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At least 253 records · Page 14Linked to original sources
PubMed1982

Mechanism of tobacco mosaic virus assembly: role of subunit and larger aggregate protein.

Tobacco mosaic virus (TMV) was reconstituted from the RNA of a common strain (OM) and the protein of a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is a member of the tobamovirus group. In 0.25 M phosphate buffer at 25 degrees C, CGMMV-W protein existed mainly as 21S aggregates. When this protein was mixed with OM RNA, complexes of short rods were formed but further elongation did not occur. After the addition of subunits in 0.1 M phosphate buffer at 25 degrees C, elongation to the 5' end of the RNA proceeded as fast as in the case of reconstitution with the usual equilibrium "disk preparation" of OM protein, to give 260-nm intermediates in the first 5-7 min. The results proved that the rapid elongation we previously observed in the reconstitution of TMV-OM following the assembly initiation is the outcome of preferential incorporation of TMV subunit protein. Either preformed 21S aggregate or the subunit of CGMMV protein was added to the 260-nm intermediate. Elongation to the 3' end of the RNA was investigated in 0.1 M phosphate buffer at 25 degrees C by measuring the distribution of rod length and the RNase-resistant infectivity. The results showed that the 21S aggregate is kinetically favored as the protein source during the slow elongation process.

Kinetics↗
PubMedDate not supplied

[Specific and nonspecific adhesion in the process of retinal cellular aggregation in chick embryos].

A study of aggregation of the retinal cells of 8 and 14 day old chick embryos has revealed two phases in this process. The first phase includes the decrease in the concentration of single cells and the increase in the concentration of aggregates. During the second phase the concentration of aggregates falls at the expense of fusion of smaller aggregates into larger ones. The rate of aggregation at both these phases increases with the initial density of cells and decreases with the age of donor embryos and at a suboptimal temperature of cultivation. Aggregation during the first phase does not depend on the presence in the culture medium of divalent cations and colchicine, the level of protein and RNA synthesis in the cells, whereas aggregation during the second phase depends on all these factors. Comparison of these results with the published data suggests that the retinal cell aggregation during the second phase, unlike the first one, is based on the specific adhesiveness of the cells, which is realized via adhesion molecules resynthesized at the cell surface.

Animals↗
PubMed1973

Gene expression during the development of Bacillus subtilis bacteriophage phi29. II. Resolution of viral-specific ribonucleic acid molecules.

The ribonucleic acid (RNA) specified by bacteriophage phi29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 x 10(6), 0.44 x 10(6), 0.37 x 10(6), 0.25 x 10(6), 0.09 x 10(6), and 0.04 x 10(6), accounting for 35% of the coding capacity of phi29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 x 10(6) were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 x 10(6)) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 x 10(6), 0.93 x 10(6), and 0.07 x 10(6), appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.

Bacillus subtilis↗
PubMed1991

CD40 signaling activates CD11a/CD18 (LFA-1)-mediated adhesion in B cells.

Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed.

Animals↗
PubMed2004

Stress granule assembly is mediated by prion-like aggregation of TIA-1.

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.

Amino Acid Sequence↗
PubMed1997

Progesterone regulates osteopontin expression in human trophoblasts: a model of paracrine control in the placenta?

Osteopontin (OPN), a matrix glycosylated phosphoprotein, has been proposed to play a role(s) in basic cellular processes, such as neovascularization and tissue remodeling, which are essential to placental morphogenesis and embryo implantation. We have shown OPN to be expressed by cytotrophoblasts of the chorionic villus, and a putative progesterone regulatory element in the OPN promoter suggests hormonal regulatory control. This led us to test the hypothesis that progesterone regulates OPN expression in human cytotrophoblasts. Cytotrophoblasts isolated from human placentas were treated with combinations of progesterone, RU486, and/or aminoglutethimide, and their expression of OPN was assessed by Northern hybridization and immunocytochemistry. The expression of OPN messenger RNA (mRNA) declined as trophoblasts aggregated, but rebounded at later times when syncytia and mononuclear cytotrophoblasts coexisted in culture. Progesterone increased OPN mRNA expression by aggregating mononuclear cytotrophoblasts. Aminoglutethimide suppression of endogenous steroidogenesis by syncytiotrophoblasts inhibited OPN expression, whereas the addition of exogenous progesterone to cells treated with aminoglutethimide reversed this inhibitory effect. These observations were confirmed at the protein level by immunocytochemistry. Treatment of cytotrophoblasts with both progesterone and RU486 inhibited the up-regulatory effect on OPN mRNA associated with exposure to progesterone alone, further confirming a direct effect of progesterone. We conclude that progesterone up-regulates OPN expression in human cytotrophoblasts, and we propose that in vivo, progesterone secretion by syncytiotrophoblasts regulates the expression of OPN by the underlying cytotrophoblasts. As the receptors for OPN, alpha(v) integrins, are expressed by syncytiotrophoblasts, we postulate that these paracrine regulatory mechanisms contribute to the adhesive and/or signaling events between the two trophoblast cell types of the chorionic villus.

Aminoglutethimide↗
PubMed1999

RNA:pseudouridine synthetase Pus1 from Saccharomyces cerevisiae: oligomerization property and stoichiometry of the complex with yeast tRNA(Phe).

Yeast RNA:pseudouridine synthetase Pus1 catalyzes the formation of pseudouridines in tRNAs. We report here the quaternary structure of purified recombinant Pus1 in solution. At low concentration, in the absence of tRNA, Pus1 oligomerizes while at high concentration it precipitates. This oligomerization/aggregation can be prevented by addition of dodecyl-beta-D-maltoside or of yeast tRNA(Phe). The detergent does not significantly interfere with substrate binding or with activity of Pus1. The stoichiometry of the Pus1/tRNA(Phe) complex is 1/1. We conclude that the detergent covers an hydrophobic region of the RNA binding pocket responsible for Pus1 aggregation.

Biopolymers↗
PubMed1974

Ovalbumin messenger RNA: evidence that the initial product of transcription is the same size as polysomal ovalbumin messenger.

The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.

Animals↗
PubMed1982

Interaction between 1,25-dihydroxyvitamin D3 receptors and intestinal nuclei. Binding to nuclear constituents in vitro.

The molecular action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to involve its localization within the nucleus of target cells, a process mediated by intracellular receptors. This report probes both the association between chick intestinal 1,25(OH)2D3 receptors and purified homologous nuclei and the interaction between this receptor and nucleic acids. 1,25(OH)2D3 receptors bound to purified nuclei in a apparently saturable manner (Kd = 2.2-4.8 X 10(-10) M) under conditions of intermediate ionic strength and constant protein concentration. Nuclear binding was hormone-dependent; whereas receptor-hormone complex (Rs) binds to nuclei under the ionic conditions employed here (greater than 70%), hormone-free (R0) receptors do not bind (less than 10%). Binding was localized to the nuclear chromatin fraction and was extremely sensitive to KCl concentration both in the incubation medium and during postincubation treatment of nuclei. The interaction appeared to be temperature-independent, suggesting the lack of a classic activation event characteristic of most steroid receptors. Partial digestion of intestinal nuclei with DNase I eliminated subsequent receptor binding by greater than 95%, pointing to the involvement of DNA in the binding interaction. In turn, receptors were found to bind to both DNA and RNA, a characteristic independent of receptor aggregation, but sensitive to disruption with increasing ionic strength buffers. Elution of both Rs and R0 from DNA appeared identical (0.28 M KCl), whereas the strength of interaction with RNA was much less (0.12 M KCl). Thus, while there appeared to be a fundamental difference between R0 and Rs, such that only the binding of receptor-hormone complex to nuclei was allowed under the conditions employed here, this characteristic was not observed during DNA binding. Nevertheless, the possibility exists that the in vivo interaction between 1,25(OH)2D3 receptor and nuclei involves DNA and that this nuclear constituent may be the ultimate site of action of this unique sterol hormone.

Animals↗
PubMed1989

Formation and function of accessory nuclei in the oocytes of the bird louse, Eomenacanthus stramineus (Insecta, Mallophaga). I. Ultrastructural and histochemical studies.

In the oocytes of Eomenacanthus stramineus accessory nuclei arise by budding from the nuclear envelope. It is suggested that microtubules and the thick layer of the nuclear lamina are involved in this process. Newly formed accessory nuclei contain aggregations of fibrillogranular material. These aggregations are slightly Feulgen positive, RNA negative and stain positively with the Ag-NOR method. During later developmental stages one dense, RNA-positive inclusion appears in each accessory nucleus. These inclusions consist of an Ag-NOR-positive cortical layer and an Ag-NOR-negative core. The function of accessory nuclei in the species investigated is discussed in the light of these results.

Actins↗
PubMed1999

Identification of IPNV-specified components released from productively infected RTG-2 cells following massive cytopathic effect.

Rainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

Animals↗
PubMed2004

MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1.

In myotonic dystrophy 1 (DM1), aggregation of the mutant DMPK RNA into RNA-protein complexes containing MBNL1 and MBNL2 has been linked to aberrant splicing of the insulin receptor (IR) RNA. In a parallel line of investigation, elevated levels of CUG-binding protein (CUG-BP) have been shown to result in altered IR splicing in DM1. The relative importance of MBNL1, MBNL2, and CUG-BP in DM1 pathogenesis is, however, unclear. Here we have demonstrated that either small interfering RNA-mediated down-regulation of MBNL1 and MBNL2 or the overexpression of CUG-BP in normal myoblasts results in abnormal IR splicing. Our results suggest that CUG-BP regulates the equilibrium of splice site selection by antagonizing the facilitatory activity of MBNL1 and MBNL2 on IR exon 11 splicing in a dose-dependent manner. We have shown that CUG-BP levels are elevated in DM1 cells by mechanisms that are independent of MBNL1 and MBNL2 loss. Importantly, rescue experiments in DM1 myoblasts demonstrated that loss of MBNL1 function is the key event, whereas the overexpression of CUG-BP plays a secondary role in the aberrant alternative splicing of IR RNA in DM1. Small interfering RNA-mediated down-regulation of MBNL1, MBNL2, and CUG-BP in DM1 myoblasts demonstrated that MBNL1 plays a critical role in the maintenance of DM1 focus integrity. Thus, these experiments demonstrate that sequestration of MBNL1 by the expanded CUG repeats is the primary determinant of both DM1 focus formation and the abnormal splicing of the IR RNA in DM1 myoblasts. The data therefore support MBNL1-mediated therapy for DM1.

Alternative Splicing↗
PubMed2002

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase.

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson-Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.

Adenosine Triphosphatases↗
PubMed1994

Comparative studies on tryptophan binding to hepatic nuclear envelopes in Sprague-Dawley and Lewis rats.

Since Lewis rats are susceptible to many inflammatory diseases and have been used in an experimental model of the eosinophilia-myalgia syndrome, we investigated whether Lewis rats would respond to L-tryptophan as have Sprague-Dawley rats reported earlier. In this comparative study using females of both strains, we observed a decrease in the affinity of in vitro L-tryptophan binding to hepatic nuclei and nuclear envelopes of Lewis rats compared with Sprague-Dawley rats. However, in vivo stimulatory effects of administering L-tryptophan on hepatic polyribosomal aggregation, protein synthesis, and nuclear RNA release were similar in both strains. In vitro [3H]tryptophan binding to hepatic nuclear envelopes, using L-tryptophan implicated in cases of the eosinophilia-myalgia syndrome, revealed less specific binding than when using nonimplicated L-tryptophan in both strains. The possible significance of the quantitative difference in the binding affinity of L-tryptophan to hepatic nuclei of Lewis rats compared with those of Sprague-Dawley rats is as yet undetermined.

Animals↗
PubMed1992

Thioredoxins from Dictyostelium discoideum are a developmentally regulated multigene family.

Thioredoxins are low molecular weight proteins which serve as hydrogen donors in a wide variety of redox reactions via reversible formation of a disulfide bridge between two neighboring cysteins. We present data demonstrating that in Dictyostelium discoideum thioredoxins constitute a highly conserved multigene family. We have isolated cDNA clones coding for three different Dictyostelium thioredoxins which show 80% mutual identity. Analysis of genomic Southern blots suggests the presence of additional genes. Except for the active site (Trp-Cys-Gly-Pro-Cys), there are only a few amino acid identities with thioredoxins from other organisms. Identity scores do not exceed 43%, the value found with the human lymphocyte protein. DdTRX1 was expressed in Escherichia coli, purified, and shown to have thioredoxin activity, as judged by its capacity to activate the NADP-malate dehydrogenase. Due to its life cycle, during which individual amoebae form a multicellular fruiting body, Dictyostelium is used to study developmental processes such as cell-type differentiation and regulation of gene expression. Transcript levels of Dictyostelium thioredoxins were regulated during the developmental cycle. Low levels of mRNAs could be detected during growth. After the onset of development, where essentially no cell divisions take place, message levels increased with maximal expression during aggregation. In later multicellular stages, RNA levels declined again. The same expression pattern could be seen for all cloned thioredoxins. Protein levels paralleled this time course with a delay of several hours as judged by Western blot and activity measurements.

Amino Acid Sequence↗
PubMed1985

In situ localization of actin mRNA in Dictyostelium discoideum aggregates.

By in situ hybridization of a labeled cloned DNA to cellular RNA, we have studied the distribution of actin mRNA in differentiating Dictyostelium discoideum aggregates. In migrating pseudoplasmodia, the mRNA is distributed uniformly. However, in culminating aggregates (sorogens) actin mRNA is enriched in stalk cells and in the cells at the anterior tip. Thus, the preferential synthesis of actin in prestalk and stalk cells, previously reported, is due at least partially to increased levels of actin mRNA.

Actins↗
PubMed1988

Nonenzymatic isolation and culture of adult islets from atrophic pancreata of copper-deficient rats: a morphologic analysis.

The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated, as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating effects on islet function.

Animals↗