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Cerebral ischemia induces neuronal expression of novel VL30 mouse retrotransposons bound to polyribosomes.

Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S (VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species (BVL-1-like and mVL30-1-like) were bound to polyribosomes and induced by ischemia. These VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c-fos. Expression of VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3'-RACE-PCR experiments yielded two unique sequences (VL30x-1 and VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of VL30 promoter sequence (U3 region) resulted in the identification of two large VL30 subgroups. VL30x-1 and VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes BVL-1 and mVL30-1. The promoter regions of VL30x-1 and VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.

Animals↗

Translation is required to remove Y14 from mRNAs in the cytoplasm.

BACKGROUND: Y14 is an RNA binding protein which is part of a multiprotein complex, the exon-exon junction complex (EJC), that assembles on the exon-exon junctions of mRNAs produced by splicing. The position-specific binding of Y14 persists on mRNAs after their export to the cytoplasm. Thus, Y14, together with its interacting proteins, has the capacity to communicate to the cytoplasm the processing history of the mRNA, including the position of the removed introns, information that is likely to be important for defining premature termination codons. How Y14 and other components of the EJC are removed from mRNAs into the cytoplasm has not been determined. RESULTS: We show that Y14 but not another EJC component, Aly/REF, is present in polysome profile fractions containing one ribosome per mRNA. Using reporter constructs in an in vitro splicing/translation-coupled system, we show that Y14 remains associated with untranslated mRNAs but is removed from translationally active mRNAs. Importantly, mRNAs whose translation in vivo is prevented by the presence of strong secondary 5' UTR structure retain Y14 in the cytoplasm. CONCLUSIONS: These findings indicate that Y14 remains associated with mRNAs in the cytoplasm until they are translated, and translation is required to remove Y14 from mRNAs. Thus, the process of translation removes the splicing-dependent EJC protein imprints, which most likely function in the surveillance of mRNAs to define premature termination codons and possibly also in modulating the translation activity of cytoplasmic mRNAs.

Cytoplasm↗

Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis.

A PCR-based DNA amplification method was applied to genetically distinguish the popular dietary supplement Hypericum perforatum L. (common St. John's Wort) from other related Hypericum species. Nuclear ribosomal gene sequences of the internal transcribed spacer (ITS) region were analyzed for 50 Hypericum taxa native to the Old and New Worlds, representing 11 of the 36 currently accepted taxonomic sections. This study provides a genetic method for authentication of commercial H. perforatum preparations. In addition, these data allow a preliminary assessment of phylogenetic relationships within the genus, revealing three strongly supported monophyletic clades, plus several secondary monophyletic groupings. Using ITS gene sequences, we were able to distinguish H. perforatum from all other species of Hypericum included in this study.

DNA, Plant↗

Ultrastructure of axonal reaction in red nucleus of cat.

Described here are ultrastructural changes in neurons of feline red nucleus exhibiting axon reaction after unilateral rubropsinal tratotomy at the C-2 level and surviving 2 to 65 days. Ultrastructural alterations included neurofilamentous hyperplasia; proliferation of smooth ER; temporary disappearance of organized granular ER with partial substitution by haphazardly arranged, broad cisternal profiles; loss of rosette ribosomes and occurrence of single ribonucleoprotein granules or an intercisternal amorphous density; increased numbers of subsurface cisterns and allied structures, often disposed in stacks; vesiculation and vacuolation of Golgi cisternae; prevalence of autophagic bodies derived in part from Golgi complexes; probable mitochondrial hyperplasia and various qualitative changes in these organelles; an increase in lipofuscin. Dendritic changes paralleled those of perikarya save that proliferation of subsurface cisterns and autophagic bodies was absent. Abnormalities of myelinated axons and boutons occurred and may have originated from retrograde degeneration of cortical neurons induced by lateral funiculotomy. Some perikarya were devoid of axosomatic boutons. Ultrastructural changes varied with the length of postoperative survival and were, at least partly, reversible. Chromatolysis was detectable light microscopically before ultrastructural abnormality appeared. The bearing of transneuronal mechanisms on axon reaction of central neurons and the protective effect of section of axons beyond the site of origin of collaterals are discussed.

Animals↗

Temperature characteristics and adaptive potential of wheat ribosomes.

The translational efficiency of wheat ribosomes was studied as a function of an in vivo temperature pretreatment of wheat seedlings (Triticum aestivum L.). Ribosomes were isolated from heat-pretreated (36 degrees C) and reference (4 degrees C, 20 degrees C) wheat seedlings. The efficiency of the ribosomes in translating polyuridylic acid was assayed. Ribosomes from heat-pretreated seedlings exhibit a threefold enhanced incorporation rate of phenylalanine as compared to ribosomes from wheat seedlings adapted to 20 or 4 degrees C. This difference develops within 24 hours after onset of the heat treatment of seedlings following a 3 hour lag phase. The temperature induced changes can be traced back to the cytoplasmic ribosomes, since cycloheximide inhibits translation almost completely. Thermal inactivation of ribosomes occurs at 45 degrees C, irrespective of the temperature pretreatment of the wheat seedlings. Specific differences in the yield of ribosomes, in the polyribosomal profiles, and in the apparent Arrhenius' activation energy of protein synthesis were observed depending on the age and the temperature pretreatments. The results presented here are considered an important molecular correlation to phenotypical temperature adaptation of in vivo protein synthesis in wheat (M Weidner, C Mathée, FK Schmitz 1982 Plant Physiol 69: 1281-1288).

Journal Article↗

A Japanese case of tinea corporis caused by Arthroderma benhamiae.

We report a case of tinea corporis in Japan caused by Arthroderma benhamiae. A 53-year-old female scientist, who had been working on dermatophytes in a laboratory, noticed pruritic erythema on the outer corner of her left lower eyelid. She used a steroid ointment for three days, but the lesion continued to expand. When she visited our clinic, the erythema was 15 mm in diameter and clearly demarcated with a slightly depressed center. A scale from the periphery of the erythema was positive with direct KOH examination, and T mentagrophytes was isolated from the lesion. The erythema was successfully treated with topical application of butenafine hydrochloride. The isolate was mated with a (+) strain of the Americano-European race of A. benhamiae. Using the most sensitive molecular typing method, restriction exzyme analysis of the non-transcribed spacer region of the ribosomal DNA, the restriction profile of the isolate was the same as that of strains used in her laboratory but different from those of any Japanese isolates associated with pet animals. The results suggest that the patient became infected during her experiment.

Arthrodermataceae↗

Effect of sheep urine deposition on the bacterial community structure in an acidic upland grassland soil.

The effect of the addition of synthetic sheep urine (SSU) and plant species on the bacterial community composition of upland acidic grasslands was studied using a microcosm approach. Low, medium, and high concentrations of SSU were applied to pots containing plant species typical of both unimproved (Agrostis capillaris) and agriculturally improved (Lolium perenne) grasslands, and harvests were carried out 10 days and 50 days after the addition of SSU. SSU application significantly increased both soil pH (P < 0.005), with pH values ranging from pH 5.4 (zero SSU) to pH 6.4 (high SSU), and microbial activity (P < 0.005), with treatment with medium and high levels of SSU displaying significantly higher microbial activity (triphenylformazan dehydrogenase activity) than treatment of soil with zero or low concentrations of SSU. Microbial biomass, however, was not significantly altered by any of the SSU applications. Plant species alone had no effect on microbial biomass or activity. Bacterial community structure was profiled using bacterial automated ribosomal intergenic spacer analysis. Multidimensional scaling plots indicated that applications of high concentrations of SSU significantly altered the bacterial community composition in the presence of plant species but at different times: 10 days after application of high concentrations of SSU, the bacterial community composition of L. perenne-planted soils differed significantly from those of any other soils, whereas in the case of A. capillaris-planted soils, the bacterial community composition was different 50 days after treatment with high concentrations of SSU. Canonical correspondence analysis also highlighted the importance of interactions between SSU addition, plant species, and time in the bacterial community structure. This study has shown that the response of plants and bacterial communities to sheep urine deposition in grasslands is dependent on both the grass species present and the concentration of SSU applied, which may have important ecological consequences for agricultural grasslands.

Agrostis↗

Sequencing bands of ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking.

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to beta-proteobacteria (Ralstonia-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.

Adaptation, Physiological↗

[Ultrastructural of the neurons and synaptic architectonics of the central nucleus of the inferior colliculi in the cat brain].

In the central nucleus of the cat's brain inferior colliculus, three classes of neurons were observed. 1. Large relay neurons were distinguished by the abundance of cytoplasmic organelles. A considerable part of their perikaryon was covered by presynaptic terminals. 2. Medium sized lucent neurons having small amount of organelles. 3. Dark cells of rare occurrence in the central nucleus, which according to the number of axo-somatic synapses may be placed between type I and II neurons. In the central nucleus four type of profiles containing synaptic vesicles were distinguished. Large polymorphic endings of specific afferent fibers (type I) and small neurons (type II) contained round synaptic vesicles, while type III irregular axon terminals contained flattened and polymorphic ones. Type I, II and III terminals always appeared to be presynaptic components of synapses. Type IV terminals were found to be rare and except the polymorphic synaptic vesicles contained profiles of endoplasmic reticulum, ribosomes, microtubules, that enables them to be attributed to dendrites. Type IV terminals may be either presynaptic, or postsynaptic components of synapses.

Animals↗

Cyclosporin-induced gingival hyperplasia: an ultrastructural study of the oral epithelial prickle cells.

In this study the ultrastructural characteristics of the prickle cells of the human gingival epithelium after cyclosporin-A treatment are described. The prickle cell layer shows hyperplasia, along with the well known hyperplasia of the connective tissue components. The prickle cells show large numbers of free ribosomes, rough endoplasmic reticulum profiles and well developed Golgi complexes. The intercellular space is usually large and the neighboring cells are joined by desmosomes. In many cases the intercellular space is almost diminished and the cells are joined by a lot of desmosomes. Occasionally, a single cilium was observed to project into the intercellular space but the functional significance of this observation is unknown. The tonofilaments exhibit a specific arrangement in the cytoplasm of the prickle cells and it is believed that they contribute to the formation of a 'tonofilament network' which, along with the desmosomes, provide strength to the epithelium. Possible mechanisms regarding the gingiva overgrowth are discussed.

Actin Cytoskeleton↗

Studies on species specificity of proteins in Ascaris lumbricoides and Ascaris suum.

The separate identity of A. lumbricoides and A. suum was confirmed by means of the disc electrophoresis method in that differences were established in the protein profile of somatic and ribosomal proteins. In addition differences were found in the representation of lipoproteins, glycoproteins, and in the activity of LDH, SDH, peroxidase, esterase and alkalic phosphatase.

Animals↗

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics.

Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.

Anti-Bacterial Agents↗

Ribosomal complexes from an extremely halophilic bacterium and the role of cations.

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.

Ammonium Chloride↗

Lysosomal degradation of cell organelles. II. Ultrastructural analysis of uptake and digestion of intravenously injected microsomes and ribosomes by Kupffer cells.

Rough and smooth microsomes, "mixed" or total microsomes, and ribosomes were isolated from one single rat liver and subsequently injected intravenously into a series of inbred rats. The uptake and the degradation of the injected organelles by Kupffer cells were followed by means of electron microscopic analysis. By 1 minute after injection, microsomes were seen attached to the surface of Kupffer cells separated by a gap of 200 to 300 A. No attachment to hepatocytes, fat-storing cells, or endothelial cells was seen. By 5 and 10 minutes, most microsomes were phagocytosed and sequestered in large numbers within single membrane-enclosed vacuoles or phagosomes. The engulfment proceeded by two mechanisms: (1) most frequently, flaplike processes of cytoplasm embraced aggregates of microsomes, concomitant with the formation of indention of the cytoplasm; (2) occasionally, single microsomal profiles were taken up by bristle-coated endocytic vacuoles. Ribosomes were also seen penetrating into the wormlike structures (micropinocytosis vermiformis) at the cell surface. At 30 minutes after injection, clear signs of alteration were noted starting with vesicle aggregation, clumping, and elongation of the microsomal profiles. The ribosomes were quickly stripped from their microsomal membranes and marginated to the inside of the vacuoles but separated from the limiting membrane by a distance of 200 to 300 A. By 1 and 2 hours, disruption of the vesicles into membrane fragments and formation of dense material in and between the profiles occurred. By 8 hours it was difficult to recognize the degradation products as membrane derivatives. The digestive vacuoles retained their size at this time interval. Typical pentalaminar structures were observed. By 14 to 24 hours the digestive vacuoles became electron lucent and appeared to shrink, and in addition to containing various types of granular material, many were laden with lipid-like droplets presumed to be conglomerates of phospholipid remnants. Rough microsomes, when compared to smooth microsomes, gave rise to more granular material within the digestive vacuoles. Ribosomes were still identifiable 24 hours after injection, indicative of a somewhat slower rate of degradation. Accumulation of various types of lipid-like droplets in the "residual bodies" was typical after microsomal injections. It is concluded that although microsomes appear to be phagocytosed at a quicker rate than mitochondria, they are digested within the lysosomal apparatus of the Kupffer cells at a somewhat slower rate. This especially seems to be the case for ribosomes. Heterophagy of microsomes is one source of residual bodies.

Animals↗

Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species with ambiguous biochemical profile from a renal transplant recipient.

Traditional ways of identification of bacteria by phenotypic characteristics cannot be used for non-cultivable organisms and organisms with unusual biochemical profiles. In this study, an Enterobacteriaceae was isolated in pure growth from the mid-stream urine of a 67-year old renal transplant recipient with urinary tract infection. Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The Vitek system (GNI+) showed that it was 18% Leclercia adecarboxylata and 55% Klebsiella ozaenae; whereas the API system (20E) showed that it was 99.8% Rahnella aquatilis. 16S ribosomal RNA gene sequencing showed that there was 7 base differences between the isolate and Enterobacter cloacae, 18 base differences between the isolate and Enterobacter asburiae, 17 base differences between the isolate and Enterobacter cancerogenus, 35 base differences between the isolate and K. ozaenae, 27 base differences between the isolate and L. adecarboxylata, and 72 base differences between the isolate and R. aquatilis, indicating that the isolate most closely resembled a strain of E. cloacae. Identification of the organism in this study is important, as the choice of antibiotics would be radically different. In this case, cephalosporins should be avoided regardless of in-vitro susceptibility as cephalosporins are well-known to select for AmpC derepressed mutants in Enterobacter, and previous administration of third-generation cephalosporins is more likely to be associated with multidrug resistant Enterobacter isolates than is administration of antibiotics that do not include a third-generation cephalosporin.

Aged↗

Comparision of the structure and function of polysomal and helical ribosomes from Entamoeba invadens.

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis. Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromoatoid body) of cysts diappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments of mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.

Adenosine Triphosphate↗

Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling.

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the alpha subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.

Alcohol Oxidoreductases↗

Structural variation in the 16S-23S rRNA intergenic spacers of Vibrio parahaemolyticus.

The 16S-23S rRNA intergenic spacers (IGS) of Vibrio parahaemolyticus were PCR-amplified and cloned with pT7Blue T vector. A total of six clones were isolated dependent on size difference. The clones were different with respect to both the number and the composition of the tRNA genes included, and were designated IGS-0, IGS-E, IGS-IA, IGS-AE, IGS-EKV and IGS-EKAV. IGS-EKAV included the cluster of tRNA(Glu)-tRNA(Lys)-tRNA(Ala)-tRNA(Val); IGS-EKV, tRNA(Glu)-tRNA(Lys)-tRNA(Val); IGS-AE, tRNA(Ala)-tRNA(Glu); IGS-IA, tRNA(Ile)-tRNA(Ala); and IGS-E, tRNA(Glu). IGS-0 had no tRNA gene. Some similarities were found in the nucleotide sequence of the non-coding regions flanked by the tRNA genes. The structure difference found in the spacers is meaningful for elucidating the evolutionary line of each ribosomal RNA operon and the profile is applicable as a molecular marker of the bacterium.

Cloning, Molecular↗