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PubMed1997

Prenucleolar bodies contain coilin and are assembled in Xenopus egg extract depleted of specific nucleolar proteins and U3 RNA.

Nuclei assembled in Xenopus egg extract contain numerous spherical aggregations or nuclear bodies. Previously we have shown that they closely resemble prenucleolar bodies (PNBs), both at the compositional and ultrastructural level. Subsequently, coilin was also identified and for this reason they were called coiled bodies. Here we present morphological and immunocytochemical evidence that the in vitro nuclear bodies resemble authentic PNBs and are different from coiled bodies. In particular we show that coilin, previously considered as the defining protein constituent of coiled bodies, is also present in PNBs of cultured cells. In contrast, the PNB-associated nucleolar proteins nucleolin and B23/NO38 are not detectable in coiled bodies and may thus serve as suitable markers for PNBs. Our results suggest that PNBs are primary assembly structures which contribute to the formation of both nucleoli and coiled bodies and thus offer an explanation for the frequently observed structural association of coiled bodies with nucleoli. To gain some insight into the assembly process of PNBs in vitro, specific nucleolar proteins were removed from Xenopus egg extract. Quite surprisingly, the immuno-depleted extracts still promoted the assembly of nuclear bodies which lacked either fibrillarin, nucleolin, xNopp180 or B23/NO38. Only after fibrillarin-depletion fewer PNBs were seen as compared to controls. Digestion of the extract with RNase followed by northern blot analysis revealed that U3 small nucleolar RNA is not required for the formation and structural maintenance of PNBs in vitro.

Animals↗
PubMed2000

Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions.

Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. Here we show that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila melanogaster. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. Our results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity.

Animals↗
PubMed1989

Sequence, packing and nanometer scale structure in STM images of nucleic acids under water.

Scanning tunneling microscope (STM) images of random-sequence nucleic acid polymers under water show internal structure which depends strongly on the packing density of the polymer. Images of dense aggregates have a semicrystalline order with the individual polymers adopting simple periodic structures. Loose aggregates (or isolated molecules) show structural variability with considerable local bending and curving on a nanometer scale. It is not clear to what extent this structure is induced by the operation of the microscope. In order to investigate the possibility that the structure is sequence directed, we have imaged various DNA and RNA polymers at low packing densities. We present results here for random sequence DNA, poly(dAT).poly(dAT), poly(dA).poly(dT), poly(dCG).poly(dCG) and for random sequence RNA and poly(U). In contrast to loose aggregates of the random sequence material, the homopolymers show few sharp bends. Furthermore, the homopolymers appear to yield characteristic backbone patterns, usually at resolutions in excess of that obtained with random sequence polymers. The random sequence polymers show much more evidence of image distortion due to tip-molecule interactions, suggesting that they are, on average, mechanically less stable in the STM tunnel-gap than the homopolymers. Thus, while some of the structure observed in STM images is a consequence of tip-molecule interactions, it is related to sequence-directed properties of the polymer.

DNA↗
PubMed2002

The case for extending storage and secretion functions of human mast cell granules to include synthesis.

Ultrastructural studies using standard procedures have for years indicated close associations of ribosomes and secretory granules in human mast cells. These descriptive studies have informed new studies, using established and new ultrastructural methods based on different principles, designed to investigate the possible role of RNA metabolism in secretory granules of human mast cells. In aggregate, these studies indicate human mast cell secretory granule associations with ribosomes, the protein synthetic machine of cells, with ribosomal proteins, with RNA, with poly(A)-positive mRNA and with various long-lived, or short-lived, uridine-rich, and poly(A)-poor RNA species with key roles in RNA processing and splicing. These studies indicate that secretory-storage granules in human mast cells may also be synthetic granules.

Cytoplasmic Granules↗
PubMed1972

Cytochalasin B prevents specific sorting of reaggregating embryonic cells.

The effect of cytochalasin B on specific sorting during reaggregation of embryonic chick heart and neural retina cells was studied. At a dose that did not measurably affect uptake of precursors of protein and RNA synthesis, ratios of potassium to sodium ions, and nonspecific aggregation, cytochalasin B disrupted the formation of the characteristic pattern of islands of heart cells within a retinal continuum.

Amino Acids↗
PubMed1997

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (> or = 2 microM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na+ and Mg2+ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions.

Amino Acid Sequence↗
PubMed1988

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus.

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.

Capsid Proteins↗
PubMed1982

Cellular commitment for post-gastrular increase in alkaline phosphatase activity in Xenopus laevis development.

Xenopus embryos were dissociated into cells and cultured in Ca2+-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (3H) thymidine and (3H) leucin proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (3H) uridine was not impaired. Such as increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.

Alkaline Phosphatase↗
PubMed1981

Bacteriolysis of Veillonella alcalescens by lysozyme and inorganic anions present in saliva.

Veillonella alcalescens subsp. dispar was grown in a synthetic medium containing either radiolabeled thymidine or uridine to monitor cell lysis by assay of the release of deoxyribonucleic acid or ribonucleic acid (RNA), respectively. Biochemical analyses demonstrated that, although human or hen egg white lysozymes alone did not release deoxyribonucleic acid or RNA, the nucleic acids were liberated in equal amounts from lysozyme-treated cells by the addition of low concentrations of the sodium salts of HCO-3, SCN-, Cl-, and F-, RNA release was dependent on enzyme and anion concentration. Human lysozyme was more potent than hen egg white lysozyme, and bicarbonate was the most effective anion in promoting bacteriolysis. Surprisingly, ultrastructural analyses differed from biochemical results. Lysozyme alone caused lysis in approximately 40% of the cell population. Detailed ultrastructural examination revealed aggregated cytoplasmic components which appeared as small clumps, explaining why nucleic acids were not measurable in the biochemical assays. In reaction mixtures containing lysozyme plus inorganic salts, electron microscopy results were compatible with biochemical data. Ultrastructural studies demonstrated that the addition of inorganic salts to lysozyme-treated cells resulted in the solubilization of the protoplasmic aggregates of lysed cells, presumably freeing the complexed RNA, and in the rapid lysis of the remaining cells (approximately 60%). These data suggest that electron microscopy must be used in conjunction with biochemical assays to assess lytic damage of bacterial cells.

Anions↗
PubMed1985

Elongation in the major direction of tobacco mosaic virus assembly.

Butler and Lomonossoff [Butler, P. J. G. & Lomonossoff, G. P. (1978) J. Mol. Biol. 126, 877-882] claim that the elongation in the major direction (3'-->5') proceeds by incorporation of disk protein in tobacco mosaic virus (TMV) assembly. The strongest argument they have for this theory is the periodicity of 50 or 100 nucleotides that they observed in the banding pattern of the protected RNAs during the first few minutes of the assembly reaction. We repeated their experiment using TMV-OM (a common Japanese strain) disk protein and TMV-OM RNA. We observed a banding pattern similar to theirs, but we found the long protected RNA at 6 min to be from the 260-nm intermediate particle rather than from the full-length TMV RNA. We also carried out the assembly reaction between TMV-OM disk protein, as well as cucumber green mottle mosaic virus (CGMMV) protein, and three strains of TMV RNAs. During the course of each assembly reaction, we examined the banding patterns. We demonstrated that the banding pattern of the protected RNA differs depending on what kind of RNA is used, rather than on what kind of aggregational state the protein is in. Specifically, the similar banding pattern observed for CGMMV subunit protein was also observed for TMV disk protein in the assembly reaction with TMV (OM) RNA. We showed previously that the assembly reaction between CGMMV protein and TMV RNA proceeds by incorporation of CGMMV subunit protein. This strongly indicates that the banding pattern of the protected RNA does not arise from the stepwise addition of the 20S disk protein.

Journal Article↗
PubMed2005

[Correlation of GLC-82 lung carcinoma cell aggregation in suspension culture to the activation of protein kinases FAK, AKT, ERK, and SRC].

BACKGROUND & OBJECTIVE: Our previous study revealed that tyrosine kinase FAK can partly mediate the aggregation of tumor cells in suspension culture, therefore, suppress cell apoptosis and promote cell proliferation. However, the downstream pathway of FAK in the formation of cell aggregation is unclear. This study was to investigate the roles of FAK and its potential downstream molecules ERK, AKT, and SRC in mediating aggregation of lung adenocarcinoma GLC-82 cells in suspension culture. METHODS: Morphology of the aggregations of GLC-82 cells and normal lung HBE cells in polyHEMA suspension culture was observed. Cell apoptosis was studied by DNA electrophoresis on agarose gel. The ability of cell transformation was studied by colony formation assay. The phosphorylation of the protein kinases was investigated by Western blot. FAK was silenced by RNA interference. RESULTS: GLC-82 cells survived and aggregated in suspension culture; while apoptosis occurred in HBE cells which formed no aggregation in suspension culture. Phosphorylation levels of FAK, ERK, AKT, and SRC were related to GLC-82 cell aggregation. FAK silencing partly blocked the formation of GLC-82 cell aggregation, and decreased the phosphorylation levels of ERK and AKT; the colony formation rate in soft agar was significantly lower in GLC-82 cells with FAK silencing than in GLC-82 cells without FAK silencing [(12.2+/-1.1)% vs. (3.6+/-0.7)%, P=0.001]. CONCLUSION: The formation of GLC-82 cell aggregation is partly mediated by FAK signal pathway, and ERK and AKT are the downstream molecules of FAK.

Adenocarcinoma↗
PubMed1980

Studies on the influence of tryptophan and related compounds on hepatic polyribosomes and protein synthesis in the rat.

The effect of the administration of L-tryptophan or tryptophan-related compounds on rat liver RNA and protein metabolism was investigated. The five biochemical parameters studied were polyribosomal aggregation, protein synthesis in vitro, synthesis of cytoplasmic poly(A)-mRNA, release of labeled nuclear RNA in vitro (nuclear and cell sap effects) and nuclear envelope nucleoside triphosphatase activity. The administration of L-tryptophan to overnight fasted rats revealed a rapid stimulation in all of the five parameters. Also, some of the tryptophan-related compounds, especially 5-hydroxy-DL-tryptophan, 3-indolepyruvic acid, indole or 3-hydroxyanthranilic acid, revealed stimulation in many of the parameters. However, when rats were pretreated with puromycin to inhibit protein synthesis, only the administration of L-tryptophan was still able to stimulate significantly the hepatic polyribosomes, in vitro protein synthesis, in vitro nuclear RNA release (involving nuclei but not cell sap) and nuclear envelope nucleoside triphosphatase activity. The effects by the other tryptophan-related compounds became inhibited. The results emphasize the unique action of L-tryptophan on hepatic protein synthesis.

3-Hydroxyanthranilic Acid↗
PubMed1990

Assembly of Xenopus transcription factor III A-5S RNA complex.

The regulation of Xenopus 5S rRNA gene expression involves multiple protein factors, among which is transcription factor III A (TFIIIA). This factor can be isolated as a protein-RNA complex. The assembly behavior of this complex was studied by sedimentation velocity and gel electrophoresis at pH 7.5 and 23 degrees C. The reaction boundary was monitored by the absorbance at 260 nm; thus, the shape of the boundary reflects mainly RNA or RNA-containing complexes. Values for the weight-average sedimentation coefficient (S20,w) change with protein-RNA concentration. At low concentrations, values of S20,w increase with increasing concentration. The extrapolated value for S20,w at infinite dilution is 5.2 S, the same value for free Xenopus rRNA under the same experimentation conditions. Furthermore, the same value of S20,w at a specific RNA concentration can be obtained either by dilution of a concentrated sample or by concentrating a diluted one. These results indicate that complex formation can be described by a reversible process. When the data are analyzed by computer fitting, the simplest model that fits the sedimentation data is that TFIIIA and RNA form a 1:1 complex which self-aggregates to a dimer. The sedimentation coefficients (S020,w) of PR and (PR)2 are 7.5 and 10.6 S, respectively, where PR and (PR)2 are the 1:1 TFIIIA-RNA complex and its dimer, respectively. The protein-RNA interaction was also investigated by gel electrophoresis. The resolved components were identified by differential staining for protein and RNA on a single gel. One band corresponding to free 5S rRNA was detected in addition to two bands which stained for both protein and nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.

OBJECTIVE: To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes. METHODS: Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined. RESULTS: The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01). CONCLUSION: Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.

Aggrecans↗
PubMed1996

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

Capsid↗
PubMed2002

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains.

Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.

Cell Nucleus↗
PubMed2006

pH-dependent self-assembly of polyalanine peptides.

Polyalanine expansions in the nuclear RNA-binding protein PABP2 induce misfolding and aggregation of the protein into insoluble inclusions in muscle tissues and cell nuclei, leading to the disease oculopharyngeal muscular dystrophy (OPMD). We have explored the effect of solvent conditions and alanine repeat number on the propensity of fibril formation in this protein deposition disease. Three peptides mimicking the N-terminal polyalanine segment of PABP2, having the generic sequence Ac-Lys-Met-(Ala)(n)-Gly-Tyr with n = 7, 11, and 17 (referred to as 7-ala, 11-ala, and 17-ala, respectively), were synthesized and their conformational properties studied as a function of pH. In strongly alkaline medium (pH >10), the two longer peptides (11-ala and 17-ala, but not 7-ala) showed remarkable enhancement of beta-sheet content and formed fibrils after incubation for 1-2 weeks at room temperature. Fluorescence studies suggested that tyrosyl radicals produced at high pH cross-linked to form dityrosine, which provided added stabilization for fibril growth. The kinetic progress curves for fibril formation, obtained by ThT fluorescence assay, showed exponential increase with time after an initial quiescent period (lag time) and an eventual saturation phase, all of which are indicative of a nucleation-controlled polymerization mechanism for fibrillation. Hierarchical self-assembly of the peptides led to the formation of striking fractal-shaped growth patterns on substrates, raising the possibility of designing novel materials using these peptides.

Benzothiazoles↗
PubMed1992

p53 mutations in spontaneously immortalized 3T12 but not 3T3 mouse embryo cells.

We have asked whether p53 mutations are involved in the process of spontaneous immortalization of mouse embryo cells. Cells from Swiss mouse embryos were used to prepare 3T3 and 3T12 lines according to the protocol of Todaro & Green [(1963). J. Cell Biol., 17, 299-313]. After the cells emerged from crisis, p53 sequences were amplified by polymerase chain reaction (PCR) from both RNA and DNA. The sequence of the aggregated cDNA from each of six 3T3 lines showed no evidence of mutation. PCR-amplified p53 cDNA from two 3T3 lines was cloned, and individual clones in M13mp19 were partially sequenced. One cell ine showed a single, non-coding nucleotide change in 2/8 independent clones. Nine cDNA clones from the second 3T3 lines were sequenced, and no single nucleotide changes appeared more than once. The mutations which appeared only once were not detected in clones of genomic DNA. Since these apparent mutations are probably reverse transcriptase or Taq polymerase errors, we conclude that both the 3T3 lines contained only wild-type p53. In two out of three independent 3T12 lines however, missense mutations were readily observed in the aggregate cDNA sequence. Restriction fragment length polymorphism and Southern blot analyses of the genomic DNA indicated that these cells were homozygous for the mutations. The p53 protein molecules in four cell lines were analysed by immunoprecipitation: one 3T12 line showed the pattern of antibody reactivity characteristic of some p53 mutants, while the others displayed the wild-type pattern. We conclude that p53 mutations arise and are strongly selected for during immortalization according to the 3T12 but not the 3T3 protocol.

Amino Acid Sequence↗