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PubMed2000

A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease.

We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m(7)G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure and poly(A) degradation at the 3' end of the RNA.

Adenosine Monophosphate↗
PubMed1991

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.

Acetaldehyde↗
PubMed1980

Poly(A) polymerase activity in ethionine-intoxicated rats: the relative effectiveness of disaggregated and intact polyribosomes as primers for poly(A) polymerase.

Ethionine intoxication causes a change in the metabolism of poly(A) sequences on the 3' OH terminus of mRNA in rat liver in vivo. In an attempt to determine the factors responsible for these changes, nuclear and cytoplasmic poly(A) polymerase activities and the state of the primer were examined in vitro. Requirements for optimal enzyme activities were determined. The nuclear and cytoplasmic enzymes had different K+, Mn2+, and poly(A) primer optima. The levels of nuclear and cytoplasmic poly(A) polymerase activity were shown to decrease following ethionine intoxication. Poly(A)+ RNA isolated from the livers of saline- and ethionine-treated rats served equally well as primers for the cytoplasmic poly(A) polymerase. Disaggregated polysomes were seven times more effective as primers than were intact polysomes. The results suggest that the mRNP particle which is released from polysomes as a result of ethionine intoxication functions better as a poly(A) polymerase primer than does the intact polysome.

Animals↗
PubMed2003

Synthesis and characterization of poly(L-alanine)-block-poly(ethylene glycol) monomethyl ether as amphiphilic biodegradable co-polymers.

Di-block co-polymers of poly(L-alanine) with poly(ethylene glycol) monomethyl ether (MPEG) were synthesized as amphiphilic biodegradable co-polymers. The ring-opening polymerization of N-carboxy-L-alanine anhydride (NCA) in dichloromethane was initiated by amino-terminated poly(ethylene glycol) monomethyl ether (MPEG-NH2, M(n) = 2000) to afford poly(L-alanine)-block-MPEG. The weight ratio of two blocks in the co-polymers could be altered by adjusting the feeding ratio of NCA to MPEG-NH2. Their chemical structures were characterized on the basis of infrared spectrometry and nuclear magnetic resonance. According to circular dichroism measurement, the poly(L-alanine) chain on the co-polymers in an aqueous medium had a alpha-helix conformation. Two melting points from MPEG block and poly(L-alanine), respectively, could be observed in differential scanning calorimetry curves of the co-polymers, suggesting that a micro-domain phase separation appeared in their bulky states. The co-polymers could take up some water and the capacity was dependent on the ratio of poly(L-alanine) block to MPEG. Such co-polymers might be useful in drug-delivery systems and other biomedical applications.

Biocompatible Materials↗
PubMed2004

Biomedical applications of chemically and microbiologically synthesized poly(glutamic acid) and poly(lysine).

This review article deals with the synthesis, physiochemical properties, and potential biomedical applications of two homo-poly amino acids. Poly-alpha-glutamic acid (alpha-PGA) and poly-alpha-lysine (alpha-PL) were synthesized by chemical synthesis. poly-gamma-glutamic acid (gamma-PGA) and poly-epsilon-lysine (epsilon-PL) were naturally occurring bio-materials that were produced by microbial fermentation. Poly(glutamic acid) (PGA) and poly(lysine) (PL) are water soluble, biodegradable, edible and nontoxic toward humans and the environment. As a result, they are suitable for various applications and have recently attracted considerable interest of the chemical industry. The distinguished features of PGA and PL also make them promising candidates for biomedical applications. The applications of PGA and PL in the areas of biomedical materials, drug delivery carriers and biological adhesives have been studied extensively and will be discussed in this review.

Adhesiveness↗
PubMed2003

Biosynthesis of poly (3-mercaptopropionate) and poly (3-mercaptopropionate-co-3-hydroxybutyrate) with recombinant Escherichia coli.

Polythioesters newly emerged as a type of novel polymer and they have showed great potential for application in industries. In this study, genes of butyrate kinase (buk) and phosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and poly (3-hydroxybutyrate) (PHB) synthase gene from Thiocapsa pfennigii were used for construction of a metabolic pathway to synthesize the polythioesters. When 3-mercaptopropionate and 3-hydroxybutyrate were fed, poly (3-mercaptopropoinate) [poly (3MP)] and poly(3-mercaptopropionate-co-3-hydroxybutyrate) [poly(3MP-co-3HB)] were synthesized by recombinant Escherichia coli JM109 (pBPP1) harboring the constructed metabolic pathway. Results indicated clearly that all these genes are necessary for the synthesis of poly(3MP) and poly(3MP-co-3HB).

3-Hydroxybutyric Acid↗
PubMed1994

In vitro translation of an intact mRNA coding for a poly(U), poly(C) specific ribonuclease isolated from six-day-old larvae of Ceratitis capitata by a modified extraction procedure.

Intact ribonucleic acid was prepared from six-day-old larvae of Ceratitis capitata, by enriching the guanidinium thiocyanate extraction procedure with a specific mixture for the active ribonuclease inhibition. RNA obtained by this means was then used as a source for the identification of mRNA coding for poly(U), poly(C) specific ribonuclease. The isolated poly(A+) RNA was translated in a cell-free protein synthesizing system. The presence of a poly(U), poly(C) ribonuclease among the newly synthesized products was detected by immunoprecipitation with anti-rabbit polyclonal antibodies against poly(U), poly(C) ribonuclease.

Animals↗
PubMed1989

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.

Base Sequence↗
PubMed1988

The role of a template sugar-phosphate backbone in the ribosomal decoding mechanism. Comparative study of poly(U) and poly(dT) template activity.

To study the role of a template sugar-phosphate backbone in the ribosomal decoding process, poly(U), poly(dT) and poly(dU)-directed cell-free amino acid incorporation was investigated under the influence of neomycin and high concentrations of Mg2+. The specificity of a factor-dependent translation system of Escherichia coli was shown to change according to the principle: "either ribo- or deoxyribopolynucleotide messenger". Poly(dT) is shown to be effectively translated in the absence of elongation factors, both at low (2 degrees C) and high (37 degrees C) temperature. Neomycin inhibits factor-free poly(dT) translation. Little or no poly(U) translation is observed in this system. A chromatographic analysis of the oligophenylalanine residues synthesized seems to show that translocation is the main step responsible for ribosome specificity to the ribo- or deoxyribopolynucleotide template in both factor-dependent and factor-free translation systems.

Cell-Free System↗
PubMed1976

Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells.

An antibody has been prepared which is highly specific for poly(adenosine diphosphate-ribose). Neither poly(A), DNA, nor a variety of adenine-containing nucleosides or nucleotides were effective in competing with poly(ADP-ribose) for binding to the antibody. Of all compounds tested, only adenosine diphosphate-ribose competed for binding to the antibody. Unlabeled poly(adenosine diphosphate-ribose) was about 10 000 times more effective in competing with labeled polymer for antibody binding than was adenosine diphosphate-ribose. Using the antibody, the amount of poly(adenosine diphosphate-ribose) was found to increase from early S phase to a peak at mid S with a second, even larger increase seen at the S-G2 transition point in synchronously dividing HeLa cells. Pulse labeling of the polymer with [2-3H]adenosine was also maximal at the same time points. Changes in the levels of poly(adenosine diphosphate-ribose) polymerase activity measured in isolated nuclei coincided with the changes in amounts of polymer present in intact cells during progression from S phase into G2.

Adenine Nucleotides↗
PubMed1975

Poly(8-aminoguanylic acid): formation of ordered self-structures and interaction with poly(cytidylic acid).

Poly(8-aminoguanylic acid) has in neutral solution a novel ordered structure of high stability. The 8-amino group permits formation of three hydrogen bonds between two residues along the "top", or long axis, of the purines. The usual hydrogen bonding protons and Watson-Crick pairing sites are not involved in the association. The bonding scheme has a twofold rotation axis and is hemiprotonated at N(7). Poly(8NH2G) is converted by alkaline titration (pK = 9.7) to a quite different ordered structure, which is the favored form over the range approximately pH 10-11. The bonding scheme appears to be composed of a planar, tetrameric array of guanine residues, in which the 8-amino group does not participate in interbase hydrogen bonding. Poly (8NH2G) does not interact with poly(C) in neutral solution because of the high stability of the hemiprotonated G-G self-structure. Titration to the alkaline plateau, however, permits ready formation of a two-stranded Watson-Crick helix. In contrast to the monomer 8NH2GMP, poly(8NH2G) does not form a triple helix with poly(C) under any conditions. The properties of the ordered structures are interpreted in terms of a strong tendency of the 8-amino group to form a third interbase hydrogen bond, when this possibility is not prevented by high pH.

Binding Sites↗
PubMed2003

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Amino Acid Sequence↗
PubMed1989

Structure of the beta-form of poly d(A).poly d(U).

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.

DNA↗
PubMed1994

A controlled clinical trial with a specifically configured RNA drug, poly(I).poly(C12U), in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a physically debilitating illness associated with immunologic abnormalities, viral reactivation, and impairment of cognition. In a randomized, multicenter, placebo-controlled, double-blind study of 92 patients meeting the CFS case definition of the Centers for Disease Control and Prevention, the response of several laboratory and clinical variables to an antiviral and immunomodulatory drug, poly(I).poly(C12U), was determined. Measures of clinical response included Karnofsky performance score, a cognition scale derived from a self-administered instrument assessing symptomatology (SCL-90-R), an activities of daily living scale, and exercise treadmill performance. After 24 weeks, patients receiving poly(I).poly(C12U) had higher scores for both global performance and perceived cognition than did patients receiving placebo. In particular, patients given poly(I).poly(C12U) had increased Karnofsky performance scores (P < .03), exhibited a greater ability to do work during exercise treadmill testing (P = .01), displayed an enhanced capacity to perform the activities of daily living (P < .04), had a reduced cognitive deficit (P = .05), and required less use of other medications (P < .05).

Activities of Daily Living↗
PubMed1980

Thermal stability of poly(A) and poly(U) complexes in H2O and D2O: isotopic effects on critical temperatures and transition widths.

This is a study of the effect of total and partial deuteration of solvent on critical temperatures and profiles of all four reactions occurring in poly(A) x n poly(U) (n = 1 or 2) aqueous systems. The study was done at observational times not longer than hydrogen exchange times at base pairs in helically ordered structures, and it was extended to a wide range of salt concentrations at neutral pH. The dependence of stability of polymer helical order on hydrogen mass does not appear to be merely attributable to the stronger intrahelical deuterium bonding. Substituting Deuterium for Hydrogen implies a probably predominant modulation of the entrophy term of polymer-solvent interactions. Effects of deuteration on the width of the 2(poly(A) x poly (U)) leads to poly(A) x 2poly(U)+poly(A) interconversion reaction were also observed. They bear on the role of polymer-solvent interaction on pattern recognition leading to formation of ordered structures. They also bear on the role of the same interaction on the "breathing" of ordered structures of this type.

Deuterium↗
PubMed2005

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats.

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.

Aging↗
PubMed2004

The interferon inducer ampligen [poly(I)-poly(C12U)] markedly protects mice against coxsackie B3 virus-induced myocarditis.

Viral replication, as well as an immunopathological component, is assumed to be involved in coxsackie B virus-induced myocarditis. We evaluated the efficacy of the interferon inducer Ampligen on coxsackie B3 virus-induced myocarditis in C3H/HeNHsd mice. The efficacy of Ampligen was compared with that of the interferon inducer poly(inosinic acid)-poly(cytidylic acid) [poly(IC)], alpha interferon 2b (INTRON A), and pegylated alpha interferon 2b (PEG-INTRON-alpha-2b). Ampligen at 20 mg/kg of body weight/day was able to reduce the severity of virus-induced myocarditis, as assessed by morphometric analysis, by 98% (P = 3.0 x 10(-8)). When poly(IC) was administered at 15 mg/kg/day, it reduced the severity of virus-induced myocarditis by 93% (P = 5.6 x 10(-5)). Alpha interferon 2b (1 x 10(5) U/day) and pegylated alpha interferon 2b (5 x 10(5) U/day) were less effective and reduced the severity of virus-induced myocarditis by 66% (P = 0.0009) and 78% (P = 0.0002), respectively. The observed efficacies of Ampligen and poly(IC) were corroborated by the observation that the drugs also markedly reduced the virus titers in the heart, as detected by (i) quantitative real-time reverse transcription-PCR and (ii) titration for infectious virus content. Whereas the electrocardiograms for untreated mice with myocarditis were severely disturbed, the electrocardiographic parameters were normalized in Ampligen- and poly(IC)-treated mice. Even when start of treatment with Ampligen was delayed until day 2 postinfection, a time at which lesions had already appeared in untreated control animals, a marked protective effect on the development of viral myocarditis (as assessed at day 6 postinfection) was still noted.

Animals↗
PubMed1990

Inhibition of poly(ADP-ribosyl)ation by overexpressing the poly(ADP-ribose) polymerase DNA-binding domain in mammalian cells.

Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.

Animals↗