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PubMed1977

HeLa cell poly(A)- mRNA codes for a subset of poly(A)+ mRNA-directed proteins with an actin as a major product.

Poly(A)+ and poly(A)- mRNA from HeLa cells were separated and translated in heterologous messenger-dependent protein synthesizing systems. Two-dimensional electrophoretic analysis revealed three classes of polypeptides. At the level of detectability in the electropherograms, a small number (about 10) of proteins were detected only among the poly(A)- mRNA products, a larger number (about 40) were produced by both poly(A)- and poly(A)+ mRNA, and a large number of polypeptides were found exclusively in the poly(A)+ mRNA products. The major product of both poly(A)+ and poly(A)- mRNA was shown to be the beta form of actin.Previous cross hybridization measurements suggested little homology between poly(A)+ and poly(A)- mRNA populations. In view of the apparent identity of many poly(A)- products with those of poly(A)+, the homology between poly(A)+ and poly(A)- mRNA sequences was examined in greater detail. cDNA complementary to only the most abundant poly(A)+ message sequences was prepared. About 10% of this cDNA hybridized to abundant sequences in the poly(A)- fraction. This corresponded to only 2% of the total mass of poly(A)+ mRNA and accounted for the failure to detect cross hybridization in previous experiments. Thus, a small number of poly(A)+ sequences appear to be present in relatively high concentration in poly(A)- mRNA as evidenced by both the translation products and the cross hybridization results.

Actins↗
PubMed1978

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.

Circular Dichroism↗
PubMed1991

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.

4-Hydroxyaminoquinoline-1-oxide↗
PubMed1993

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.

2',5'-Oligoadenylate Synthetase↗
PubMed1993

Biologic effects after a single dose of poly(I):poly(C12U) in healthy volunteers.

Poly(I):poly(C12U) (mismatched double-stranded RNA; atvogen), an interferon inducer, is active against human immunodeficiency virus in vitro. To determine the extent and duration of the biologic effects of poly(I):poly(C12U), we administered a single dose of the drug to healthy volunteers in a randomized, double-blind, placebo-controlled 2-week crossover study. We analyzed blood for alpha and gamma interferons, neopterin, 2',5'-oligoadenylate synthetase, lymphocyte surface markers, lymphocyte proliferation after exposure to soluble antigens and mitogens, and natural killer cell activity. Minimal biologic effects were observed after administration of a single 200-mg dose to four volunteers; therefore, the dose was increased to 600 mg in 10 subjects. Only neopterin levels and symptoms were greater after administration of 600 mg of poly(I):poly(C12U) than after administration of placebo (Wilcoxon signed rank sum test, P = 0.06). A definite response in 2',5'-oligoadenylate synthetase activity, however, was seen in a few subjects. Neither alpha nor gamma interferon was detectable in serum after poly(I):poly(C12U) dosing. The neopterin changes after administration of poly(I):poly(C12U) were similar at both poly(I):poly(C12U) dose levels, with an early decrease at 6 h, a peak at 1 day, and a gradual decrease toward the baseline over the following 3 days. A mild flu-like syndrome occurred in one-half of the subjects following administration of poly(I):poly(C12U) and in only one subject following administration of placebo. This syndrome resolved within 16 h after poly(I):poly(C12U) dosing. We conclude that poly(I):poly(C12U) does not induce measurable levels of interferon and causes only minimal biologic or toxic effects among those parameters measured after administration of a single dose in the 200- to 600-mg dose range in health volunteers.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Selective inhibition of hepatitis B virus and human immunodeficiency virus sequence-promoted gene expression by cotransfected poly(I):poly(C).

The transient expression of hepatitis B virus (HBV) surface and "eJ" antigens caused by transfection of human hepatoblastoma HepG2 cells with HBV DNA was markedly inhibited by cotransfection with poly(I):poly(C). Cotransfection with poly(I):poly(C) also inhibited the expression of bacterial chloramphenicol acetyltransferase (CAT) gene which was under the control of either the HBV core promoter or the human immunodeficiency virus (HIV-1) long terminal repeat. This inhibition was much more pronounced on the expression of HBV-promoted CAT than HIV-promoted CAT. The uptake of reporter plasmid was not affected by cotransfected poly(I):poly(C). The inhibition was found to be at the steady-state CAT mRNA level and appeared to be specific for HBV and HIV regulatory sequences since CAT expression directed by other viral and cellular regulatory sequences was not inhibited. Cotransfection with a mixture of equal amounts of poly(I) and poly(C) had similar inhibitory effects whereas cotransfection with poly(l) or poly(C) alone, or other double-stranded ribo- or deoxyribonucleotides, did not have such strong effects. The addition of poly(l):poly(C) to the culture medium of cells transfected with these reporter plasmids caused little inhibition. Transfection with poly(l):poly(C) induced a minimal amount of intracellular interferon-alpha in HepG2 cells which may be involved in selective inhibition of HBV-and HIV-1-directed gene expression. 2-Aminopurine, an inhibitor of double-stranded RNA activated protein kinase known to block interferon gene induction by poly(l):poly(C), partially reversed the poly(l):poly(C)-induced inhibitory effect on HBV-CAT expression.

2',5'-Oligoadenylate Synthetase↗
PubMed1982

Age-dependent gene induction in quail oviduct. XV. Alterations of the poly(A)-associated protein pattern and of the poly(A) chain length of mRNA.

The effect of ageing on polyadenylate [poly(A)] metabolism of mRNA was studied in two age groups of female quails: mature (250-320 days' old) and senescent animals (3-3.5 years' old). In introductory experiments it was shown that poly(A)-associated proteins can not be recovered from cytosol by affinity chromatography. We isolated the poly(A)-associated proteins from polyribosomal poly(A)-ribonucleoprotein complex [poly(A)-RNP] and radioactively labeled them with dansyl chloride. Three main protein species were identified with molecular masses of 48000 (P48), 35000 (P35) and 24000 (P24). During ageing the percentage portion of P48 in poly(A)-RNP from liver (mitotic tissue) and from oviduct or heart (post-mitotic tissue) is reduced at the expense of P35 and P24. Quantitative analyses revealed that the amount of poly(A)-RNP in the different organs decreases significantly with age if the values are based on DNA. The protein content in poly(A)-RNP was found to be reduced especially in post-mitotic tissue. From this finding we assume that the number of poly(A)-associated protein molecules per poly(A) stretch drops from approximately 4.7 molecules (mature oviduct) to 1.9 molecules (senescent oviduct). Control experiments revealed that free, non-polyribosomal poly(A)-RNP accounts only for 10% of total poly(A)-RNP. The size of the poly(A) segment of mRNA decreases with age. After labeling with [3H] dimethylsulfate, the poly(A) stretch from mature oviduct was found to consist mainly of 120-180 AMP units, and those from mature liver and mature heart of 110 and 100, respectively. In organs from senescent animals the percentage of shorter poly(A) stretches is enlarged; on the average, poly(A)-70 chains were detected. These results support the assumption that age-dependent changes occur also on the post-transcriptional level during the maturation steps of poly(A)(-) hnRNA to poly(A)-(+) mRNA.

Aging↗
PubMedDate not supplied

Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove.

The structure of poly(dA).poly(dT) currently arouses great interest, mainly because dAn.dTn stretches are associated with considerable DNA bending. Until recently the heteronomous DNA described by Arnott et al., with the poly(dA) and poly(dT) chains in A and B conformations respectively, was the only detailed model of this structure. Following our earlier studies of the interaction of DNA and monovalent ions, we examined the X-ray diffraction of the bivalent Ca2+ salt of poly(dA).poly(dT) (Ca-poly(dA).poly(dT)) and found no sign of a heteronomous structure: Ca-poly(dA).poly(dT) in fibres shows fully equivalent B-type conformations of the opposite sugar-phosphate chains. A revision of the structure of the sodium salt, Na-poly(dA).poly(dT), based on this result, yields only a slightly heteronomous structure with each chain in a B-type conformation, which is in much better agreement with the experimental data underlying the original heteronomous model. Both structures, Ca- and Na-poly(dA).poly(dT), have a minor groove narrower than that of the B form: this peculiarity seems to be very important for the interaction of poly(dA).poly(dT) and biologically significant molecules (including proteins and antibiotics). The specific base-pair positions in poly(dA).poly(dT) may account for the DNA bending adjacent to dAn.dTn tracts.

Calcium↗
PubMed1987

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs.

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.

Animals↗
PubMed2000

[Analysis of poly(dA).poly(dT) structure by Raman spectroscopy and lattice dynamics].

Poly(dA).poly(dT) is a kind of DNA which one strand contains adenine(A) bases, and the other only thymine(T) bases. This DNA possesses flexible structure and the X-ray diffraction for poly(dA).poly(dT) fiber gives three different structures. So it is of interest to study poly(dA).poly(dT) structure in solution. In this paper, Raman spectrum of poly(dA).poly(dT) in 0.2 mol.L-1 NaCl solution was recorded over the spectral range 750-1000 cm-1. The Raman bands at 817 cm-1 and 843 cm-1 exist simultaneously. This implied that the secondary structure of poly(dA).poly(dT) is neither A-conformation nor B-conformation. Normal mode analysis of heteronomous secondary structure from poly(dA).poly(dT) fibers was carried out by the lattice dynamics. Normal modes were assigned by potential energy distribution (PED). The calculated frequencies are good agreement with the observed Raman spectrum. This indicated that poly(dA).poly(dT) in solution probably has the same structure as poly(dA).poly(dT) fiber, namely the poly(dA) chain has the C-3'-endo ring pucker while the poly(dT) chain has the C-2'-endo ring pucker.

DNA↗
PubMed1976

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.

Autoanalysis↗
PubMed1981

Induction of the Z conformation in poly(dG-dC).poly(dG-dC) by binding of N-2-acetylaminofluorene to guanine residues.

Poly(dG-dC).poly(dG-dC) and poly(dG).poly(dC) were modified by treatment with N-acetoxy-N-2-acetylaminofluorene, and their conformations were examined by circular dichroism and susceptibility to nuclease S1 digestion. A sample of poly(dG-dC).poly(dG-dC) modified to an extent of 28% with acetylaminofluorene (AAF) at the C(8) position of the deoxyguanosine residues showed a circular dichroism spectrum that had the characteristics of the Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. A sample of poly(dG-dC).poly(dG-dC) modified only 3% by AAF showed a spectrum characteristic of the B form of DNA. However, it was converted to the Z form at ethanol concentrations lower than required to convert unmodified poly(dG-dC).poly(dG-dC) from the B to the Z form. Poly(dG).poly(dC), which does not undergo the B-to-Z transition at high ethanol concentrations, did not show any large conformational changes with high AAF modification. Susceptibility to digestion with nuclease S1 also suggested differences in the conformations of the two modified polynucleotides. Poly(dG-dC).poly(dG-dC) modified by AAF to an extent of 28% was almost completely resistant to nuclease S1 digestion. However, both poly(dG).poly(dC) and DNA modified to similar levels by AAF were highly susceptible to nuclease S1 digestion. Two different conformations for AAF-modified deoxyguanosine are proposed, depending on whether its position is in alternating purine-pyrimidine sequences or in random-sequence DNA.

2-Acetylaminofluorene↗
PubMed1990

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.

Ammonium Chloride↗
PubMed1987

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.

Base Sequence↗
PubMed1976

Relationship between the molecular size of poly I-poly C and its biological activity.

Seven polyinosinic-polycytidylic acid (poly I-poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon-inducing activity in mice. The increase in size of poly I-poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I-poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD50 of poly I-poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I-poly C in culture cells were examined. The resistance-inducing activity of poly I-poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon-inducing activity in RK13 was not appreciably influenced by the size of poly I-poly C, especially in the presence of DEAE-dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I-poly C upon the resistance-inducing and interferon-inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE-dextran.

Animals↗
PubMedDate not supplied

Conjugates of cis-4-hydroxy-L-proline and poly(PEG-Lys), a water soluble poly(ether urethane): synthesis and evaluation of antifibrotic effects in vitro and in vivo.

Synthetic approaches for the preparation of macromolecular conjugates of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) were explored, and the efficacy of the conjugates in inhibiting collagen accumulation was investigated in vitro and in vivo. In one approach, poly(PEG-Lys), an alternating copolymer of poly(ethylene glycol) (PEG) and lysine, was used as the carrier. To prepare pendent chain systems, cHyp was attached to poly(PEG-Lys) through an amide linkage [poly(PEG-Lys-cHyp amide)] or through an ester linkage [poly(PEG-Lys-cHyp ester)]. In an alternative approach, cHyp was incorporated into the backbone of a linear copolymer consisting of PEG, succinic acid, and cHyp units [poly(PEG-succinate-cHyp)]. Bioactivity in vitro was assessed by the ability of the cHyp conjugates to inhibit growth of cultured smooth muscle cells (SMC) and rat lung fibroblasts (RLF). Cell numbers were compared to control experiments in the presence of biologically inactive trans-4-hydroxy-L-proline (tHyp). After a 5 day period, the presence of 8 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp amide) resulted in a 47% reduction in the number of SMC (p < 0.05), the presence of 36 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp ester) resulted in a 38% reduction in the number of SMC (p < 0.05), while the presence of 118 micrograms/mL of cHyp delivered by poly(PEG-succinate-cHyp) resulted in a 31% reduction in the number of cells (p < 0.05). An identical trend was observed for the inhibition of RLF growth. In general, poly(PEG-Lys-cHyp amide) was most active, followed by poly(PEG-Lys-cHyp ester) and the backbone system, poly(PEG-succinate-cHyp). Specifically, poly(PEG-Lys-cHyp amide) was over 100-fold more active in inhibiting cell growth than free cHyp. Bioactivity in vivo was evaluated by measuring collagen accumulation in subcutaneously implanted poly(vinyl alcohol) sponges in rats. Among the tested conjugates, poly(PEG-Lys-cHyp amide) was most active, reducing collagen accumulation in the sponge by 33% after 14 days relative to controls (p < 0.05). This result indicates that the covalent attachment of cHyp to poly(PEG-Lys) carries may be a useful strategy for the local inhibition of collagen accumulation in tissues.

Animals↗
PubMed1980

Conformation of poly(dG-dC) . poly(dG-dC) modified by the carcinogens N-acetoxy-N-acetyl-2-aminofluorene and N-hydroxy-N-2-aminofluorene.

Poly(dG-dC) . poly(dG-dC) was modified by reaction with N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF). Two samples with 6.6% and 8.5% modified bases were prepared. The modified bases are randomly distributed along the polymer chain, as deduced from competition experiments between antibodies against N-2-(guanosin-8-yl)-acetylaminofluorene, modified poly(dG-dC) . poly(dG-dC), and modified DNAs. Circular dichroism studies show that poly(dG-dC) . poly(dG-dC) modified by N-AcO-AAF is much more sensitive to the addition of alcohol than poly(dG-dC) . poly(dG-dC). In about 50% (vol/vol) alcohol, both polynucleotides have the same conformation, which is the Z form or a Z-like form. Moreover, in low salt and in the absence of alcohol, poly(dG-dC) . poly(dG-dC) modified by N-AcO-AAF is partially in the Z form. Poly(dG-dC) . poly(dG-dC) modified by N-hydroxy-N-2-aminofluorene can also adopt the Z form, but the transition is induced at a higher percentage than that of poly(dG-dC) . poly(dG-dC) modified by N-AcO-AAF. In low salt and in the absence of alcohol, no Z form was detected in poly(dG-dC) . poly(dG-dC) modified by N-hydroxy-N-2-aminofluorene.

2-Acetylaminofluorene↗
PubMed1984

An immunochemical examination of acetylaminofluorene-modified poly(dG-dC) X poly(dG-dC) in the Z-conformation.

Immunization of rabbits with a complex of methylated bovine serum albumin and N-2-acetylaminofluorene (AAF)-modified poly(dG-dC) X poly(dG-dC), a polynucleotide that can assume the Z-DNA conformation, yielded several populations of antibodies specific for Z-DNA determinants. The Z-DNA determinants were analyzed by examination of the antisera and of antibody preparations purified on immunoadsorbents. The following was found: AAF-poly(dG-dC) X poly(dG-dC) shared Z-DNA determinants in common with poly(dG-dC) X poly(dG-dC) in 3.0 M NaCl, poly(dG-m5dC) X poly(dG-m5dC) in 1.5 M NaCl, and brominated poly(dG-dC) X poly(dG-dC) in 0.2, 1.5, and 3.0 M NaCl. Included among the antibodies induced by these determinants was a subpopulation whose reaction with brominated poly(dG-dC) X poly(dG-dC) was sensitive to increased ionic strength. Another distinct population of antibodies recognized determinants present on AAF-poly(dG-dC) X poly(dG-dC) but not on the other Z-DNAs. Only a small portion of this population was specific for the AAF moiety; the greater part appeared to recognize Z-DNA-associated conformational characteristics that were unique to AAF-poly(dG-dC) X poly(dG-dC). These findings are consistent with the existence of a continuum of Z-DNA determinants, which might be capable of functioning as recognition signals for regulatory DNA-binding proteins.

2-Acetylaminofluorene↗