolving pathogens raises many issues and challenges concerning efficient control measures.

Alnus↗

PubMed1978

Ultrastructure of gastric leiomyosarcoma.

Samples of gastric leiomyosarcomas from two male patients (69 years and 57 years of age) were studied by electron microscopy. The tumour cells contained abundant 50--90 A thick microfilaments and microtubules. Another cell type, regarded as immature leiomyosarcoma cell, contained numerous profiles of granular endoplasmic reticulum, free ribosomes and a well developed Golgi apparatus. There were intercellular junctions of zonula adherens-type between adjacent tumour cells. It was concluded that electron microscopy offers a valuable aid in the diagnosis of gastric neoplasms of smooth muscle origin.

Aged↗

PubMed1987

Establishment of a new human thyroid medullary carcinoma cell line. Morphological studies.

A new human medullary carcinoma cell line has been established from a thyroid tumor removed from a 76-year-old female patient. The cultured cells grew in suspension, formed round islands and did not attach to the plastic dish. The doubling time of 48 h is the shortest recorded for C-cell lines. Ultrastructural studies disclosed that the cells had a few short profiles of rough endoplasmic reticulum, numerous ribosomes and polyribosomes, a poorly developed Golgi apparatus and small secretory granules (75 nm in mean diameter). Immunohistochemical staining for somatostatin was positive. These results show that, compared with previously established C-cell lines, this cell line has a rapid growth rate, is morphologically less differentiated, but retains a hormone production potential.

Carcinoma↗

PubMed1983

Morphological observations on the parathyroid of the opossum (Didelphis virginiana).

Parathyroid III of the opossum, Didelphis virginiana, lies on the medial surface of the internal carotid artery at its origin just above the bifurcation of the common carotid artery. It is limited externally by a capsule and is subdivided into lobule-like structures by connective tissue septae. The parenchyma consists solely of chief (principal) cells, arranged in irregular cords and clumps supported by a delicate vascular connective tissue. Ultrastructurally, the chief cells are polyhedral in shape and have centrally placed nuclei. The cytoplasm contains small, randomly distributed, rod-shaped mitochondria, scattered profiles of granular endoplasmic reticulum, free ribosomes, and small perinuclear Golgi complexes. Secretory granules are small, electron-dense, and appear to be limited by a delicate membrane. The granules are related both to the Golgi complex and the basal cell membrane. Capillaries that lie immediately adjacent to the chief cells show an extensively fenestrated endothelium.

Animals↗

PubMedDate not supplied

Competitive exclusion of campylobacters from poultry with K-bacteria and Broilact.

The competitive exclusion (CE) product (Broilact) which is effective against Salmonellas, was found to be inactive against campylobacters. Microecological concepts were applied in the search of a new competitive flora and two novel strains ('K-bacteria') were isolated. These strains resembled campylobacters but differed from them in morphology, enzyme profiles (API), cellular fatty acid profiles and when tested with a ribosomal RNA hybridization probe (Gene-Trak). Two-week laboratory trials on broiler chickens showed that CE treatment may protect the birds against campylobacters but revealed the need for facultatively anaerobic bacteria in establishing a protective flora. A 5-week pilot scale trial was carried out. The trial involved 1800 newly hatched chicks in 30 groups. K-bacteria and Broilact, which provided the necessary facultatively anaerobic bacteria, were administered to some of the birds in the first drinking water. A seeder bird technique was used to challenge experimental and control birds with Campylobacter jejuni biotype 2 (broiler origin). Three seeder birds were placed in each group of 60 birds. Groups were sampled weekly for campylobacters and finally at the slaughterhouse. From each group, the caecal contents of two birds were examined quantitatively for campylobacters. The performance of the birds was also monitored during the trial. The results showed a 1.5 week delay in the onset of campylobacter infection in treated chicks and a consistently lower level of colonization in comparison with control birds. At slaughter, levels of carriage in caecal contents of treated birds were 1.5-2.0 log10 units lower than those of controls, despite apparent stress from harvesting and transportation. The treatment had no economically important effects on the performance of the birds during rearing.

Animals↗

PubMed1993

Persistent degenerative state of non-pyramidal neurons in the CA1 region of the gerbil hippocampus following transient forebrain ischemia.

Morphological changes in the neurons of the gerbil hippocampus following 5 min of forebrain ischemia were examined using light and electron microscopy. Although non-pyramidal neurons in the CA1 region of the hippocampus survived through the full length of the observation period, up to six weeks after ischemia, they consistently demonstrated degenerative changes distinct from those of the well-known "delayed neuronal death" of CA1 pyramidal cells. When examined with the light microscope, CA1 non-pyramidal neurons were found to be shrunken and their nuclei and cytoplasm were hyperchromatic between seven days and six weeks after ischemia. When examined with the electron microscope, postischemic non-pyramidal neurons were found to have markedly electron-dense profiles; their cytoplasm contained numerous free ribosomes and heterogeneous smaller granular substances, the latter also filling the nuclei. However, there was no loss of ribosomes from the rough endoplasmic reticulum, and mitochondrial cristae were preserved, suggesting that these neurons were viable. CA1 non-pyramidal neurons were studied immunohistochemically using three types of monoclonal antibodies, one each against parvalbumin, a nonphosphorylated epitope on the 168,000 mol. wt and 200,000 mol. wt subunits of neurofilament proteins, and microtubule-associated protein 2. CA1 non-pyramidal neurons lost immunoreactivity to these neuron-specific substances six weeks after ischemia, suggesting that these degenerating cells lacked certain types of normal neuronal activity. We conclude that non-pyramidal neurons in the hippocampal CA1 region survive transient ischemia but undergo degenerative changes following complete loss of CA1 pyramidal cells. These changes may be due to depletion of presumptive target-derived trophic factors within the non-pyramidal neurons.

Animals↗

PubMed2004

Ocurrence of the antibiotic producing bacterium Burkholderia sp. in colonies of the leaf-cutting ant Atta sexdens rubropilosa.

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.

Animals↗

PubMed1997

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Animals↗

PubMedDate not supplied

Isolation and characterisation of a new antagonistic Burkholderia strain from the rhizosphere of healthy tomato plants.

A new Burkholderia strain (PVFi5A) which exhibits antagonism towards many bacterial and fungal plant pathogens has been partially characterised. This strain was isolated from the rhizosphere of tomato plants and was referred to the Burkholderia cepacia complex on the basis of cultural, morphological and biochemical characteristics, including determination of the 16S ribosomal DNA sequence and fatty acid profile. Strain PVFi5A is a Gram-negative, aerobic bacterium, oxidase- and catalase-positive, motile with a polar tuft of flagella, able to grow on a variety of media without producing diffusible pigments; it is avirulent to onion, able to grow at 41 degrees C and resistant to several antibiotic substances. Its fatty acid profile contains the hydroxy acids 18:1 20H, 14:0 3OH and 16:0 3OH, but not the hydroxy acids 16:0 2OH. The antagonistic activity of strain PVFi5A is due to its production of various, as yet unidentified, antimicrobial compounds, one or more of which may differ from those reported previously for certain 'B. cepacia' strains. The ability of PVFi5A to suppress the growth of important bacterial and fungal phytopathogens makes this strain a potential biocontrol agent.

Anti-Bacterial Agents↗

PubMed2004

Twelve C2H2 zinc-finger genes on human chromosome 19 can be each translated into the same type of protein after frameshifts.

We report a discovery that, of the 226 C2H2 zinc-finger (C2H2-ZNF) genes on human chromosome 19, 12 genes each have two open reading frames (ORFs) that are in different reading frames but that can be translated into the same type of C2H2-ZNF proteins. We came to this observation after using standard tools in an original manner. First, we found that the both ORFs of such a gene contained the same type of significant C2H2-ZNF domain with e-values of e-2 or better. Second, the both ORFs had a promoter, a transcription start site, a start codon, a Kozak pattern and a poly(A) site; hence, each of them can be viewed as a gene in terms of a gene's primary structure. Third, both the ORFs matched not only human C2H2-ZNF expressed sequence tags (ESTs) but also human C2H2-ZNF proteins with e-values of e-50 or better. This indicates that the both ORFs can be transcribed and translated into the same zinc-finger proteins. More importantly, we observed that the phenomenon-a DNA can be translated into the same type of proteins after a frameshift-also occurred in a set of 160 human C2H2-ZNF ESTs and in a set of nine cDNAs of human C2H2-ZNF proteins. This observation based on the two sets of wet-experimental data much strengthened our confidence on the discovery. Our discovery is useful in the deeper understanding of a gene's regulatory mechanism to maintain its function.

Base Sequence↗

PubMed1975

Protein synthesis in liver, muscle, and brain of rats fed a high tyrosine-low protein diet.

Effects of feeding rats a high tyrosine-low protein diet on protein synthesis in liver, muscle, and brain were investigated both in vitro and in vivo. Tissue tyrosine concentrations of rats consuming the high tyrosine diet for 7 days were substantially elevated; this was accompanied by severe growth retardation. In the livers of rats fed the high tyrosine diet for 6 days, polysomal profiles showed a shift toward heavier ribosomal aggregates, while in muscle and brain, extensive disaggregation of polysomes occurred. In an in vitro amino acid incorporating system, the activities of both the microsomal and pH 5 enzyme fractions isolated from the livers of the high tyrosine animals that had been fed the diet for 6 weeks were elevated. On the other hand, the capacity of muscle or brain ribosomal preparations to incorporate [14-C] leucine was much reduced. Similar results were obtained in a study of [14-C] leucine incorporation in vivo in which rats were force-fed two meals and killed at various times after the last feeding. In rats fed the high tyrosine diet, incorporation of leucine into liver increased progressively; this was accompanied by a gradual decrease in leucine incorporation into muscle. In contrast, leucine incorporation into brain was immediately suppressed. In view of the apparently paradoxical effect of a high tyrosine load on protein synthesis in the liver, rates of the anabolic and catabolic phases of protein turnover in animals fed a high tyrosine diet were determined from radioactivity measurements made after pulse labeling them with [14-C] bicarbonate. Results indicated that the rates of both synthesis and degradation of liver proteins were elevated over control values. Differences in the effects of a toxic load of tyrosine on protein synthesis in the tissues examined could be the consequence of altered metabolic or hormonal balance as a result of nutritional stress.

Amino Acids↗

PubMed2007

Role of Pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (Gossypium hirsutum) grown in the field.

AIMS: To investigate the aetiology of seed and boll rot of cotton grown in South Carolina (SC). METHODS AND RESULTS: Bacteria were isolated from diseased locules of cotton bolls collected in a field in SC, USA and tested for the ability to cause comparable disease symptoms in greenhouse grown cotton fruit. Spontaneously generated rifampicin-resistant (Rif(r)) mutants of the isolates were used in confirmatory pathogenicity tests. Resistance to the antibiotic was both stable and effective in differentiating between an inoculated Rif(r) strain, rifampicin-sensitive contaminants and/or endophytes. A series of inoculation methods was tested at various boll developmental stages and at different fruiting nodes on the plant. Field disease symptoms were reproduced by inoculating bolls at 2 weeks postanthesis with bacterial suspensions ranging from 10(3) to 10(6) CFU ml(-1). Pathogenic isolates were categorized as Pantoea agglomerans on the basis of phenotype testing, fatty acid profiling (similarity index = 0.94), and 16s ribosomal DNA sequence analysis (99% nucleotide identity). CONCLUSIONS: Pantoea agglomerans isolates from field-collected immature, diseased cotton caused comparable infection symptoms in greenhouse produced cotton fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: In 1999, significant yield losses in SC cotton resulted from a previously unobserved seed and boll rot that has since been reported in other southeastern states. This study demonstrated a role of P. agglomerans in producing opportunistic bacterial seed and boll rot of cotton.

Anti-Bacterial Agents↗

PubMed2005

Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field.

AIMS: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. METHODS AND RESULTS: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (> or =3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (> or =2) antibiotics than corresponding isolates from control plots. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.

Agriculture↗

PubMed2003

Highly resistant metabolically deficient dwarf mutant of Escherichia coli is the cause of a chronic urinary tract infection.

We present the case of a 68-year-old diabetic woman who has been suffering from chronic urinary tract infections, recurring over a period of at least 5 years, caused by a slowly growing metabolically deficient dwarf mutant (MDD) of Escherichia coli. This MDD strain was auxotrophic for histidine, was resistant to multiple antibiotics, and showed atypical growth behavior. Colonies were tiny on routine media but were able to revert to normal growth after extended incubation. This strain was identified as E. coli by 16S ribosomal DNA sequencing, and virulence factor profiles were determined by PCR. Seven MDD isolates collected over the 5-year period were grown from midstream urine to significant colony counts and shown to belong to the same clonal group by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. These MDDs were repeatedly misidentified by biochemical methods due to their slow growth and atypical colony morphology. This case highlights the importance of recognizing MDDs of Enterobacteriaceae in patients with chronic infections. To our knowledge this is the first report of an MDD of E. coli causing a chronic urinary tract infection.

Aged↗

PubMed1993

Restriction fragment length polymorphisms (RFLPs) of medicinal plants and crude drugs. I. RFLP probes allow clear identification of Duboisia interspecific hybrid genotypes in both fresh and dried tissues.

A simple and efficient method for identification of Duboisia leichhardtii F. Muell, D. myoporoides R. BR. and their interspecific hybrid was established by analyzing restriction fragment length polymorphism (RFLP) profiles using non-radioactive rice ribosomal DNA as a probe. This procedure was shown to be applicable to both fresh and dried samples, and also suitable for detection of somatic hybrid at the callus stage after protoplast fusion.

DNA↗

PubMed2002

Atomic force microscope-based dissection of human metaphase chromosomes and high resolutional imaging by carbon nanotube tip.

The present study was performed to introduce a novel chromosome dissection method employing atomic force microscopy (AFM) in a dynamic force mode for the chemical or molecular biological analysis of tiny chromosomal fragments. After AFM observation of human chromosomes prepared for light microscopy, a region of interest was dissected by increasing the loading force in a series of single-line scans of the target portion by controlling it with the amplitude reference of the tip in a dynamic force mode. The marker gene of the nucleolar organizing region (NOR) was amplified by our designed primers for 5.8S ribosomal DNA. After the dissection, topographic profiles in the section were then obtained with a carbon nanotube (CNT) probe in ambient condition. These results are discussed in relation to a fundamental technology for chromosomal analysis.

Chromosomes, Human↗