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PubMed1990

High mobility group proteins 1 and 2 bind preferentially to brominated poly(dG-dC).poly(dG-dC) in the Z-DNA conformation but not to other types of Z-DNA.

Three proteins from bull testis, previously thought to be Z-DNA-binding proteins but recently found to recognize brominated poly(dG-dC). poly(dG-dC) by criteria different from the Z-conformation, were partially sequenced. Of these, the 31 kDa protein was identified as a member of the high mobility group 2 protein family, and the 33 kDa protein as a member of the high mobility group 1 protein family. Both proteins had molecular weights approximately 30% higher than expected, indicating considerable posttranslational modification. In contrast, the 58 kDa protein remained unidentified for lack of any significant homology with known protein sequences.

Amino Acid Sequence↗
PubMed1995

An A-form of poly[d(A-C)].poly[d(G-T)] induced by mercury (II) as studied by UV and FTIR spectroscopies.

The conformational changes of poly[d(A-C)].poly[d(G-T)] induced by Hg(ClO4)2 in aqueous solution have been studied using UV absorption and fourth derivative spectrophotometries, and FTIR spectroscopy. The UV absorption and fourth derivative spectra reflect changes in the polynucleotide stacking interactions as a result of the metal-polynucleotide interaction. The fourth derivative spectra do not indicate a Z-form either at low or at high metal-to-polynucleotide ratios. Furthermore, the infrared spectrum at high metal-to-polynucleotide ratio (r = 1.2; r = [Hg(ClO4])2/[nucleotide] molar ratio) has the main features of an A-form, in contrast with previous CD studies which proposed that the polynucleotide adopts a Z-form under these conditions. The nature of a different conformation of the polynucleotide induced at low r-ratios (r < or = 0.2) is discussed.

DNA↗
PubMed1993

B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of basic oligopeptides.

The effect of basic oligopeptides (Lys-Ala-Ala)n and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in aqueous solution and in methanol-water mixtures was investigated by c.d. spectroscopy. In aqueous solution dimers and higher oligomers induce a transition to the Z conformation. These temperature dependent transitions are consistent with positive delta HB-Z values which depend on peptide composition. In the absence of peptides no transition can be observed. In the presence of peptides B-Z transition can be induced by small amount of methanol. The temperature dependence of this transition is consistent with a small, but definitely negative delta HB-Z. At high methanol concentration a transition to Z' type conformation was observed. In subcritical methanol concentrations B-Z transition can be induced by the addition of peptides. In this case the delta HB-Z values are again very small, but definitely positive. The effects of bulky hydrophobic peptide side chains, of the presence of methanol and the temperature dependences are consistent with an important contribution of hydrophobic interactions in maintaining the stability of the Z DNA-peptide complex.

Amino Acid Sequence↗
PubMed1991

Effect of plasma glow, glutaraldehyde and carbodiimide treatments on the enzymic degradation of poly (L-lactic acid) and poly (gamma-benzyl-L-glutamate) films.

The hydrolytic and enzymic degradation of poly(L-lactic acid) (PLA) and poly(gamma-benzyl L-glutamate) (PBGA) films, together with a series of surface treatments, were studied, as a function of exposure time. The degradation of these polymers was monitored by weight loss, contact angle, pH changes and tensile strength studies. Glutaraldehyde treatment retained the maximum strength of PLA in buffer, followed by carbodiimide, compared with control films. On the other hand, plasma glow reversed the effect. The ability of alpha-chymotrypsin, carboxypeptidase, ficin, esterase, bromelain and leucine aminopeptidase to modulate the degradation of PLA and PBGA was also investigated. Addition of these enzymes to the polymer-buffer system reduced the tensile strength of these polymers variably. Among the six enzymes studied, leucine aminopeptidase showed the highest enzymic effect on the degradation of the glutaraldehyde-treated and bare PLA or bare PBGA films. However, glutaraldehyde-cross-linked PLA demonstrated maximum stability in buffers or in all other enzyme systems studied compared with bare PLA. It is conceivable that surface treatments on these polymers might have altered their physical and chemical configuration and the subsequent degradation properties. Surface modifications may provide new ways of controlling the biodegradation of polymers for a variety of biomedical applications.

Biocompatible Materials↗
PubMed1992

Heparin-poly(ethylene glycol)-poly(vinyl alcohol) hydrogel: preparation and assessment of thrombogenicity.

Heparin was immobilized on to poly(vinyl alcohol) hydrogel (PVA) through the free isocyanate end-group on a poly(ethylene glycol) (PEG2000) which had been previously covalently linked to the hydrogel via a urethane moiety. The intention was to reduce the platelet reactivity of the PVA while also suppressing fibrin formation. Elemental nitrogen analysis revealed that the total amount of bound heparin was 19 +/- 7 mumol/g of dried gel. An increase in the in vitro whole blood clotting time of PVA was observed. This was attributed to bound heparin, as the elution rate of heparin from the gel (23 pmol/m2 min) was too low to produce a significant bulk concentration to interfere with fibrin formation. Ex vivo assessment using a chronic canine A-V shunt showed that the bound heparin hydrogel had no effect on the drop in the number of platelets induced by PVA hydrogel, but increased the fractional rate of platelet destruction from approximately 0.35/d to an average value of 0.42/d.

Animals↗
PubMed1992

Grafting reactions and heparin adsorption of poly(amidoamine)-grafted poly(urethane amide)s.

Differently terminated poly(amidoamine) (PAA) oligomers were grafted on the surface of poly(ether urethane amide)s (PEUAm), with fumaric or maleic acid moieties. The grafting reaction was Michael-type addition of amino groups to activated double bonds in the PEUAm backbone. PAAs having primary amino, or secondary amino end-groups were directly grafted on the surface of PEUAm sheets. For vinyl-terminated chains an alpha, omega amino-polyether spacer was introduced initially, following the same addition mechanism. Ungrafted and grafted materials were characterized, besides other analytical techniques, by ATR FT-IR spectroscopy. The heparin adsorption on PEAUm films was analysed after its elution from heparinized samples, quantified by coagulation tests (aPTT), and related to the presence of the PAAs chains grafted on to the surface. Results indicate that PAA-grafted PEUAm elastomeric biomaterials, display enhanced heparin adsorption abilities.

Adsorption↗
PubMed1993

Interfacial photopolymerization of poly(ethylene glycol)-based hydrogels upon alginate-poly(l-lysine) microcapsules for enhanced biocompatibility.

The biocompatibility of microcapsules made by the co-acervation of alginate and poly(l-lysine) (PLL) was enhanced by coating the surface of these microcapsules with a poly(ethylene glycol) (PEG)-based hydrogel. The hydrogel was formed by an interfacial photopolymerization technique using visible light from an argon ion laser. The light absorbing chromophore, eosin Y, was immobilized on the microcapsule surface. This restricted the formation of the PEG hydrogel to the surface of the microcapsule. The presence of the PEG gel on the surface was confirmed by fluorescent dextran entrapment, by direct visualization after dissolution of the underlying membrane and by electron spectroscopy for chemical analysis. The biological response of such microcapsules was evaluated by intraperitoneal implantation in mice. The PEG-coated microcapsules were found to be less inflammatory and were seen not to elicit a fibrotic response, as was the case with alginate-PLL microcapsules.

Alginates↗
PubMed1994

Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture.

Biodegradable foams of hydrophobic polymers can be efficiently wet by two-step immersion in ethanol and water, which overcomes the hindered entry of water into air-filled pores. Ethanol readily enters into the porous polymer, after which it is diluted and replaced by water. This method was evaluated for porous disks of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) foams of copolymer ratios 85:15 and 50:50. For PLLA disks of 0.88 porosity and 1730 microns thickness, prewetting with ethanol for 1 h increased the percentage of void volume filled with water after 48 h from 23 to 79%. The same enhanced entry of water was also observed for prewet PLGA 85:15 disks of 0.86 porosity and 1300 microns thickness, which exhibited an increase from 59 to 97% void volume occupied by water. Furthermore, the water entry even after 1 h was very close to its plateau value for all prewet polymers tested. In recent studies, this method has been useful in uniformly seeding three-dimensional biodegradable polymer substrates for cell and tissue culture.

Culture Techniques↗
PubMed1996

Degradation behaviour of block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments.

The degradation behaviour of a new class of multi-block copolymers containing poly(D,L-lactic-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) segments was studied under conditions mimicking those found in biological fluids. The dissolution times of the new PLGA-PEG multi-block copolymers mainly depend on the length of the PEG segments present in their structure, i.e., on their PEG content on a weight/weight basis. At higher PEG contents, partially degraded PLGA segments are brought in solution by attached PEG segments. In all cases, the dissolution rates decrease by increasing the total surface area of the specimens tested.

Adsorption↗
PubMed1996

Effect of cross-linking agents on the dynamic mechanical properties of hydrogel blends of poly(acrylic acid)-poly(vinyl alcohol-vinyl acetate).

A range of hydrogels were prepared by blending aqueous solutions of poly(vinyl alcohol-vinyl acetate) with poly(acrylic acid) in various proportions. The effects of two cross-linking agents (glyoxal and glutaraldehyde) and subsequent thermal treatment on the properties of the blends are discussed. Dynamic mechanical analysis (DMA) of the xerogels indicated complete miscibility of the various blends which was evident from the appearance of a single glass transition temperature (Tg) in the presence of either glyoxal or glutaraldehyde at all thermal treatments studied. A 50/50% wt/wt blend was found to have the highest storage modulus and was thus selected for further study. Hydrogels prepared with glutaraldehyde without subsequent thermal treatment exhibited higher storage modulus values than those prepared using glyoxal when tested isothermally at 20 degrees C in a water bath. A further increase in the storage modulus was observed when these hydrogels were thermally treated at 120 or 150 degrees C. In a non-isothermal study on the cross-linked hydrogels, no variation in storage modulus was observed. Broad peaks were observed in tan delta plots, these peaks shifting towards higher frequencies as the degree of cross-linking increased in the hydrogel.

Acrylates↗
PubMed1996

In vitro biocompatibility of bioresorbable polymers: poly(L, DL-lactide) and poly(L-lactide-co-glycolide).

The acute toxicity of two degradable polymers, a 70:30 poly (L-D, L-lactide) (PLDLA) and a 90:10 poly(L-lactide-co-glycolide) (PLGA), was evaluated by the agar diffusion test and the filter test with L929 mouse fibroblasts. Extracts of the materials prepared in phosphate-buffered saline at 37 and 70 degrees C were assessed for mitochondrial succinate dehydrogenase activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of BALB 3T3 cells. Both materials revealed no signs of cytotoxicity during the agar diffusions and filter tests. In the MTT and BrdU assays PLDLA and PLGA showed similar results. Cells treated with extracts prepared at 37 degrees C caused slight stimulation of mitochondrial activity. In contrast, cells incubated with the 70 degrees C media revealed a concentration-dependent decrease of mitochondrial activity. DNA synthesis was significantly decreased by the 37 degrees C extracts. As in the MTT assay, the effect of the extracts prepared at 70 degrees C was significantly greater. From these in vitro results it is suggested that PLDLA and PLGA have satisfactory biocompatibility. High concentrations of the degradation products, however, had a toxic influence on the cell culture systems used.

3T3 Cells↗
PubMed1992

Do intramedullary rods of self-reinforced poly-L-lactide or poly-DL/L-lactide cause lactic acid acidosis in rabbits?

Intramedullary rods of self-reinforced poly-DL/L-lactic acid or poly-L-lactic acid were implanted in both femurs of ten rabbits for 60 weeks. Levels of arterial blood L- and D-lactate, pyruvate, pH, pCO2, base excess, standard bicarbonate, pO2 and O2-saturation were determined before implantation and 1, 3, 6, 12, 24, 36, 48 and 60 weeks after implantation. No significant increase in blood L- and D-lactate level was observed at any follow-up. Also, the blood acid-base balance remained normal throughout the study. Further studies on larger implants and on larger animals with follow-ups up to 5 years are recommended.

Acid-Base Equilibrium↗
PubMed1996

Poly(A) metabolism in Xenopus laevis embryos: substrate-specific and default poly(A) nuclease activities are mediated by two distinct complexes.

The metabolism of the poly(A) tail is a process important for the translational regulation of maternal mRNAs in Xenopus laevis oocytes and early embryos. Two poly(A) nuclease (PAN) activities have been described in Xenopus embryo or activated egg extracts (Legagneux et al (1995) RNA 1, 1001-1008). These activities (default PAN and EgPAN) are distinguishable by their deadenylation kinetics and their substrate specificities. In this report, we show that these activities display different sensitivities to biochemical treatments. Urea and, to a lesser extent, spermidine, inhibit EgPAN at concentrations which have no effect on default PAN. Heparin activates default PAN but inhibits EgPAN. When extracts are fractionated by ultracentrifugation, the default activity is recovered in one unique fraction, whereas two fractions must be combined to reconstitute the EgPAN activity. Moreover, these two deadenylation activities are separable by size exclusion chromatography under native conditions. We conclude that these two deadenylation activities are mediated by two protein complexes.

Animals↗
PubMed2003

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.

DNA↗
PubMed2004

Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs.

Methoxy poly(ethylene glycol)-poly(lactide) copolymer (MPEG-PLA) was synthesized and used to make nanoparticles by the nanoprecipitation method for clinical administration of antineoplastic drugs. Paclitaxel was used as a prototype drug due to its excellent efficacy and commercially great success. The size and size distribution, surface morphology, surface charge and surface chemistry of the paclitaxel-loaded nanoparticles were then investigated by laser light scattering, atomic force microscopy, zeta-potential analyzer and X-ray photoelectron spectroscopy (XPS). The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography. The effects of various formulation parameters were evaluated. The prepared nanoparticles were found of spherical shape with size less than 100 nm. Zeta potential measurement and XPS analysis demonstrated the presence of PEG layer on the particle surface. Viscosity of the organic phase was found to be one of the main process factors for the size determination. The EE was found to be greatly influenced by the drug loading. The drug release pattern was biphasic with a fast release rate followed by a slow one. The particle suspension exhibited good steric stability in vitro. Such a nanoparticle formulation of paclitaxel can be expected to have long-circulating effects in circulation.

Absorbable Implants↗
PubMed2004

Preparation, characterization, and in vitro evaluation of physostigmine-loaded poly(ortho ester) and poly(ortho ester)/poly(D,L-lactide-co-glycolide) blend microspheres fabricated by spray drying.

The physostigmine-loaded poly(ortho ester) (POE), poly(dl-lactide-co-glycolide) (PLGA) and POE/PLGA blend microspheres were fabricated by a spray drying technique. The in vitro degradation of, and physostigmine release from, the microspheres were investigated. SEM analysis showed that the POE and POE/PLGA blend particles were spherical. They were better dispersed when compared to the pure PLGA microspheres. Two glass transition temperature ( Tg ) values of the POE/PLGA blend microspheres were observed due to the phase separation of POE and PLGA in the blend system. XPS analysis proved that POE dominated the surfaces of POE/PLGA blend microspheres, indicating that the blend microspheres were coated with POE. The encapsulation efficiencies of all the microspheres were more than 95%. The incorporation of physostigmine reduced the Tg value of microspheres. The Tg value of the degrading microspheres increased with the release of physostigmine. For instance, POE blank microspheres and physostigmine-loaded POE microspheres had a Tg value of 67 degrees C and 48 degrees C, respectively. After 19 days in vitro incubation, Tg of the degrading POE microspheres increased to 55 degrees C. Weight loss studies showed that the degradation of the blend microspheres was accelerated with the presence of PLGA because its degradation products catalyzed the degradation of both POE and PLGA. The release rate of physostigmine increased with increase of PLGA content in the blend microspheres. The initial burst release of physostigmine was effectively suppressed by introducing POE to the blend microspheres. However, there was an optimized weight ratio of POE to PLGA (85:15 in weight), below which a high initial burst was induced. The POE/PLGA blend microspheres may make a good drug delivery system.

Body Fluids↗