Stability of triple-helical poly(dT)-poly(dA)-poly(dT) DNA with counterions

Structural conformation of triple-helical poly(dT)-poly(dA)-poly(dT) has been a very controversial issue recently. Earlier investigations, based on fiber diffraction data and molecular modeling, indicated an A-form conformation with C'3-endo sugar pucker. On the other hand, Raman, solution infrared spectral, and NMR studies show a B-form structure with C'2-endo sugars. In accordance with these experimental results, a theoretical model with B-form, C'2-endo sugars was proposed in 1993. In the present work we investigate the dynamics and stability of the two conformations within the effective local field approach applied to the normal mode calculations for the system. The presence of counterions was explicitly taken into account. Stable equilibrium positions for the counterions were calculated by analyzing the normal mode dynamics and free energy of the system. The breathing modes of the triple helix are shifted to higher frequencies over those of the double helix by 4-16 cm-1. The characteristic marker band for the B conformation at 835 cm-1 is split up into two marker bands at 830 and 835 cm-1. A detailed comparison of the normal modes and the free energies indicates that the B-form structure, with C'2-endo sugar pucker, is more stable than the A-form structure. The normal modes and the corresponding dipole moments are found to be in close agreement with recent spectroscopic findings.     

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.     

Effect of flanking sequences on the right- to left-handed transition of a (dA-dT)n tract in supercoiled DNA

Alternating (dA-dT)n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni2+ ions and high NaCl concentration (Nejedly, K., Klysik, J. and Palecek, E., FEBS Lett. 243, 313-317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)n tract. In particular, the presence of 5'-homopyrimidine (C3) and 3'-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left-handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT)n sequence.     

Poly(2-aminoadenylic acid): interaction with poly(uridylic acid)

Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.     

Electrophoresis of DNA sequencing fragments at elevated temperature in capillaries filled with poly(N-acryloylaminopropanol) gels

The performance of poly(N-acryloylaminopropanol) (poly AAP) gel columns, proved to be stable during electrophoresis at elevated temperature, was investigated. The column manufacturing procedure included the preparation of a coating of the inner wall of the fused silica capillary column with linear poly(AAP). Then, a mixture of the AAP monomer, the cross-linker dihydroxyethylenebisacrylamide (DHEBA) and linear poly(AAP) was introduced into the column and in situ polymerized (for preparation of linear gel columns, the addition of DHEBA was omitted). The poly(AAP) columns were first evaluated by electrophoresis of oligonucleotides at room temperature and at 50 degrees C, utilizing 260 nm UV-absorbance detection. In a further evaluation of column performance, samples of T-terminated DNA Sanger fragments from the bacteria Moraxella were separated at 200 V/cm electrical field strength, utilizing a 488 nm argon ion laser and a confocal optical setup for laser-induced fluorescence (LIF) detection. A temperature increase from 25 degrees C to 50 degrees C effectively released a compression of DNA bands. However, for cross-linked poly(AAP) gel columns, the elevated temperature resulted in a considerable reduction of the DNA sequence reading length. When a linear poly(AAP) column was utilized, no detrimental effect of elevated temperature on the separation could be observed.     

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-t rimethylammoniumethyl-amine)phenyl]porphy rin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine )phenyl]porphyrin (TthetaF4TAP) has been synthesized. The binding interactions of TthetaF4TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TthetaF4TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TthetaF4TAP binds to calf thymus DNA, [poly(dA-dT)]2, and [poly(dG-dC)]2 in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TthetaF4TAP/CT DNA resembled that of TthetaF4TAP/[poly(dA-dT)]2, suggesting that TthetaF4TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TthetaF4TAP binds preferentially to [poly(dA-dT)]2 over [poly (dG-dC)]2. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TthetaF4TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TthetaF4TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation.     

Binding of 2-azaanthraquinone derivatives to DNA and their interference with the activity of DNA topoisomerases in vitro

We have investigated the binding ability to DNA of compounds belonging to the 2-azaanthraquinone-type structure and have examined the effect on the activity of DNA gyrase as well as on mammalian topoisomerases in vitro. Using different biophysical techniques it was found that one of these ligands, 9-((2-dimethylamino)ethyl)amino)-6-hydroxy-7-methoxy-5, 10-dihydroxybenzo[g]isoquinoline-5,10-dione (TPL-I), is an intercalating DNA binding agent, whereas the parent compound tolypocladin (TPL) and a derivative (TPL-II) showed almost no similar affinity to DNA. CD measurements demonstrated a significant and selective binding tendency of TPL-I to alternating purine/pyrimidine sequences with some preference for poly(dA-dT). poly(dA-dT). Tm values were increased of the ligand complex with the alternating AT-containing duplex polymer. The binding to various DNAs was characterized by CD and visible absorption spectral changes. From the latter, different binding constants of 6.2 x 10(5) and 1.5 x 10(5) M-1 were obtained for poly(dA-dT).poly(dA-dT) and poly(dA). poly(dT), respectively. Sedimentation measurements with supercoiled pBR322 plasmid DNA clearly indicated an intercalative binding mechanism associated with an unwinding angle of about 18 degrees. These results suggest that the intercalative binding of TPL-I is promoted by the 2-(dimethylamino)ethylamino group substituted on carbon 9 of the anthraquinone system. The cytotoxic compound TPL-I, but not TPL or TPL-II, effectively inhibited the DNA supercoiling reaction of DNA gyrase and the activity of mammalian topoisomerases I and II as measured by the relaxation assay. TPL-I affects the cleavage reaction of topoisomerases on a single site located in alternating purine-pyrimidine sequence regions. The inhibitory potency of TPL-I can be ascribed to a blocking of cleavage sites on the DNA substrate, which correlates with the sequence preference of the ligand.     

Inhibition of entry of HIV into cells by poly(A).poly(U)

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A).poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A).poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I).poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A).poly(U) or other polyanions. These results confirm the potential of poly(A).poly(U) as an antiviral drug against HIV infection.     

Temperature effects on the structure of poly(dA).poly(dT): an X-ray fiber diffraction study

X-ray fiber diffraction of poly(dA).poly(dT) subjected to variation in the relative humidity, has allowed us to demonstrate the effects of temperature on the conformation of the polynucleotide. When the temperature of the poly(dA).poly(dT) is greater than 30 degrees C and the relative humidity near 80%, a new diffraction pattern is obtained. We observe a transition between the classical alpha B' form of poly(dA).poly(dT) and a double helical structure, B*, which remains stable at a temperature up to 70 degrees C. This new conformation of poly(dA).poly(dT) is a right-handed double helix with 11.4 nucleotide pairs per turn and a pitch of 36.7 A.     

Synthesis and luminescence properties of CdSe:Eu NPs and their surface polymerization of poly(MMA-co-MQ)

CdSe:Eu nanoparticles(NPs) were synthesized using an oil phase method and the substitution of Cd~(2+)with Eu3+ was confirmed by XRD,TEM,UV-Vis absorption and fluorescence emission analyses.The CdSe:Eu NPs are monodispersed and uniform spherical particles with a diameter of 3.2 nm,bigger than the pure CdSe NPs(2.3 nm),but with a similar cubic structure as CdSe NPs.Compared with those of pure CdSe NPs,both emission spectrum and absorption spectrum of CdSe:Eu NPs are red-shifted.The CdSe:Eu NPs are incorporated into poly(MMA-co-MQ) to afford poly(MMA-co-MQ)-CdSe:Eu NPs with the cubic structure and particle size(～3-4 nm) similar to those of CdSe:Eu NPs.The TEM imaging suggests that the CdSe:Eu NPs are uniformly dispersed in poly(MMA-co-MQ,) without any obvious aggregation.The fluorescent emission peak and absorption peak of poly(MMA-co-MQ)-CdSe:Eu NPs are between those of CdSe:Eu NPs and poly(MMA-co-MQ),possibly due to the energy transfer caused by the interactions of Cd or Eu atoms on the surfaces of CdSe:Eu NPs with the N and O atoms of poly(MMA-co-MQ).These CdSe:Eu and poly(MMA-co-MQ)-CdSe:Eu NPs with tunable photoluminescence properties can be potentially used for the fabrication of optical and optoelectronic devices.     

Injectable microcapsules prepared with biodegradable poly(alpha-hydroxy) acids for prolonged release of drugs

In this paper, microencapsulation techniques for the preparation of drug-containing monolithic microcapsules for prolonged release using biodegradable poly(alpha-hydroxy) acids, such as polylactic acid, poly(lactide-co-glycolide) and copoly(lactic/glycolic) acid are reviewed. Phase separation, solvent evaporation, and spray drying procedures are discussed. In order to achieve controlled-release formulations of highly water-soluble drugs that are entrapped efficiently, various manufacturing techniques and procedures have been developed. Degradation of poly(alpha-hydroxy) acids is altered by the copolymer ratio and molecular weight of the polymer used to make microcapsules and the amounts of released microencapsulated drugs correlate almost linearly with polymer degradation, indicating that controlled-release formulations, which release drugs over different times, can be prepared using suitable poly(alpha-hydroxy) acids with different degradation rates.     

Tumor accumulation of poly(vinyl alcohol) of different sizes after intravenous injection

The body distribution and tumor accumulation of polymers were evaluated using poly(vinyl alcohol) (PVA) which has the simplest chemical structure among water-soluble polymers, similar to poly(ethylene glycol). To study the effect of polymer size on the tumor accumulation, we used not only water-soluble PVA with different molecular weights but also PVA microgels prepared through gamma-irradiation of aqueous PVA solutions. The PVA specimens in the aqueous solution were intravenously injected to mice carrying a tumor mass at their footpad. Both types of PVA (water-soluble and microgel) of larger size were retained in the blood circulation for longer time periods and excreted more slowly from the kidney than those of smaller size. The plasma half-life period of PVA became longer with increasing size both for the water-soluble and microgel PVA, indicating that the body fate of PVA is governed only by the size. Both the water-soluble PVA and PVA microgels were accumulated in tumor tissue to a significantly greater extent than in normal tissue. The size dependence of the plasma half-life period and tumor accumulation was similar between the water-soluble PVA and the PVA microgels and the tumor accumulation became maximum around the size of 60 nm both for water-soluble and microgel PVA. A pharmacokinetical study demonstrated that the tumor uptake rate index of PVA decreased with the increase in PVA size. On the other hand, the greater the size, the larger the value of the area under the blood concentration-time curve (AUC). In addition, the PVA around 60 nm in diameter showed the smallest liver clearance. It was concluded that the balance between the uptake rate and the AUC as well as the liver clearance resulted in the maximum accumulation of PVA with the size of 60 nm.     

Long circulating biodegradable poly(phosphazene) nanoparticles surface modified with poly(phosphazene)-poly(ethylene oxide) copolymer

The biodistribution of biodegradable poly(organo phosphazene) nanoparticles surface modified by adsorption of a novel poly(organo phosphazene)-poly(ethylene oxide) copolymer with a 5000 M(W) PEO chain (PF-PEO[5000]), following intravenous administration in rats and rabbits, is described. The data are compared to the biodistribution of poly(organo phosphazene) and poly(lactide-co-glycolide) nanoparticles coated with a tetrafunctional copolymer of poly(ethylene oxide)-poly(propylene oxide) ethylenediamine, commercially available as Poloxamine 908. This copolymer has a PEO chain of the same size as the poly(organo phosphazene)-PEO derivative used. The results in the rat model reveal that poly(organo phosphazene) nanoparticles with a Poloxamine 908 coating were mainly captured by the liver, although a retardation in clearance from the systemic circulation was seen. In contrast, the poly(organo phosphazene) nanoparticles coated with PF-PEO(5000) showed a prolonged blood circulating profile, with only a small amount of the nanoparticles sequestered by the liver. This indicates the importance of the nature of both the anchoring group and the particle surface on the biological performances of the system. Study of the biodistribution of the PF-PEO(5000)-coated poly(organo phosphazene) nanoparticles in the rabbit model also indicated a prolonged systemic circulation lifetime and reduced liver uptake, whereby a significant amount of the administered nanoparticles was targeted to the bone marrow.     

Joint molecular modeling and spectroscopic studies of DNA complexes of a bis(arginyl) conjugate of a tricationic porphyrin designed to target the major groove

To target selectively the major groove of double-stranded B DNA, we have designed and synthesized a bis(arginyl) conjugate of a tricationic porphyrin (BAP). Its binding energies with a series of double-stranded dodecanucleotides, having in common a central d(CpG)2 intercalation site were compared. The theoretical results indicated a significant energy preference favoring major groove over minor groove binding and a preferential binding to a sequence encompassing the palindrome GGCGCC encountered in the Primary Binding Site of the HIV-1 retrovirus. Spectroscopic studies were carried out on the complexes of BAP with poly(dG-dC) and poly(dA-dT) and a series of oligonucleotide duplexes having either a GGCGCC, CCCGGG, or TACGTA sequence. The results of UV-visible and circular dichroism spectroscopies indicated that intercalation of the porphyrin takes place in poly(dG-dC) and all the oligonucleotides. Thermal denaturation studies showed that BAP increased significantly the melting temperature of the oligonucleotides having the GGCGCC sequence, whereas it produced only a negligible stabilization of sequences having CCCGGG or TACGTA in place of GGCGCC. This indicates a preferential binding of BAP to GGCGCC, fully consistent with the theoretical predictions. IR spectroscopy on d(GGCGCC)2 indicated that the guanine absorption bands, C6=O6 and N7-C8-H, were shifted by the binding of BAP, indicative of the interactions of the arginine arms in the major groove. Thus, the de novo designed compound BAP constitutes one of the very rare intercalators which, similar to the antitumor drugs mitoxantrone and ditercalinium, binds DNA in the major groove rather than in the minor groove.     

Mode of action of the anti-AIDS compound poly(I).poly(C12U) (Ampligen): activator of 2',5'-oligoadenylate synthetase and double-stranded RNA-dependent kinase

The mismatched double-stranded RNA (dsRNA), poly(I).poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 micrograms/ml (80 micrograms/ml) of poly(I).poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I).poly(C12U) markedly extends the duration of the transient increase of 2',5'-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I).poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I).poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I).poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (> or = 3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I).poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10(-7) to 10(-6) grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I).poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I).poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.     

Isolation of cDNA encoding full-length rat (Rattus norvegicus) poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP-ribose). Poly(ADP-ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation. We established the complete cDNA sequence of rat poly(ADP-ribose) polymerase by RT-PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules of poly(ADP-ribose) polymerase.     

Elastase-induced hydrolysis of synthetic solid substrates: poly(ester-urea-urethane) and poly(ether-urea-urethane)

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) were incubated with two radiolabelled model poly(urethane), a poly(ester-urea-urethane) containing [14C]toluene diisocyanate ([14C]TDI), poly(caprolactone)(PCL) and ethylenediamine (ED), and a poly(ether-urea-urethane) containing [14C]TDI, poly(tetramethylene oxide) (PTMO) and ED. Ten-fold more radioactive carbon was released when PPE was incubated with [14C]TDI/PCL/ED than when HNE was used. The PPE-induced radioactive carbon release was significantly reduced by a specific elastase inhibitor. Ten-fold less radioactive carbon was released when [14C]TDI/PTMO/ED was incubated with PPE as compared to [14C]TDI/PCL/ED. Since neutrophils, which contain elastolytic activity, are present during the inflammatory response, the stability of biomaterials used in implanted devices may be affected.     

Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae

Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.