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At least 667 records · Page 37Linked to original sources
PubMed1985

Evidence for a role of calmodulin in the regulation of prolactin gene expression.

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.

Animals↗
PubMed2000

Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation.

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.

Aggrecans↗
PubMed1990

Active RNA polymerase I is fixed within the nucleus of HeLa cells.

We have investigated whether active RNA polymerase I, the enzyme responsible for transcribing ribosomal RNA, is immobilized by attachment to a large subnuclear structure in HeLa cells. As unphysiological salt concentrations induce artifacts, we have used isotonic conditions throughout the preparative and analytic procedures. Cells are encapsulated in agarose microbeads and lysed in Triton and a 'physiological' buffer; then soluble proteins and RNA diffuse out through the agarose pores to leave encapsulated chromatin. This can be manipulated without aggregation but is accessible to molecular probes; it retains the replicational and transcriptional activities of the living cell. After treatment with a restriction endonuclease, most chromatin can be removed from beads by electrophoresis: then active ribosomal genes and polymerase I remain behind. Active ribosomal genes are very accessible to nuclease digestion whilst the rest are even more inaccessible than inactive globin genes. Our observations confirm the complex organization of rDNA within nucleoli and are compatible with transcription occurring at fixed sites. A model for transcription involving an attached polymerase is presented.

Cell Nucleus↗
PubMed2002

Role of dimerization in KH/RNA complexes: the example of Nova KH3.

The K homology module, one of the most common RNA-binding motifs, is present in multiple copies in both prokaryotic and eukaryotic regulatory proteins. Increasing evidence suggests that self-aggregation of KH modules has a functional role. We have used a combination of techniques to characterize the behavior in solution of the third KH domain of Nova-1, a paradigmatic KH protein. The possibility of working on the isolated module allowed us to observe specifically the homodimerization and RNA-binding properties of KH domains. We provide conclusive evidence that self-association of Nova-1 KH3 occurs in solution even in the absence of RNA. Homodimerization involves a specific protein/protein interface. We also studied the dynamical behavior of Nova-1 KH3 in isolation and in complex with RNA. These data provide a model for the mechanism of KH/RNA recognition and suggest functional implications of dimerization in KH complexes. We discuss our findings in the context of the whole KH family and suggest a generalized mode of interaction.

Amino Acid Motifs↗
PubMed2000

Detection of beta-amyloid peptide aggregation using DNA electrophoresis.

DNA could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of beta(1-40) and beta(25-30). DNA from different sources such as lambda phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric beta(1-40) and beta(25-35) as well as by the aggregated beta(1-40) and beta(25-35). The association of DNA with the aggregated beta-amyloid peptides could occur at pH 4-9. The inhibitory action of hemin on beta-amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of beta-amyloid aggregation as well as for testing of agents that might modulate beta-amyloid aggregation.

Amyloid beta-Peptides↗
PubMed1983

RNA-protein interactions in some small plant viruses.

The structure of the three quasi-equivalent protein subunits A, B and C of the spherical, T = 3 southern bean mosaic virus (SBMV) have been carefully built in accordance with a refined electron density map of the complete virus. The lower electron density in the RNA portion of the map could not be explicitly interpreted in terms of a preferred RNA structure on which some icosahedral symmetry might have been imposed. However, the extremely basic nature of the interior surface of the coat protein must be associated with the binding and organization of the RNA. Comparison with the small spherical, T = 1 satellite tobacco necrosis virus (STNV; Liljas et al., J. Mol. Biol. 159, 93-108, 1982) and the T = 1 aggregate of alfalfa mosaic virus (AMV) protein (Fukuyama et al., J. Mol. Biol. 150, 33-41, 1981) showed similar results. The pattern of basic residues on the SBMV coat protein surface facing the RNA is able to dock a 9 base pair double-helical A-RNA structure with surprising accuracy. The basic residues are each associated with a different phosphate and the protein can make interactions with five bases in the minor groove. This may be one of a small number of ways in which the RNA interacts with SBMV coat protein. The self-assembly of SBMV has been studied in relation to the presence of the 63 basic amino-terminal coat protein sequence, pH, Ca2+ and Mg2+ ions and RNA. These results have led to a two-state model where the "relaxed" dimers initially self-assemble into 10-mer caps which nucleate the assembly of T = 1 or T = 3 capsids depending on the charge state of the carboxyl group clusters in the subunit contact region. The two-state condition of dimers in a viral coat protein extends the range of structures originally envisaged by Caspar and Klug (Cold Spring Harbor Symp. Quant. Biol. 27, 1-24, 1962).

Binding Sites↗
PubMedDate not supplied

Stimulation of 3H-uridine incorporation into ribosomal ribonucleic acids by insulin.

Ribosomal RNA was extracted from hepatic ribosomal subunits of rats treated with alloxan or with alloxan and insulin and additionally injected with 3H-uridine. Sedimentation of the extracted RNA through sucrose, gradients with registration of the optical density and with measurement of the radioactivity of rRNA reveals reduction of uridine incorporation after the induction of diabetes of diabetes mellitus. If alloxan-diabetic animals are substituted with insulin, this decrease in uridine incorporation is reversed to incorporation rates which even exceed uridine incorporation of control animals. With regard to the reduction of protein synthesis in diabetes mellitus this finding of reduced rRNA synthesis is suggested to be an additional factor in the reduction of ribosomal aggregation.

Animals↗
PubMed2006

Repair of methyl lesions in DNA and RNA by oxidative demethylation.

It was established several decades ago that it is crucial for all organisms to repair their DNA to maintain genome integrity and numerous proteins are dedicated to this purpose. However, it is becoming increasingly clear that it is also important to prevent and repair lesions in the macromolecules encoded by the DNA, i.e. RNA and protein. Many neurological disorders such as Alzheimer's disease and Parkinson's disease are associated with the aggregation of defective, misfolded proteins, and several mechanisms exist to prevent such aggregation, both through direct protein repair and through the elimination and repair of faulty or damaged RNAs. A few years ago, it was discovered that the E. coli AlkB protein represented an iron and 2-oxoglutarate dependent oxygenase capable of repairing methyl lesions in DNA by a novel mechanism, termed oxidative demethylation. Furthermore, it was found that both human and bacterial AlkB proteins were able to demethylate lesions also in RNA, thus representing the first example of RNA repair. In the present review, recent findings on the AlkB mechanism, as well as on RNA damage in general, will be discussed.

Animals↗
PubMed1997

Agrin-deficient myotube retains its acetylcholine receptor aggregation ability when challenged with agrin.

Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.

Acetylcholinesterase↗
PubMed1991

Isolation of two genes encoding putative protein kinases regulated during Dictyostelium discoideum development.

Two Dictyostelium discoideum protein kinase(PK)-encoding cDNAs (Dd PK1 and Dd PK2) have been isolated by hybridization with an oligodeoxyribonucleotide derived from a highly conserved region of eukaryotic PKs. The two nucleotide (nt) sequences encode new putative serine/threonine-specific PKs. Dd PK1 is a partial cDNA covering the entire catalytic domain. The derived amino acid (aa) sequence is about 30% identical to both cAMP-dependent protein kinase (cAPK) and protein kinase C. The Dd PK2 sequence was extended through the isolation of a genomic fragment encoding a complete putative protein. A single intron is present, as deduced from sequence comparison with the cDNA. The catalytic domain appears more closely related to the catalytic subunit of cAPK (54% sequence identity). However, our nt sequence potentially codes for a much larger protein (648 vs. about 350 aa for most cAPKs) with a N-terminal half containing long homopolymers of threonines, glutamines and asparagines. Similar repeats occur at the C terminus of Dd PK1, Dd PK1 is expressed in vegetatively growing cells and during development. Dd PK1 RNA decreases after 6 h of starvation to re-accumulate once the cells have aggregated. Dd PK2 transcripts, present at a low amount in growing cells, rise upon starvation. A switch to a shorter form of transcripts occurs between 3 and 6 h into development.

Amino Acid Sequence↗
PubMed1983

Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein.

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.

Capsid↗
PubMed1995

The CD40 ligand expressed by human B cells costimulates B cell responses.

The possibility that activated B cells might express a ligand for CD40 that was of functional importance for B cell responses was examined by using highly purified human peripheral blood B cells, as well as a variety of B lymphoblastoid cell lines and hybridomas. Following stimulation with the combination of a calcium ionophore and a phorbol ester, human B cells bound a soluble fusion protein containing the extracellular portion of CD40 and the Fc region of IgG1 (CD40.Ig). A variety of B cell lines and hybridomas also bound CD40.Ig, either constitutively or after activation. In addition, CD40.Ig specifically immunoprecipitated a 33-kDa glycoprotein from surface 125I-labeled activated B cells. The nucleotide sequence of the coding region of the CD40 ligand mRNA amplified by RT-PCR from activated T cells and B cell lines was identical. The CD40 ligand expressed on human B cells was important functionally because homotypic aggregation of CD40 ligand-expressing B cells was inhibited by the CD40.Ig construct. Additionally, RNA and DNA synthesis as well as Ig production by polyclonally activated, highly purified peripheral B cells and a variety of B cell lines were inhibited significantly by the CD40.Ig construct. Finally, B cell lines expressing the CD40 ligand induced Ig production from resting normal B cells in a CD40-dependent manner. These results indicate that human B cells express a ligand for CD40 that is identical with that expressed by activated T cells and that the B cell-expressed CD40 ligand plays an important role in facilitating responses of activated B cells.

Adult↗
PubMed2000

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification.

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment.

Actins↗
PubMed1995

The functional integration of a polytopic membrane protein of Escherichia coli is dependent on the bacterial signal-recognition particle.

In eukaryotes, the cotranslational targeting of proteins to the endoplasmic reticular membrane is initially mediated by the signal-recognition particle (SRP), a ribonucleoprotein complex consisting of the 7SL RNA and six protein subunits. Since the discovery of sequence homology between (a) the Escherichia coli 4.5S RNA (Ffs) and 7SL RNA, and (b) the E. coli P48 (Ffh) and SRP 54-kDa subunit, more evidence has been obtained that E. coli also possesses an SRP-type pathway that acts in the translocation of secreted proteins. Such a pathway could possibly be involved in the cotranslational integration of hydrophobic membrane proteins that cannot be effectively targeted post-translationally due to folding and aggregation. In this study, we report that disruption of the E. coli SRP complex with a dominant lethal 4.5S RNA mutant in vivo prevents functional membrane integration of the E. coli lactose permease (LacY). Likewise, depletion of the P48 (Ffh) protein also results in a decrease in the amount of functional LacY inserted into the E. coli plasma membrane. In direct contrast, inhibition of SecA function does not affect LacY integration. These results suggest a major function of the bacterial SRP in the targeting and subsequent integration of hydrophobic membrane proteins as opposed to SecA mediating the post-translational targeting of secretory proteins.

Escherichia coli↗
PubMed1990

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients.

PCR DNA amplification of IgH genes was performed on liver biopsy samples of 42 unselected hepatitis C virus (HCV)-positive patients. Genotypic analysis and signal amplification by branched DNA were used to characterize and quantitate HCV RNA genomic sequences. Intraportal lymphoid follicle-like structures were isolated from surrounding hepatocytes by microdissection technique. IgH VDJ PCR products were cloned and sequenced. IgH VDJ gene rearrangements were detected in the liver of 26 (62%) patients. Unequivocal monoclonal or oligoclonal patterns of B cell expansions were found in 14 (33.3%) and 12 (28.6%) patients, respectively. Patients with intrahepatic B cell monoclonal expansions showed liver HCV RNA levels higher than those with oligoclonal or polyclonal features (1106.4 +/- 593.5 vs 677.3 +/- 424.3 vs 406.2 +/- 354.3 pg HCV RNA/g tissue; p = 0.048 and p = 0.001, respectively). Although a single dominant band was obtained with total DNA, characterization of DNA recovered from intraportal inflammatory aggregates resulted in the detection of multiple IgH VDJ gene rearrangements, pointing to an oligoclonal pattern of lymphoproliferation. Cloning and sequence analyses showed that B cell clonalities were differently distributed in adjacent portal tracts of the same liver area. In addition, HCV RNA genomic sequences could be consistently amplified from each of the portal inflammatory aggregates examined. These data support the concept that in chronic HCV infection the intrahepatic B cell repertoire is frequently clonally restricted and that HCV may have a direct role in sustaining in situ B cell proliferation.

Adult↗
PubMed1987

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.

Animals↗
PubMed1986

Evidence for the role of glycosylation of proteins in the tryptophan-induced stimulation of nucleocytoplasmic translocation of mRNA in rat liver.

Tryptophan induces a rapid stimulation of polyribosomal aggregation and protein synthesis in the livers of rats or mice. This stimulatory effect of tryptophan correlates with increased levels of mRNA in the cytoplasm of the liver and is related to enhanced translocation of nuclear mRNA into the cytoplasm. In the present study the possible role of glycoproteins of the rat liver, particularly those in the nuclear membranes and in the cytosol, in relation to the effect of tryptophan on enhanced nucleocytoplasmic translocation of mRNA was investigated. In the standard treatment regimen, tryptophan (30 mg/100 gm of body weight) was administered by stomach tube 10 minutes before killing. The following results were noted in the liver: Administration of tryptophan increased [14C]glucosamine incorporation into proteins of the subcellular fractions, particularly those of the soluble, nucleus and nuclear membrane. In vivo treatment of rats with tryptophan increased the in vitro [14C]orotate-labeled nuclear RNA release and [3H]tryptophan binding to proteins of the nuclei or cytosols. Treatment of cytosol proteins with alpha-mannosidase or beta-galactosidase, but not with neuraminidase, inhibited these increases. After treatment of cytosol fractions on a concanavalin A agarose column, the effluents showed decreased activity and the eluates showed increased activity in in vitro nuclear RNA release and in tryptophan binding to proteins of the control and, particularly, of the tryptophan-treated animals. Administration of tryptophan increased the polyribosomal aggregation, the in vitro [14C]leucine incorporation into protein, and the in vitro nuclear RNA release by cytosol. However, tunicamycin pretreatment of rats prevented these increases due to tryptophan. The data of this study suggest that glycosylated proteins, one or more, are the active components whereby tryptophan acts upon the liver.

Animals↗