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Results for “Poly(A) selection”

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PubMed2004

Factorial design, physicochemical characterisation and activity of ciprofloxacin-PLGA nanoparticles.

Poly(lactide-co-glycolide) nanoparticles incorporating ciprofloxacin HCl were prepared by means of a W/O/W emulsification solvent evaporation method. The stabiliser selected was poly(vinylalcohol). A 2(4) full factorial design based on four independent variables was used to plan the experiments and the variable parameters were the number of homogenisation cycles, addition of boric acid to the inner water phase containing the drug, ciprofloxacin concentration in the inner water phase and oil:outer water phase ratio. The effects of these parameters on the particle size, zeta potential, drug loading efficiency and drug release were investigated. Also the effect of gamma irradiation on the particle size and drug release was evaluated and DSC and XRD analyses of the compounds and the nanoparticles were performed. The activity on two series of microorganisms, Pseudomonas aeruginosa and Staphylococcus aureus, was examined.

Anti-Bacterial Agents↗

PubMed1979

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.

Animals↗

PubMed1985

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.

Animals↗

PubMed2001

Application of linear solvation energy relationships to polymeric pseudostationary phases in micellar electrokinetic chromatography.

Polymerized sodium 11-acrylamidoundecanoate (poly(Na 11-AAU)) was used as a pseudostationary phase (PSP) for micellar electrokinetic chromatography to separate uncharged compounds. The polymer PSP showed signifcantly different solute migration behaviors from conventional micelles including sodium dodecyl sulfate and poly (sodium 10-undecylenate), giving high separation efficiencies (>200000 theoretical plates/m). Linear solvation energy relationships were used to evaluate and characterize the chemical interactions that influence the retention behavior in the poly (Na 11-AAU) micellar system. It was found that the solute volume and solute hydrogen bond basicity mainly influenced the retention. The characteristic feature of the poly (Na 11-AAU) micellar system is that the micelle has a significantly higher capacity for dipole-dipole and dipole-induced dipole interactions as well as a slightly higher capacity for electron pair interactions than the aqueous phase. Due to its unique selectivity, the poly(Na 11-AAU) micellar system would become an attractive new option for selectivity optimization on methods development.

Chromatography↗

PubMed2006

Evaluation of the separation characteristics of application-specific (pesticides and dioxins) open-tubular columns for gas chromatography.

The solvation parameter model is used to characterize the retention properties of four application-specific open-tubular columns (Rtx-CLPesticides, Rtx-OPPesticides, Rtx-Dioxin and Rtx-Dioxin2) at five equally spaced temperatures over the range 60-140 degrees C. Cluster analysis is used to compare the system constants to a database of forty open-tubular columns characterized according to the same method. System constants differences and retention factor correlation plots are then used to determine selectivity differences between the application-specific columns and their nearest neighbors identified by cluster analysis. The Rtx-CLPesticides and Rtx-OPPesticides columns are shown to belong to the selectivity group containing poly(dimethylmethyltrifluoroprpylsiloxane) stationary phases with Rtx-OPPesticides having a similar selectivity to a poly(dimethylmethyltrifluoropropylsiloxane) stationary phase containing 20% methyltrifluoropropylsiloxane monomer (DB-200) and Rtx-CLPesticides separation properties for a stationary phase containing less than 20% methyltrifluoropropylsiloxane monomer. The Rtx-Dioxin and Rtx-Dioxin2 columns are located in the selectivity group dominated by the poly(dimethyldiphenylsiloxane) stationary phases containing less than 20% diphenylsiloxane monomer. The Rtx-Dioxin and Rtx-Dioxin2 columns are shown to be selectivity equivalent to a (5% phenyl) carborane-siloxane copolymer stationary phase (Stx-500) and a second generation silarylene-siloxane copolymer stationary phase containing dimethylsiloxane and diphenylsiloxane monomers (DB-XLB), respectively.

Chromatography, Gas↗

PubMed2004

Inhibition of poly(ADP-ribose) glycohydrolase by gallotannin selectively up-regulates expression of proinflammatory genes.

Poly(ADP-ribose)-polymerase-1 (PARP-1) and poly(ADP-ribose) (PAR) are emerging key regulators of chromatin superstructure and transcriptional activation. Accordingly, both genetic inactivation of PARP-1 and pharmacological inhibition of PAR formation impair the expression of several genes, including those of the inflammatory response. In this study, we asked whether poly(ADP-ribose) glycohydrolase (PARG), the sole depoly(ADP-ribosyl)ating enzyme identified so far, also regulates gene expression. We report the novel finding that inhibition of PARG by gallotannin triggered nuclear accumulation of PAR and concomitant PAR-dependent expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), but not of interleukin-1beta and tumor necrosis factor-alpha, in cultured RAW 264.7 macrophages. Remarkably, silencing of PARG by means of small interfering RNA selectively impaired gallotannin-induced expression of iNOS and COX-2. Consistent with a PAR-dependent transcriptional activation, increases of iNOS and COX-2 transcripts were not caused by activation of transcription factors such as nuclear factor-kappaB, activator protein-1, signal transducer and activator of transcription-1 or interferon regulatory factor-1, nor by mRNA stabilization. Overall, our data provide the first evidence that pharmacological inhibition of PARG leads to PAR-dependent alteration of gene expression profiles in macrophages.

Animals↗

PubMed1994

Evaluation of an ammonium ionophore for use in poly(vinyl chloride) membrane ion-selective electrodes: solvent mediator effects.

A macrotetrolide nonactin-based ammonium ionophore (AI) preparation containing 25% nonactin optimized for use in liquid membrane ion-selective electrodes was investigated for its possible use in poly(vinyl chloride) (PVC) matrix membrane-type electrodes. Nine different plasticizing solvent mediators were employed in the ammonium ion-selective membranes with PVC and the AI preparation. Near Nernstian responses were observed in aqueous solution with electrodes fabricated with dioctyl sebacate-, 2-nitrophenyl phenyl ether-, dibutyl sebacate-, trioctyl phosphate-, dioctyl adipate-, dioctyl phenyl phosphonate- and 2-nitrophenyl octyl ether-plasticized membranes. The responses of these electrodes were found to improve in the presence of a background concentration of non-interfering calcium ions.

Chemical Phenomena↗

PubMed2002

Apoptotic and genotoxic effects of a methyl sulfonate ester that selectively generates N3-methyladenine and poly(ADP-ribose) polymerase inhibitors in normal peripheral blood lymphocytes.

UNLABELLED: Selective N3-adenine methylation represents a novel strategy for tumors with a phenotype of poor responsiveness to a number of anticancer agents currently used in the clinic. Resistance to N3-methyladenine-inducing agents, such as MeOSO(2)(CH(2))(2)-lexitropsin (Me-Lex), is due to high levels of N-methylpurine glycosylase (MPG). However, tumor cells with high MPG activity can be rendered susceptible to Me-Lex using poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. PURPOSE: To evaluate the potential toxicity of Me-Lex, used as single agent or combined with PARP-1 inhibitors, in normal peripheral blood lymphocytes (PBL). METHODS: PBL either resting or activated with phytohemagglutinin (PHA), obtained from healthy donors, were treated with graded concentrations of Me-Lex with or without PARP-1 inhibitor (3-aminobenzamide, AB, or NU1025, NU). MPG activity, apoptosis and sister chromatid exchanges (SCE) were evaluated. RESULTS: (a) Me-Lex was cytotoxic mainly in PHA-activated PBL with low MPG activity; (b) combined treatment with Me-Lex and AB induced apoptotic effects as early as 24 h after drug exposure both in non-stimulated and PHA-activated PBL. When concentrations of PARP-1 inhibitors (25 microM NU and 4 m M AB) that produced a twofold increase in Me-Lex cytotoxicity in tumor cells were compared, NU induced a less-pronounced increase in apoptosis in PBL treated with Me-Lex; (c) Me-Lex at concentrations that allowed cytogenetic analysis did not induce a significant number of SCE; (d) PARP-1 inhibitors provoked a dose-dependent increase in SCE, but 25 microM NU was devoid of genotoxic effects and did not significantly increase SCE in PBL treated with Me-Lex. CONCLUSIONS: Me-Lex showed preferential cytotoxicity against mitogen-activated PBL. Our results also indicated that for each PARP-1 inhibitor it is necessary to define the concentration devoid of genotoxic effects in normal cells, but still capable of enhancing the efficacy of DNA-damaging agents in tumor cells.

Alkylating Agents↗

PubMed1981

Nucleosomes will not form on double-stranded RNa or over poly(dA).poly(dT) tracts in recombinant DNA.

We have been unable to "force" double-stranded RNA to fold into nucleosome-like structures using several different histone-RNA "reconstitution" procedures. Even if the histones are first stabilized in octameric form by dimethylsuberimidate cross-linking they are still unable to form specific complexes with the RNA. Moreover double-stranded RNA is unable to induce histones to assemble into octamers although we confirm that the non-nucleic acid homopolymer, polyglutamic acid, has this ability. We have also determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule. Thus nucleosomes cannot fold DNA containing an 80 base pair poly(dA).poly(dT) segment but a 20 base pair segment can be accommodated in nucleosomes fairly well. Segments of intermediate length can be accommodated but are clearly selected against. Poly(dA).poly(dT) differs only slightly from natural DNA in helix structure. Therefore either this homopolymer resists folding, or nucleosomes are very exacting in the nucleic acid steroid parameters they will tolerate. Such constraints may be relevant to nucleosome positioning in chromatin.

Animals↗

PubMed1996

The immunogenic and pathogenic potential of short poly(C) tract Mengo viruses.

We have shown previously that genetically engineered Mengo viruses with artificial deletions in their 5' noncoding polyribocytidylic acid (poly(C)) tracts are highly attenuated for the natural murine host and also for other animals such as baboons, macaques, and domestic pigs. The present report further characterizes select short poly(C) tract Mengo viruses in the natural murine host. A positive correlation was found between the length of the poly(C) tract and murine virulence, as measured by virus brain titers and brain lesion scores after infection. Histological examination of brain tissue collected from infected animals clearly showed that the short poly(C) tract viruses did not induce the devastating pathological effects characteristic of animals inoculated with wild-type virus. Instead, the short-tract Mengo viruses proved excellent immunological agents. A dose of only 100 plaque-forming units of vMC24 (poly(C) tract: C13UC10), injected subcutaneously, protected 80% of recipient animals against a normally lethal dose of encephalomyocarditis virus. The protection was long-lived, and animals similarly immunized with vMCo virus (poly(C) tract: Co) still had protective neutralizing antibody titers up to 16 months after inoculation. In addition, the short-tract viruses proved genetically stable, in that the vMC24 virus did not yield detectable pathogenic revertants even after multiple, forced passages in 4-week-old mice. These studies suggest that Mengo viruses containing deletions in their poly(C) tracts are biologically safe and potent immunogens and imply that they may have uses as cardiovirus vaccines.

Animals↗

PubMed1978

Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells.

The structure of the 5'-termini has been investigated in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 microgram/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA.

Base Sequence↗

PubMed1979

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.

Animals↗

PubMed1976

The translational capacity of deadenylated ovalbumin messenger RNA.

We present evidence that the poly(A) sequence at the 3' end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system. Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact. It is shown that the stability of the mRNA in a cell free system is not appreciably affected by this procedure. Measurements of the size of ovalbumin-synthesizing polysomes, rate of peptide elongation, and number of rounds of translation per messenger show a generally reduced efficiency for deadenylated mRNA compared to native mRNA. No comparable difference was observed in experiments with a wheat germ cell-free system, which gives few rounds of translation per mRNA. This indicates that the effect results from a lowering of the efficiency of reinitiation on deadenylated mRNA.

Base Sequence↗

PubMed2006

Macroporous condensed poly(tetrafluoroethylene). I. In vivo inflammatory response and healing characteristics.

This study was designed to determine whether the novel spatial geometry of macroporous condensed poly(tetrafluoroethylene) (cPTFE) favorably affects the in vivo repair process. Specifically, the macroporous surface geometry and the reduced material thickness contribute to better healing characteristics. For this purpose, three other materials used for abdominal wall repair were selected, expanded poly(tetrafluoroethylene) (ePTFE), low-weight poly(propylene) (lwPP), and high-weight poly(propylene) (hwPP). Samples of each material (1 x 2 cm, n = 4) were implanted subcutaneously in rats for 7, 28, or 56 days. After sacrificing the animals, at each time point, the tissue implant sites were subjected to morphometric analysis and evaluation of inflammatory and wound-healing tissue characteristics. Although the fibrous capsule thickness did not significantly vary among the four materials (p > 0.05), cPTFE consistently led to the most mature fibrous capsule. However, ePTFE showed the greatest tissue-material integration. Both PP materials presented various levels of tissue integration, but they were characterized by significant early inflammation and increased foreign body reaction around the mesh openings, especially for hwPP. In contrast, cPTFE did not induce extensive inflammation or elevated foreign body reaction around its mesh openings. We conclude that cPTFE combines the inherent PTFE biocompatibility with low polymer surface area (large mesh openings, reduced material thickness) leading to a better inflammatory and wound healing response compared to available materials used in abdominal wall reconstruction.

Abdominal Wall↗

PubMed2004

Conformational effect of silica-supported poly(octadecyl acrylate) on molecular-shape selectivity of polycyclic aromatic hydrocarbons in RP-HPLC.

Poly(octadecyl acrylate) with a terminal reactive group was synthesized by radical telomerization in various solvents. The polymers were grafted onto porous silica for use in RP-HPLC, and the molecular recognition ability was investigated along with the selectivity for the structural isomers of polycyclic aromatic hydrocarbons. The mechanism of selectivity was also investigated with differential scanning calorimetry and NMR spectroscopic observations.

Journal Article↗

PubMed2003

U2AF modulates poly(A) length control by the poly(A)-limiting element.

The poly(A)-limiting element (PLE) restricts the length of the poly(A) tail to <20 nt when present in the terminal exon of a pre-mRNA. We previously identified a 65 kDa protein that could be cross-linked to a functional PLE, but not to an inactive mutant element. This binding was competed by poly(U) and poly(C), but not poly(A) or poly(G). Selectivity for the pyrimidine-rich portion of the PLE was demonstrated by RNase footprinting of the binding activity in total nuclear extract. A 65 kDa protein that selectively cross-linked to the functional PLE was purified by conventional chromatography and identified as the large subunit of U2 snRNP auxiliary factor (U2AF). Overexpression of U2AF65 in cells transfected with a PLE-containing reporter construct resulted in the appearance of a population of mRNAs with heterogeneous poly(A) tails. However, this effect was lost following deletion of the C-terminal RNA recognition motifs (RRMs). A C-->G mutation following the AG dinucleotide in the PLE resulted in mRNA with poly(A) ranging from 25-50 nt. This reverted to a discrete, <20 nt poly(A) tail in cells expressing U2AF65. Our results suggest that U2AF modulates the function of the PLE, perhaps by facilitating the binding of another protein to the element.

Animals↗

PubMed1984

Interaction of Z form poly(dG-dC).poly(dG-dC) with divalent metal ions: localization of the binding sites by I.R. spectroscopy.

The secondary structures of poly(dG-dC).poly(dG-dC) in the presence of alcaline , alcaline earth and first row transition metal ions (Na+, Mg2+, Co2+, Ni2+) are investigated by infrared spectroscopy. The conformational transitions are studied as a function of the hydration of the polynucleotide and counter-ion nature and content. The use of selectively deuterated poly(dG-dC).poly(dG-dC) on the 8-carbon of guanines allows to show that a direct interaction occurs between the N7 site of guanines and the transition metal ions Co2+ and Ni2+. In the case of Mg2+, for high ion/nucleotide ratios, the interaction occurs essentially at the level of the phosphate groups. This interaction leads to a modification of the left-handed conformation. Based on the IR spectroscopy results, an explanation is proposed for the different efficiencies of these various ions to induce the B----Z transition.

Cations, Divalent↗

PubMed2001

Monodisperse plasticized poly(vinyl chloride) fluorescent microspheres for selective ionophore-based sensing and extraction.

A convenient method for the preparation of monodisperse, plasticized poly(vinyl chloride) particles based on an automated particle casting technique is described. The particles are made highly selective for a number of ions by doping them with ionophores and other active components, in complete analogy to thin-film or fiber-optic chemical sensors. The approach used here produces spheres of high monodispersity at a rate of approximately 20000 particles/s. The casting process is based on a reproducible polymer drop formation and precipitation process, and the particles are formed under very mild, nonreactive conditions. This allows one to conveniently incorporate known amounts of different active components into the polymers. As an initial example, the particles are doped with three optical sensing components, the sodium ionophore tert-butylcalix[4]arene tetraethyl ester, the H+-chromoionophore ETH 5294, and the anionic additive sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate. The particles are found to be of spherical shape with a diameter of approximately 10 microm. They respond individually and selectively to sodium according to classical optode theory, as determined by fluorescence microscopy. With a RSD of 1.6%, sensing reproducibility from particle to particle is excellent. This technique may allow the development of mass-produced chemically selective microspheres on the basis of bulk extraction processes.

Algorithms↗