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At least 613 records · Page 34Linked to original sources
PubMed1979

Studies of the replication of a bovine enterovirus RNA.

Replicative intermediate (RI), replicative form (RF) and single-stranded (SS) RNA have been isolated from BHK cells infected with a bovine enterovirus by salt precipitation and gel filtration techniques. Kinetic experiments showed that at no time up to 16 h post-infection (p.i.) did the amount of RF exceed that of RI or SS RNA. Electrophoresis of RF on 1.5% polyacrylamide-agarose gels showed that at least three species of double-stranded RNA were present, one of which was associated with an accessible poly(A)-containing tract. All of the RF was denatured by 99% dimethylsulphoxide (DMSO), although reannealling occurred rapidly when samples were returned to aqueous conditions. No evidence for circular structures in the RF molecular population was found by use of caesium sulphate density gradients containing ethidium bromide. Treatment of RI with ribonuclease produced double-stranded RNA molecules, some of which were smaller in size than intact RF. Denaturation with DMSO and analysis on 99% DMSO sucrose gradients showed that the RI did not contain single strands of greater length than virion RNA. A portion of the RI bound to poly(U)-Sepharose 4B columns. The poly(A) tracts involved were present only in the nascent RNA strands with greatest sedimentation coefficients (30 to 35S). Bovine enterovirus induced SS RNA was heterogeneous with regard to both sedimentation through sucrose gradients and mobility on acrylamide gels compared to purified virion RNA. The reason for this difference has never been satisfactorily resolved. Sedimentation through 99% DMSO-sucrose gradients showed that the heterogeneity was due to aggregation rather than any variation in chain length or conformational differences. Our results support the single-stranded template model rather than a circular model for picornavirus RNA replication.

Animals↗
PubMed2000

Interactions of the C-terminus of viral protein R with nucleic acids are modulated by its N-terminus.

The basic viral protein R (Vpr) performs several functions during the human immunodeficiency virus HIV-1 retroviral cycle, including G2 mitosis arrest and nuclear import of the preintegration complex allowing lentivirus to replicate in nondividing cells. Accordingly, this protein was found in the nucleus of infected cells. In the virus, Vpr is incorporated through interaction with both nucleocapsid protein 7 (NCp7) and p6, two small proteins encoded by the C-terminal part of the Gag precursor. NCp7 is also involved in genomic RNA encapsidation during the budding process suggesting a possible interaction of Vpr with nucleic acids, either directly or via the NCp7 intermediate. Gel shift experiments were carried out with RNA and DNA using synthetic Vpr and peptide derivatives. The results show that Vpr binds to nucleic-acid inducing aggregates. This process, which requires the C-terminal basic domain of the protein (in particular the helical 70-80 domain), is regulated by the N-terminal region of Vpr. Moreover, NCp7 was shown to enhance RNA recognition by Vpr, a feature that could be required for Vpr encapsidation and during nuclear import of the preintegration complex.

Amino Acid Sequence↗
PubMed1994

Expression and characterization of a Dictyostelium discoideum annexin.

The annexins are calcium-dependent phospholipid-binding proteins. Recently the gene encoding the homologue of a mammalian annexin has been identified in Dictyostelium discoideum. Analysis of cDNA and genomic clones showed that the transcript for Dictyostelium annexin is alternatively spliced (Greenwood, M. and Tsang, A. (1991) Biochim. Biophys. Acta 1088, 429-432; Döring, V., Schleicher, M and Noegel, A. (1991) J. Biol. Chem. 266, 17509-17515). Here, we showed that the Dictyostelium annexin DNA hybridized to two populations of transcripts. We used a recombinant annexin polypeptide to raise polyclonal antibody. Immunoblot analysis revealed that the antibody recognized two polypeptides of 48 kDa and 54 kDa in developing D. discoideum cells. The molecular sizes of these polypeptides correspond well with the expected sizes of the alternatively spliced products. The 48-kDa and 54-kDa polypeptides were purified by isoelectric focusing to more than 70% homogeneity. The partially purified proteins were found to associate with phosphatidylserine vesicles in a calcium-dependent manner. These results suggest that the 48- and 54-kDa polypeptides are the products of alternative splicing of the annexin transcripts. During development the two polypeptides accumulate at different rates to about 60 times the level detected in vegetative cells. On the other hand, RNA blot analysis showed that the level of the annexin transcripts in multicellular aggregates was about 5 times that of vegetative cells.

Alternative Splicing↗
PubMed1969

Control of tyrosine aminotransferase synthesis in tissue culture by a factor in serum.

The hormonal induction of tyrosine aminotransferase in an established line of rat hepatoma cells is enhanced by the presence of a macromolecular component of serum in the inducing medium. The addition of serum to cells previously induced in serum-free medium results in a rapid two- to threefold increase in the rate of tyrosine aminotransferase synthesis, as measured by specific radio-immunoprecipitation techniques, as well as a smaller increase in over-all protein synthesis. The stimulation of protein synthesis by serum is accompanied by an increase in the proportion of ribosomes sedimenting as polysomal aggregates. The increase in the rate of TAT synthesis is largely independent of RNA synthesis as it is insensitive to antinomycin D, suggesting that the serum acts at a site beyond gene transcription. The maximum effect of serum on TAT synthesis requires the continued presence of the hormone inducer.

Blood Proteins↗
PubMed1989

Dictyostelium discoideum contains a gene encoding a myosin I heavy chain.

We have cloned and completely sequenced a gene encoding the heavy chain of Dictyostelium myosin I. Like the myosin I molecules from Acanthamoeba, the Dictyostelium myosin I heavy chain is composed of a globular head domain fused to a 45-kDa glycine-, proline-, and alanine-rich carboxyl-terminal domain, rather than the coiled-coil rod domain of conventional myosins. Comparisons of the Dictyostelium myosin I heavy-chain amino acid sequence with those of the Acanthamoeba myosins I reveal that they are highly similar throughout, including the unconventional carboxyl-terminal domains. The Dictyostelium myosin I gene is expressed in growing cells as a 3600-nucleotide mRNA. Measurements of the steady-state level of this mRNA at different times during starvation-induced aggregation and development are consistent with a role for myosin I in chemotaxis and aggregation. Generation of Dictyostelium cells lacking myosin I by gene disruption and/or antisense RNA production should provide a way to test directly the role of this nonfilamentous myosin in cell motility. These experiments will be simplified by the fact that Southern blot analyses of Dictyostelium genomic DNA are consistent with there being a single myosin I heavy-chain gene.

Amino Acid Sequence↗
PubMed1976

The effect of epsilon-aminocaproic acid on biochemical changes in the development of the cellular slime mould Dictyostelium discoideum.

epsilon-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain AX2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of N-acetylglucosaminidase, alpha-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells.

4-Aminobenzoic Acid↗
PubMed2005

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase.

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

Acetylcholine↗
PubMedDate not supplied

[Preparation of single-chained antibodies to human ferritin in Escherichia coli].

We have cloned cDNA copies of the immunoglobulin heavy and light chain genes from hybridoma cells able to produce antibody against human ferritin. Variable segments of these genes were obtained using the polymerase chain reaction (PCR). The specific amplifications of ligase reaction products were carried out to combine the variable segments with DNA fragments coding for a peptide-linker and for a signal peptide of the cloned pelB gene of Erwinia carotovora. During the antiferritin single-chain antibody gene expression under the T7 RNA polymerase control in E. coli the processed molecules of recombinant proteins formed aggregates in periplasm. The reversible denaturation in the absence of reducing agents had allowed us to obtain single-chain antibodies with the original binding specificity toward human ferritin.

Amino Acid Sequence↗
PubMed2005

Neurodegeneration caused by the translation of nonsense: does macromolecular misfolding impair the synchrony of gene expression?

Here, it is proposed that the principal event underlying neurodegeneration occurs when cytotoxic, truncated proteins are expressed from normally-untranslated nonsense RNA and pseudogene transcripts. The proximal event occurs when a small fraction of the total pool of gene expression machinery within the cell is disrupted by rare events of macromolecular misfolding during gene expression. Macromolecular misfolding, such as beta-sheet formation of protein leading to intracellular aggregation, has been implicated in a number of neurodegerative diseases. As gene expression is a synchronised series of processes from the nucleus to the cytoplasm, should macromolecular misfolding occur in any given component of the gene expression apparatus, co-dependent gene expression processes could become disrupted. For example, should proteins misfold during their own translation, aggregates could accumulate within the translation machinery and disrupt the regulation of upstream gene expression events, such as RNA splicing or Nonsense Mediated Decay. Although only a limited amount of gene expression machinery would be affected by macromolecular misfolding, the resultant loss in fidelity could allow sufficient levels of expression of aberrant proteins from nonsense RNA and pseudogene transcripts to produce cytotoxic effects within the cell over time and ultimately lead to neurodegeneration.

Gene Expression↗
PubMed1985

Ultrasonic absorption in tobacco mosaic virus and its protein aggregates.

The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein assemblies exhibit specific structural fluctuations detected in ultrasonic experiments. As in spherical viruses, the fluctuations exhibited by the protein aggregates having a quaternary structure similar to that of the virion were modified in the virus by interaction with the RNA strand. It is unlikely that the origin for the observed effect is due either to: (1) the difference in local mobility of the segment 89 to 113 of the polypeptide chain in TMV and in the helical aggregate on the one hand, and in smaller aggregates, on the other hand; or (2) a local fluctuation associated with proton transfer reactions or ion-pair interactions. The most remarkable feature in the TMV system is the fact that the two-ring disk showed no excess of ultrasonic absorption with respect to the A-protein oligomer, while a large increase of ultrasonic absorption was observed in the rod-like aggregate that had undergone the disk-helix transition.

Hydrogen-Ion Concentration↗
PubMed1991

Transcription of a stem/loop region of a tumour-amplified sequence induces bacterial aggregation.

A sequence of human DNA, amplified in a lung tumour, was shown to induce expression-dependent effects on Escherichia coli phenotype. Previous studies have demonstrated the induction of abnormal plasmid supercoiling and bacterial aggregation in host strains harbouring various constructs encoding this region. Subcloning identified a short stretch of 50 bp crucial for these effects. This study details further characterization of the active sequence. Computer analysis of the region predicted the formation of a stem/loop structure in transcribed RNA. Transcription, in the absence of translation, of a 22 bp sequence comprising this structure was sufficient to induce bacterial aggregation. Site-directed and random mutagenesis of the sub-region were carried out in order to identify critical factors in the induction of this phenotypic shift. It was found that changes in the loop sequence modulated activity, and mutations increasing predicted stem stability produced more active constructs. The wild-type sequence induced high level aggregation in only a limited number of strains but using the mutagenesis data, a sequence was synthesized that induced high level aggregation in most E. coli strains tested.

Bacterial Adhesion↗
PubMed1988

[Effect of the heat shock reaction on the phenotypic manifestation of mutations in rifampicin resistance affecting the gene of the RNA polymerase beta-subunit under the control of the lactose promotor].

Plasmids bearing the rifampicin-resistant RNA polymerase beta-subunit gene under control of the lac promoter failed to provide Escherichia coli cells with resistance to rifampicin, despite the accumulation of large quantities of the resistant subunit. The effect proved to be connected with the aggregation of the plasmid-borne subunit. Conditions that induce synthesis of heat-shock proteins make the plasmid-containing cells resistant to rifampicin. This finding suggests that heat-shock proteins prevent the formation of protein aggregates.

DNA-Directed RNA Polymerases↗
PubMed2006

Novel sequence variants in dysferlin-deficient muscular dystrophy leading to mRNA decay and possible C2-domain misfolding.

Mutations in the gene encoding dysferlin (DYSF) cause the allelic autosomal recessive disorders limb girdle muscular dystrophy 2B and Miyoshi myopathy. It encompasses 55 exons spanning 150 kb of genomic DNA. Dysferlin is involved in membrane repair in skeletal muscle. We identified three families with novel sequence variants in DYSF. All affected family members showed limb girdle weakness and had reduced or absent dysferlin protein on immunohistochemistry. All exons of DYSF were screened by genomic sequencing. Five novel variants in DYSF were found: two missense mutations (c.895G>A and c.4022T>C), one 5' donor splice-site variant (c.855+1delG), one nonsense mutation (c.1448C>A), and a variant in the 3'UTR of DYSF (c.*107T>A). All alterations were confirmed by restriction enzyme analysis and not found in 400 control alleles. Nonsense mediated RNA decay or changes in the three-dimensional protein structure resulting in intracellular dysferlin aggregates and finally the lack of dysferlin protein were identified as consequences of the novel DYSF variants.

Adult↗
PubMed2003

Upstream signaling pathways leading to the activation of double-stranded RNA-dependent serine/threonine protein kinase in beta-amyloid peptide neurotoxicity.

One of the hallmarks of Alzheimer's disease is extracellular accumulation of senile plaques composed primarily of aggregated beta-amyloid (Abeta) peptide. Treatment of cultured neurons with Abeta peptide induces neuronal death in which apoptosis is suggested to be one of the mechanisms. We have demonstrated previously that Abeta peptide induces activation of double-stranded RNA-dependent serine/threonine protein kinase (PKR) and phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in neurons in vitro. Degenerating neurons in brain tissues from Alzheimer's disease patients also displayed high immunoreactivity for phosphorylated PKR and eIF2alpha. Our previous data have also indicated that PKR plays a significant role in mediating Abeta peptide-induced neuronal death, because neurons from PKR knockout mice and neuroblastoma SH-SY5Y cells stably transfected with dominant negative mutant of PKR are less susceptible to Abeta peptide toxicity. Therefore, it is important to understand how PKR is activated by Abeta peptide. We report here that inhibition of caspase-3 activity reduces phosphorylation of PKR and to a certain extent, cleavage of PKR and eIF2alpha in neurons exposed to Abeta peptide. Calcium release from the endoplasmic reticulum and activation of caspase-8 are the upstream signals modulating the caspase-3-mediated activation of PKR by Abeta peptide. Although in other systems HSP90 serves as a repressor for PKR, it is unlikely the candidate for caspase-3 to affect PKR activation in neurons after Abeta peptide exposure. Elucidation of the upstream pathways for PKR activation can help us to understand how this kinase participates in Abeta peptide neurotoxicity and to develop effective neuroprotective strategy.

Amyloid beta-Peptides↗
PubMed1977

Oligo(dG)12-18 aggregates result in non-homogeneity of oligo(dG)12-18.poly(C) type primer-template.

Studies on the absorption spectra of equimolar solutions of oligo(dG)12-18 and poly(C), poly(Cm) or poly(Ce) showed that only 13%, 3% and 3% of base pairs, repectively, form complexes. Upon centrifugation of oligo(dG)12-18 with a molar excess of poly(C) of poly(Cm) in an analytical and a preparative centrifuge, it was found that only a part of oligodeoxynucleotide sediments with the polynucleotide, i.e. more rapidly than oligo(dG)12-18, poly(C) or poly(Cm). Products of binding of oligo(dG)12-18 with poly(C), poly(Cm) or poly (Ce) direct the synthesis of poly (dG) by AMV reverse transcriptas in accordance with the reported characteristics of these primer-templates, as well as of the enzyme. These observations suggest that the solutions of oligo(dG)12-18 with (polyC) or its analogues, commonly used as primer-templates of RNA- and DNA-directed DNA polymerases, contain a polynucleotide, to which oligo(dG)12-18 aggregates are bound through a few nucleotides chains shorter than 12-18 residues. These chains of oligo(dG)12-18 containing the 3'-OH ends are capable of of initiating the reaction with DNA POLYMERASES.

Avian Myeloblastosis Virus↗
PubMed1980

The effect of diabetes and insulin on the polyadenylic acid-containing RNA of rat skeletal muscle.

Poly(A)-containing RNA in various fractions of RNA from rat skeletal muscle has been detected and quantitated by hybridization to [3H]poly(U). Comparison has been made between the RNA in skeletal muscle from normal adult rats, from rats 2 days after induction of diabetes with streptozotocin, and from diabetic rats killed 60 min after injection of insulin. The poly(A)-containing RNA constituted a similar proportion of the total RNA in skeletal muscle from each of the three types of rats. In diabetes there was a decrease in the proportion of skeletal muscle ribosomes sedimenting as polyribosomes, but this was reversed after rats had been injected with insulin. However these changes were not associated with any alterations in the relative amounts of poly(A)-containing RNA in ribosomes isolated from rat skeletal muscle. Diabetes did not significantly alter the size distribution of the poly(A)-containing RNA or its poly(A) segment. If it is assumed that poly(A)-containing RNA is mRNA, these results imply that insulin stimulates protein synthesis in the skeletal muscle of diabetic rats by affecting the translation of pre-existing mRNA. Sucrose density gradient analysis showed that there was less poly(A)-containing RNA in the polyribosome region from diabetic rats than there was in that from normal rats. The balance of the poly(A)-containing RNA in the ribosomes from diabetic rats (presumed to be the untranslated mRNA) was found associated with rapidly sedimenting aggregated ribosomes, but its native form has not yet been determined.

Animals↗
PubMed2003

Molecular epidemiological and clinical aspects of hepatitis D virus in a unique triple hepatitis viruses (B, C, D) endemic community in Taiwan.

The molecular epidemiological and clinical aspects of hepatitis D virus (HDV) in a unique HBV, HCV, and HDV triple virus endemic community in southern Taiwan were investigated. A total of 2,909 residents aged 45 or older were screened for hepatitis B surface antigen (HBsAg), anti-HCV antibody, and anti-HDV antibody (specifically for HBsAg-positive carriers). Factors that might be associated with HDV infection, viral nucleic acid detection, and genotyping of HBV, HCV, and HDV were investigated. The prevalence of HBsAg and anti-HCV were 12.6% (366/2,909) and 41.6% (1,227/2,909), respectively. For HBsAg carriers, 15.3% (56/366) were positive for anti-HDV assay. Living in a higher endemic district of HCV infection (odds ratio [OR] = 3.2; 95% confidence interval [CI] = 1.7-6.3), male gender (OR = 1.9; 95% CI = 1.1-3.6) and co-infection with HCV (OR = 1.8; 95% CI = 1.0-3.3) were significantly independent factors associated with HDV infection. The detection rate of HDV RNA among anti-HDV-positive patients was only 12.7% (7/55). The mean HBV titer of triple infection group was significantly lower than in the HBV/HDV co-infection group (2.23 vs 3.05 in log(10), copies/ml, P = 0.046). HCV RNA detection among the triple infection group showed 47.4% (9/19) viremia rate and viral loads of 579,121 IU/ml in median (16,803-1,551,190 IU/ml). The prevalent genotype of HBV was type B (23/25); HCV was 1b (7/9) and HDV was IIa/IIb (4/4). Only the presence of HCV RNA predicted the presence of elevated ALT significantly (OR = 25.0; 95% CI = 3.39-184.6). In conclusion, the geographical aggregation of HDV infection paralleled that of HCV infection in this community. HCV suppressed the replication of HBV among triple vital infection patients. HBV and HDV lapsed into a remission or nonreplicative phase in most cases, and HCV acted as a dominant factor in triple viral-infected individuals. Only the presence of HCV RNA was associated with elevated ALT values, but not HBV or HDV.

Female↗