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PubMed1983

Monoclonal antibodies against poly(ADP-ribose) recognize different structures of poly(ADP-ribose).

The characteristics of two monoclonal antibodies to poly(ADP-Rib) and the various structures of poly(ADP-Rib) recognized by these monoclonal antibodies have been examined. One antibody, IgG monoclonal antibody 10H, reacted with most parts of poly (ADP-Rib) molecules, and its binding was only slightly inhibited by Ado(P)-Rib-P, a monomer unit of the polymer. On hydrolysis of poly(ADP-Rib) protected by the antibody hydrolytic products, such as Ado(P)-Rib-P and Ado(P)-Rib(P)-Rib-P, were almost the same as those from control poly(ADP-Rib). The other antibody, IgM monoclonal antibody 16B, recognized only some parts of poly(ADP-Rib) molecules of larger size, and its binding was not inhibited by Ado(P)-Rib-P. Branched portions were considerably concentrated in parts protected by this antibody. These two monoclonal antibodies are suggested to recognize different structures of poly(ADP-Rib): possibly IgG antibody 10H recognizes linear portions whereas IgM antibody 16B recognizes branched portions of the polymer. These monoclonal antibodies should be useful in studies on the structures, including unknown structures and the locations of these structures in poly(ADP-Rib).

Antibodies, Monoclonal↗
PubMed2000

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail.

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.

Allosteric Regulation↗
PubMed2002

Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme from an epsilon-poly-L-lysine-producing strain of Streptomyces albulus.

An epsilon-poly-L-lysine-degrading enzyme of an epsilon-poly-L-lysine-producing strain of Streptomyces albulus was purified and characterized. The enzyme was tightly bound to the cell membrane. After solubilization with NaSCN, the enzyme was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography. The subunit molecular mass of the purified enzyme was 54 kDa. Enzyme activity was inhibited by o-phenanthroline, and could be restored in the presence of 1 mM Mg(2+), Ca(2+), Fe(3+) or Zn(2+). The mode of epsilon-poly-L-lysine degradation was of the exo-type, and the enzyme released N-terminal L-lysines one by one. The enzyme acted on various peptides possessing L-lysine residues at the N-terminus and was classified as an aminopeptidase. Epsilon-Poly-L-lysine-degrading activity was found in the membrane fraction of some other Streptomyces strains as well as that of Streptomyces albulus. Streptomyces virginiae IFO 12827 and Streptomyces norsei IFO 15452 exhibited high epsilon-poly-L-lysine-degrading activity, and both strains could produce epsilon-poly-L-lysine, indicating a correlation between the distribution of membrane-bound epsilon-poly-L-lysine-degrading enzyme and epsilon-poly-L-lysine-producing activity.

Aminopeptidases↗
PubMed1985

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences.

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.

Acetaldehyde↗
PubMed1979

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy.

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). At pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.

Animals↗
PubMed1983

Conformational transitions in the synthetic polynucleotide poly[d(G-C)] . poly[d(G-C)] double-helix.

Conditions are described for observing by X-ray fibre diffraction the A, B and S conformations of the poly[d(G-C)] . poly[d(G-C)] double-helix and also a new form designated as B". For fibres with an appropriate ionic content, transitions between these conformations can be induced by varying the relative humidity of the fibre environment. With increasing relative humidity the transitions B" leads to A leads to S leads to B occur. However, reducing the relative humidity does not result in a simple reversal of these transitions. If the relative humidity is reduced rapidly, a B leads to A transition is observed followed by an A leads to B" transition, but if it is reduced slowly, the transition is from B to S. Once the S form has been assumed, further reduction in the relative humidity does not result in a transition to the A form. The S form emerges as a particularly stable form of the poly[d(G-C)] . poly[d(G-C)] double-helix. From the point of view of its relationship to the classical A and B forms, the S form of poly[d(G-C)] . poly[d(G-C)] is shown to exhibit similarities to the D form of poly[d(A-T)] . poly[d(A-T)].

Humidity↗
PubMed1998

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)].

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.

Journal Article↗
PubMed2001

Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance.

Thin films of poly(vinyl alcohol) (PVA) polymer were prepared on a flat, nonporous, poly(styrene) support matrix by adsorption from aqueous solution and were characterized in order to investigate the nonspecific adsorption of proteins to a chromatographically relevant surface. The integrity and surface coverage of the PVA thin films were established by surface analysis and atomic force microscopy imaging. The adsorption of the PVA polymers to the poly(styrene) substrate and the nonspecific adsorption of proteins to the PVA-coated surface were monitored using surface plasmon resonance. PVA was strongly bound to the poly(styrene) surface, but the surface density of the adsorbed PVA polymers was affected substantially by the concentration, molecular weight, and degree of hydrolysis of PVA polymers used. There was evidence of increasing degrees of unfolding of the PVA polymer onto the poly(styrene) surface as the concentration of the the PVA coating solution increased. Complete PVA coverage of the poly(styrene) surface was observed at PVA concentrations of 0.1 mg/mL or greater but with significant influence of both molecular weight and degree of hydrolysis of the PVA polymers. Resistance of the PVA-coated poly(styrene) surface to the nonspecific adsorption of human serum albumin (HSA) correlated with the degree of surface coverage of the PVA. The use of anti-HSA as a probe for adsorbed HSA suggested that HSA was displacing PVA from the poly(styrene) surface at the lower PVA surface coverage. A complete barrier to nonspecific protein adsorption was observed with a PVA coating solution concentration of greater than 0.1 mg/ mL with a degree of hydrolysis of <88%.

Adsorption↗
PubMed1985

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies.

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).

Kinetics↗
PubMed2002

Equilibrium studies on the association of the nuclear poly(A) binding protein with poly(A) of different lengths.

The nuclear poly(A) binding protein (PABPN1) binds the growing poly(A) tail during pre-mRNA 3'-end processing, stimulating its elongation and controlling its final length. Here we report binding studies of PABPN1 to poly(A) in solution. Quantitative fluorescence titration was used to determine the stoichiometry, intrinsic affinity, and cooperativity of binding to a series of size-fractionated poly(A). The intrinsic association constant K(i) was about 2 x 10(6) M(-1) for oligo(A) and all size classes of poly(A). The binding of PABPN1 to poly(A) was enhanced by protein-protein interactions which were, however, weak (cooperativity parameter omega < 50). No significant change of cooperativity could be detected with increasing polynucleotide length in the range of 140-450 nucleotides. An average binding site size n of 11-14 was found for all poly(A) lengths, which is close to the minimal site size m found for binding to oligo(A). The data are discussed with respect to the previous observation of two different forms of the poly(A)-PABPN1 complex.

Animals↗
PubMed1984

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.

Circular Dichroism↗
PubMed1996

The assisted self-association of ATP4- by a poly(amino acid) [poly(Lys)] and its significance for cell organelles that contain high concentrations of nucleotides.

The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self-association of ATP via the concentration dependence of the 1H-NMR chemical shifts for H2, H8 and H1' in D2O at pD 8.4 (25 degrees C) in the range 0.0025-0.4 M in the presence and absence of poly(alpha, L-lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H.Lys)n+, whereas at pD 12.1 only approximately 10% of the epsilon-amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non-cooperative stacking. The stacking tendency follows the series: ATP4- (K = 1.3 M-1; pD 8.4) < ATP4-/poly(H.Lys)n+ (K = 11.5 M-1; pD 8.4) > ATP4-/90% poly(Lys)/10% poly(H.Lys)n+ (K = 3.1 M-1; pD 12.1). It is evident that poly(H.Lys)n+ assists the association of ATP by a factor of approximately 10, and it is suggested that, via its positively charged epsilon-ammonium groups, poly(H.Lys)n+ acts as a matrix by aligning ATP4- ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such as epinephrine or 5-hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides.

Adenosine Triphosphate↗
PubMed1971

Effect of Separate and Combined Injections of Poly rI: Poly rC and Endotoxin on Reticulo-endothelial Activity, Interferon, and Antibody Production in the Mouse.

The effect of repeated stimulation on both the interferon and antibody systems and on reticuloendothelial activity was studied by injection of endotoxin 2 days before injection of (poly rI):(poly rC). A single injection of endotoxin or (poly rI):(poly rC) increased or decreased the response in each system depending on the time of administration. If the injection of (poly rI):(poly rC) was preceded by an injection of endotoxin 2 days before, its activity was markedly reduced in all of the three systems studied. Although different doses of endotoxin were required to induce a state of hyporeactivity or tolerance to the effects of (poly rI):(poly rC) in either system, it is possible that a common mechanism underlies the hyporeactivity in all systems.

Journal Article↗
PubMed2000

Phase transfer and characterization of poly(epsilon-caprolactone) and poly(L-lactide) microspheres.

A method suitable for transfer of poly(epsilon-caprolactone) and poly(L-lactide) microspheres (synthesized by pseudoanionic dispersion polymerization of epsilon-caprolactone and L-lactide in heptane-1,4-dioxane mixed solvent) from heptane to water was developed. This method consists of treating the microspheres with KOH-ethanol in the presence of surfactants (nonionic Triton X-405, anionic sodium dodecyl sulfate (SDS), and zwitterionic ammonium sulfobetaine-2 (ASB)). Partial hydrolysis of polyesters results in the formation of hydroxyl and carboxyl groups in the surface layer of microspheres and enhances their stability in water-based media. Minimal concentrations of surfactants, needed to obtain stable suspensions of particles, were equal to 3 x 10(-2) and 6 x 10(-2), and 3 x 10(-2) mol l(-1) for Triton X-405. SDS, and ASB, respectively. In the case of poly(epsilon-caprolactone) microspheres, suspensions in water were stable for all three surfactants for pH values ranging from 3 to 11. Suspensions of poly(L-lactide) were stable in the same range of pH values only for ASB. Surface charge density determined by electrophoretic mobility varied for poly(epsilon-caprolactone) microspheres from 2.6 x 10(-7) to 8.9 x 10(-7) mol m(-2), for particles stabilized with Triton X-405 and ASB. respectively. In the case of poly(L-lactide) microspheres, surface charge density varied from 3.9 x 10(-7) (stabilizer: Triton X-405) to 7.4 x 10(-7) mol m(-2) (stabilizer: ASB). Carboxyl groups located in the surface layer of poly(L-lactide) microspheres were used for covalent immobilization of 6-aminoquinoline, a fluorophore with an amino group. Maximum surface concentration of immobilized 6-aminoquinoline was equal to 1.9 x 10(-6) mol m(-2). Poly(epsilon-caprolactone) microspheres transferred into water were loaded with ethyl salicylate. Loading up to 38% (w/w) was obtained.

Biocompatible Materials↗
PubMed1992

Comparative assessment of poly-L-aspartic and poly-L-glutamic acids as protectants against gentamicin-induced renal lysosomal phospholipidosis, phospholipiduria and cell proliferation in rats.

Coadministration of poly-L-aspartic acid (poly-L-Asp) protects rats against all measured signs of aminoglycoside nephrotoxicity. Based on in vitro and acute in vivo models, previously we hypothesized that poly-L-Asp protects by forming complexes with the drug in lysomes of proximal tubular cells. However, another closely related peptide, poly-L-glutamic acid (poly-L-Glu), could not protect against gentamicin-induced phospholipidosis and nephrotoxicity, presumably because it is susceptible to rapid hydrolysis in sysosomes in vivo. The present study expands the in vivo comparison between these two polyanions to a subacute model of rats and examines in detail the influence of these polymers on the qualitative and quantitative morphological alterations of lysosomes, phospholipiduria and proliferation of cortical cells induced by gentamicin. Our results not only demonstrated that despite a significantly higher drug cortical accumulation, the coadministration of poly-L-Asp almost completely protects against the development of all these early renal alteration but also pointed to the possibility of a mild, albeit apparently nonlethal, lysosomal thesaurismosis to develop under these conditions. In contrast, poly-L-Glu could not protect against these early renal alterations, though cortical drug accumulation was not significantly higher; however, it induced a conspicuous proliferation of peritubular interstitial cells. Therefore, the present work, taken together with the earlier results of ours as well as that of others, tends to strengthen the hypothesis that the site of action of poly-L-Asp must be in lysosomes, which are also the organelles that sequester and accumulate the drug.

Animals↗
PubMed1996

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry.

Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD+ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.

Animals↗
PubMed1997

Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease.

3'-Exonucleolytic removal of the poly(A) tail is the first and often rate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplasmic extract from HeLa cells, the poly(A) tail of mRNA was degraded from the 3'-end. In agreement with earlier in vivo observations, prominent decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for this poly(A) shortening activity was purified from calf thymus. A polypeptide of 74 kDa copurified with the activity. The deadenylating nuclease (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly dependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly(A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. At either salt concentration DAN and PAB I reconstituted poly(A) shortening with the same pattern of intermediates seen in cytoplasmic extract. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.

Animals↗
PubMed1998

Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis.

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.

3T3 Cells↗