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PubMed1974

Poly(A)-associated RNA in plants.

The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.

Animals↗

PubMed1998

In vitro changes of the nuclear AgNORs pattern induced by RNA inhibitors and 5-fluorouracil in human breast cancer cells, MCF-7 and HBC-4.

The morphological changes of argyrophilic nucleolar organizer regions (AgNORs) were studied in two human breast cancer cell lines, MCF-7 and HBC-4. Treatment with an RNA polymerase inhibitor (actinomycin D) reduced the size of AgNORs and increased the number of AgNORs. Messenger RNA polymerase inhibitor (alpha-amanitin) also increased the number of AgNORs. However, translational blocking agents closely related to ribosomal RNA (cycloheximide and anisomycin) caused a decrease in the number of AgNORs, which seemed to fuse to an aggregate around the nucleolus and formed a single large spherical AgNOR in the final stage. These changes were observed typically when cells were treated with 5-fluorouracil or 5-fluorouridine. These morphological changes in the AgNORs pattern, AgNORs aggregation, might reflect certain damage in ribosomal RNA.

Antimetabolites, Antineoplastic↗

PubMedDate not supplied

[Specific reaction between oligovaline and nucleic acids].

The DNA binding activity of trivaline dansyl hydrazide was investigated by circular dichroism, UV spectrophotometry and fluorescence methods. It is shown that these peptides in the absence of DNA can adopt statistical coil and antiparallel beta-conformation and can exist in aqueous solution as monomers, dimers and higher orders aggregates depending upon the concentration and the presence of N- and C-blocking groups. The aggregation and disaggregation processes are very slow especially for N- and C-protected peptides. Our observations show that oligopeptides in the monomeric and dimeric forms bind to double-stranded DNA and RNA whereas tetramers and higher order aggregates exhibit no DNA binding activity. The binding of monomers is a cooperative process favoring the formation of deformed antiparallel beta-structure between adjacently bound monomers. The binding constant of dimeric oligovaline species to GC-rich DNA sequences is about 5 fold higher than that found from the binding of oligovaline to poly(dA) . poly(dT). The binding of dimers takes place in the minor DNA groove as revealed from our observations that oligovaline binds to T6 phage DNA containing massive glucose and diglucose residues in the major groove. The backbone C=0 groups of oligovaline probably serve as specific reaction centres for the interaction with guanine 2-amino groups in the minor DNA groove.

Chemical Phenomena↗

PubMed2006

Physical interaction of apoptosis-inducing factor with DNA and RNA.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to single-stranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well double-stranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.

Amino Acid Sequence↗

PubMed2006

Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules.

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, we found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons.

Animals↗

PubMed1999

Evaluation of single-stranded nucleic acids as carriers in the DNA-directed assembly of macromolecules.

Current developments in nanosciences indicate that the self-assembly of macromolecules, such as proteins or metallic nanoclusters, can be conveniently achieved by means of nucleic acid hybridization. Within this context, we here report on the evaluation of single-stranded nucleic acids to be utilized as carrier backbones in DNA-directed self-assembly. A microplate solid-phase hybridization assay is described which allows rapid experimental determination of the hybridization efficiencies of various sequence stretches within a given nucleic acid carrier strand. As demonstrated for two DNA fragments of different sequence, the binding efficiencies of several oligonucleotides depend on the formation of specific secondary structure elements within the carrier molecule. A correlation of sequence-specific hybridization capability with modeled secondary structure is also obvious from experiments using the fluorescence gel-shift analysis. Electrophoretic studies on the employment of helper oligonucleotides in the formation of supramolecular conjugates of several oligonucleotide-tagged proteins indicate, that structural constraints can be minimized by disruption of intramolecular secondary structures of the carrier molecule. To estimate the influences of the chemical nature of the carrier, gel-shift experiments are carried out to compare a 170mer RNA molecule with its DNA analogue. Ternary aggregates, containing two protein components bound to the carrier, are formed with a greater efficiency on the DNA instead of the RNA carrier backbone.

Base Sequence↗

PubMed1971

In vitro aggregation of mixed embryonic kidney and nerve cells. Influence on macromolecular synthesis.

The possible role of nerve on growth of embryonic parenchymal organs such as kidney was explored by measuring macromolecular synthesis (DNA, RNA, and protein and three enzymes) in aggregates of mixed suspensions of cells from dissociated chick embryo kidney and nerve tissue. One and one-half to threefold increments in net synthesis of the three different types of macromolecules were observed in the mixed aggregates of kidney and nerve cells as compared with those of single organs or mixtures of kidney with nonneural cells. The addition of nerve-growth factor (NGF) did not significantly affect the results. Increased incorporation of label was paralleled by increases in chemically measured DNA and protein, suggesting an increase in growth in the mixed kidney-nerve aggregates compared with those of single tissues. Measurements of survival rate did not indicate increased cell stability in the mixed aggregates. The activities of three enzymes, acid phosphatase, alkaline phosphatase, and lactic dehydrogenase, were also enhanced two to four times in cultures of kidney plus nerve cells. Morphologic studies indicated a high degree of reorganization of tubular structures within the reaggregates of kidney cells alone or in those mixed with nerve. In addition, radioautographs of thymidine-(3)H-labeled cells in the aggregates showed a high level of DNA synthesis in the reformed tubular cells. Electron micrographs revealed the presence of large numbers of nerve fibers containing microtubules in the mixed cell aggregates. The data suggest a significant role for nerve in the growth processes of embryonic parenchymal organs.

Acid Phosphatase↗

PubMed1980

Short capped hnRNA precursor chains in HeLa cells: continued synthesis in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

The labeling of m7GpppN1mpN2p caps with L-[methyl-3H]methionine on short (100-500 nucleotides) heterogeneous nuclear RNA (hnRNA) chains of HeLa cells is increased 2-3 times but the labeling of caps on longer (greater than 2000 nucleotides) hnRNA chains is decreased by approximately 80% by treatment of the HeLa cells with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The experimental conditions were as follows: HeLa cells were treated with 75 muM DRB for 40 min before labeling and also during the 30-min pulse of L-[methyl-3H]methionine; actinomycin D (0.05 microgram/mL) was used to suppress ribosomal RNA synthesis. Control cells received no DRB. The RNA was separated in Me2SO gradients to ensure no aggregation. Labeling of cells with [3H]uridine for 10 min and separation of RNA by these techniques reconfirmed the findings [Tamm, I., Hand, R., & Caliguiri, L. A. (1976) J. Cell Biol. 69, 229-240; Sehgal, P. B., Darnell, J. E., Jr., & Tamm, I. (1976) Cell 9, 473-480] that 70-80% of the synthesis of hnRNA (GREATER THAN 1000 NUCLEOTIDES) IS SENSITIVE TO INHIBITIOn by DRB but that 20-30% is resistant. This analysis of the methyl-labeled caps provides evidence that DRB causes early termination of a large fraction (approximately 70-80%) of hnRNA precursor chains. In contrast to the finding of continued synthesis and accumulation of short m7GpppN1mpN2p-capped chains in the presence of DRB, the synthesis of m2,2,7GpppN1mpN2mp-capped small nuclear RNAs was inhibited by approximately 70% by DRB.

Chromatography, High Pressure Liquid↗

PubMed1980

Interaction of Escherichia coli host factor protein with Q beta ribonucleic acid.

The affinity of Escherichia coli host factor protein for a variety of ribonucleic acids (RNAs) is compared in an equilibrium competition assay with (pA)15 or (pA)27 as the common probe. Of the homopolymers tested, only polyriboadenylate [poly(rA)] binds the protein with a high affinity. At low ionic strength (0.1 M NaCl), the binding to Q beta RNA is much stronger than to the oligoadenylates, but the situation is reversed upon fragmentation of the RNA with ribonuclease T1. Increasing the ionic strength results in a drastic reduction of the affinity of host factor for Q beta RNA over a relatively narrow salt range (0.1--0.3 M NaCl). Over the same range, added salt greatly reduces the tendency of host factor hexamers to aggregate. The tight binding of host factor to Q beta RNA is proposed to result from the binding of an aggregate, which can interact with several low affinity sites on the RNA simultaneously.

Binding Sites↗

PubMed1984

U1 small nuclear RNA-like sequences in human high molecular weight RNA.

Human U1 small nuclear RNA synthesis was shown earlier to be very sensitive to UV radiation. This led us to test for the possible presence of U1 RNA-like sequences in large RNAs. Human RNA was analyzed in gel blots hybridized with U1 DNA probes. A high molecular weight, heterodisperse RNA population was detected, which hybridizes both to a U1 RNA-coding region single-stranded DNA probe, and to a U1 gene fragment that contains only 6 nucleotides of flanking sequence. These large RNAs can be hybrid selected using immobilized U1 DNA, and have an average size of several kilobases. Additional observations support the claims that the high molecular weight RNA hybridization signal is not an aggregation artifact and that it is sequence specific.

Carcinoma↗

PubMedDate not supplied

Further characterization of FcgammaRII and FcgammaRIII expression by cultured human mast cells.

BACKGROUND: We have reported that resting human mast cells exhibit minimal expression for FcgammaRI, and that interferon-gamma will upregulate this expression. The expression of FcgammaRII and FcgammaRIII by human mast cells remains to be fully examined. METHODS: To investigate FcgammaRII and FcgammaRIII expression, we determined mRNA and protein expression of FcgammaRII and FcgammaRIII in human peripheral blood CD34+ derived cultured mast cells by RT-PCR and flow cytometry. The expression of FcgammaRII and FcgammaRIII in intact and permeabilized mast cells was also compared. We measured histamine release to monitor mast cell degranulation following cross-linking of FcgammaRII. RESULTS: We found by RT-PCR that resting human mast cells exhibit mRNA for FcgammaRIIA, FcgammaRIIb1, FcgammaRIIb2 and FcgammaRIII but not FcgammaRIIC. FACS analysis of Fcgamma receptors in intact versus permeabilized mast cells showed expression of FcgammaRII to be 42.2 +/- 3.9% and this was unchanged by permeabilization. FcgammaRIII protein expression was minimal and this was also unchanged by permeabilization. Aggregation of FcgammaRII on human mast cells led to no significant degranulation as evidenced by histamine release. CONCLUSIONS: In addition to FcgammaRI expression, human mast cells express FcgammaRIIA, FcgammaRIIb1, FcgammaRIIb2 and FcgammaRIII mRNA, and significant surface expression of FcgammaRII. Aggregation of FcgammaRII on cultured human mast cells in this model was not followed by histamine release.

Cells, Cultured↗

PubMed1993

Detection of hepatitis C virus RNA in liver tissues by an in situ hybridization technique.

Hepatitis C virus (HCV) infection of cells in liver tissues was determined by detecting HCV RNA by an in situ hybridization technique using synthetic oligonucleotide probes derived from the 5'-non-coding and core regions of HCV genome. Aggregated silver grains indicating hybridization with HCV RNA were observed over the nuclei as well as the cytoplasm of hepatocytes with none on non-parenchymal cells. The specificity of the hybridization was confirmed by absence of autoradiographic signals after ribonuclease predigestion, addition of an excess of non-labeled probes, or application of an M 13 probe. The hepatocytes with HCV RNA-positive signals were scattered in the periportal and mediolobular zones of liver lobules rather than in the pericentral zones. Fifteen out of 33 biopsy specimens from patients with chronic HCV infection studied had the HCV RNA-positive hepatocytes. These cells were more frequently detected in specimens with advanced periportal, bridging and intralobular necrosis but showed no correlation with the extent of inflammatory cell infiltration. These findings suggest a close correlation between the detection of HCV RNA in hepatocytes and advanced necrosis of the specimens.

Adult↗

PubMed2004

The role of hydrophobic interactions in catalysis of RNA cleavage by 1,4-diazabicyclo[2.2.2]-octane based artificial ribonucleases.

Molecular interactions of RNA cleaving compounds-conjugates of 1,4-diazabicyclo[2.2.2.]octane substituted at the bridge position with tetradecamethylene fragment and imidazole were investigated using light scattering and small angle x-ray scattering methods. The compounds are known to efficiently cleave RNA and one source of the activity could result from micellar catalysis. It was found that the compounds indeed are capable of forming complex aggregates in solution. However, maximal efficacy of RNA cleavage by the conjugates is observed at concentrations well below the concentration required for micelle formation.

Catalysis↗

PubMed2006

Modeling polyglutamine pathogenesis in C. elegans.

A growing number of human neurodegenerative diseases are associated with disruption of cellular protein folding homeostasis, leading to the appearance of misfolded proteins and deposition of protein aggregates and inclusions. Recent years have been witness to widespread development of invertebrate systems (specifically Drosophila and Caenorhabditis elegans) to model these disorders, bringing the many advantages of such systems, particularly the power of genetic analysis in a metazoan, to bear on these problems. In this chapter, we describe our studies using the nematode, C. elegans, as a model to study polyglutamine expansions as occur in Huntington's disease and related ataxias. Using fluorescently tagged polyglutamine repeats of different lengths, we have examined the dynamics of aggregate formation both within individual cells and over time throughout the lifetime of individual organisms, identifying aging as an important physiological determinant of aggregation and toxicity. Expanding on these observations, we demonstrate that a genetic pathway regulating longevity can alter the time course of aging-related polyglutamine-mediated phenotypes. To identify novel targets and better understand how cells sense and respond to the appearance of misfolded and aggregation-prone proteins, we use a genome-wide RNA interference-based genetic screen to identify modifiers of age-dependent polyglutamine aggregation. Throughout these studies, we used fluorescence-based, live-cell biological and biophysical methods to study the behavior of these proteins in a complex multicellular environment.

Aging↗

PubMed1980

Immunohistochemical studies of cyclic guanosine monophosphate and nuclear function.

In previous immunohistochemical studies, it has been found that all nuclei contain cyclic (c)GMP, which occurs in discrete aggregates and in the nucleolus. We have studied the nature of the cGMP aggregates in isolated mouse fetal nuclei using a specific immunofluorescent technique. These aggregates correspond to the areas of condensation of DNA, demonstrable by either Felugen's or acridine orange stain. Treatment with DNAase eliminated DNA and cGMP staining. Staining for RNA, with a human anti-RNA antibody, demonstrated RNA to be distributed diffusely throughout the nucleus and not preferentially in the areas of discrete cGMP aggregates. The diffuse stain for nuclear RNA was eliminated by pretreatment with RNAase but not DNAase, but aggregates of cGMP were not affected by pretreatment with RNAase. Sites of active RNA synthesis were determined by autoradiography using [3H]uridine, and did not correspond to the aggregates of cGMP. The relationship of cGMP to nucleolar function was examined in the endothelial cells of the isthmus and ampulla of the rat fallopian tube. Previous studies have shown that in proestrous, a period of increased RNA synthesis, nucleoli detectable by staining for RNA appear in the endothelial cells lining the fallopian tube. After immunofluorescent staining, we found prominent accumulation of cGMP in the nucleoli. During other phases of the cycle, there is an absence of nucleoli detectable by staining for RNA, and an absence of nucleolar cGMP. After we treated hypophysectomized or oophorectomized rats with estrogen, which is known to increase nucleolar RMA synthesis in the fallopian tube and endometrium, nucleoli in the endothelial cells of the rat fallopian tube and uterus stained strongly for cGMP. In conclusion, our studies suggest that the discrete aggregates of nuclear cGMP are associated with a fraction of DNA uninvolved in RNA synthesis. In contrast, cGMP appears in the nucleolus during a period of increased RNA synthesis, suggesting a role for cGMP in regulating nucleolar synthesis and processing of RNA.

Animals↗

PubMed1990

Aggregation of dispersed human cytotrophoblastic cells: lessons relevant to the morphogenesis of the placenta.

The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.

Cell Aggregation↗

PubMed1980

Dexamethasone binding to chromatin, inhibition of in vitro RNA synthesis, and therapeutic effect on human lymphosarcoma.

A very high degree of specific dexamethasone binding to chromatin and a marked in vitro inhibition of RNA-synthesizing capacity of purified lymphoma cell nuclei was found to correlate closely with the very strong cytolethal effect of glucocorticoids on lymphoma cells (in this case neither B- nor T-cells) of a young patient, whose condition rapidly and unexpectedly deteriorated after a total dose fo 90 mg of Prednisone given during a two day period; and in spite of prophylactic antihyperuricaemia treatment, the patient subsequently died. In this case the amount of glucocorticoid bound specifically by lymphoma chromatin was about 400% larger than is normally found in chromatin isolated from normal human thymus cells in persons on this age. The in vitro inhibition of RNA-Synthesizing activity measured with the aggregate enzyme and with isolated nuclei from lymphoma cell-nuclei by dexamethasone correlates closely to the specific dexamethasone binding capacity of chromatin. Thus a better prediction of the therapeutic effect of such lymphosarcoma cells to glucocorticoid may be possible.

Child↗

PubMed1997

Lymphotactin gene expression in mast cells following Fc(epsilon) receptor I aggregation: modulation by TGF-beta, IL-4, dexamethasone, and cyclosporin A.

Recruitment of lymphocytes is a prominent feature of allergic inflammation. However, the mechanisms by which lymphocytes are attracted to such sites are not understood. Recently, cDNAs encoding a lymphocyte-specific chemokine, lymphotactin (Ltn), were isolated from mouse pro-T cell and human CD8+ T cell libraries, leading us to hypothesize that mast cells might also produce Ltn. Using the reverse transcriptase-PCR and Northern blot analysis, we found that the Ltn gene is inducible in C1.MC/C57.1 and murine bone marrow-cultured mast cells (BMCMC) by Fc(epsilon)RI aggregation. Activation of a human mast cell (HMC-1) or basophil cell line (KU812) similarly led to transcription of Ltn. Fc(epsilon)RI aggregation-dependent Ltn mRNA expression was detected by 1 to 2 h, maximal at 6 h, independent of de novo protein synthesis, and was inhibited by cyclosporin A and dexamethasone. Compared with macrophage inflammatory protein alpha (MIP-1alpha), Fc(epsilon)RI-dependent Ltn and MIP-1alpha mRNA levels were up-regulated by IL-4, but not IFN-gamma, although higher levels of IL-4 (100 and 1000 U/ml) inhibited Ltn expression only; and TGF-beta preferentially enhanced Fc(epsilon)RI-dependent Ltn mRNA levels, suggesting that Ltn and MIP-1alpha have shared and unique regulatory mechanisms. A rabbit polyclonal Ab against a synthetic peptide was developed for use in immunoblot analysis and detected a 15-kDa Ltn protein within mast cell pellets and in the supernatants of mast cells following Fc(epsilon)RI aggregation. Ltn is thus expressed in mast cells and may contribute to the recruitment of lymphocytes to areas of allergic inflammation.

Animals↗