Pseudomonas 2 F: Kinetics of growth and accumulation of poly - D(-)-3-hydroxybutyric acid (poly - HB)

Summary Kinetics of poly - HB accumulation in Pseudomonas 2F, a recently isolated and new strain, differ considerably from the behaviour found hitherto with other strains. After a period of carbon limitation of 1 hour, without the application of growth - limiting conditions, the accumulation of poly - HB is evidently accelerated (carbon - overcompensation).     

Laser Raman spectroscopic studies of poly(r-cytidylic acid) and poly(r-adenylic acid) complexes with poly(L-lysine) and poly(L-arginine)

Complexes of polyribocytidylic acid and polyriboadenylic acid with poly(L -lysine) and poly(L -arginine) were studied by Raman spectroscopy. The backbones of both polynucleotides are distorted by poly(L -arginine). On the other hand, poly(L -lysine) could distort the backbone of polyriboadenylic acid but not that of polyribocytidylic acid. In general, poly(L -arginine) can increase the order of the base stacking, while poly(L -lysine) causes disordering in the base stacking.     

Interactions of a 5-nitroxide-labeled poly(uridylic acid) with poly(adenylic acid)

The physicochemical properties of a high-molecular-weight spin-labeled nucleic acid, (RUGT,U)_n, synthesized by enzymatic copolymerization, were evaluated by uv and ESR spectroscopy. It was shown earlier that spin labeling of nucleic acids by chemical modification to an extent which gives a nitroxide-to-nucleotide ratio greater than 0.002 can cause noticeable lattice perturbations (A. M. Bobst, A. Hakam, P. W. Langemeier, and S. Kouidou (1979), Arch. Biochem. Biophys. 187, 339–345). The presence of RUGT, a 5-nitroxide-labeled uridine residue, in a (U)_n lattice at a $\text{RUGT}\text{U}$ ratio of 0.01 is shown here not to affect the complexation with (A)_n, since the uv melting temperature (T₀^OD) of the 2 → 1 transition and the hypochromicity changes were the same for (RUGT,U)_n· (A)_n and (U)_n·(A)_n. ESR measurements indicated that the nitroxide radical reflects the transition accurately within the error limit, although a slight destabilization of the spinlabeled segment could not be excluded. Computer simulations showed conclusively that the spin melting temperature (T_m^sp) corresponds to the temperature at which half of the spin-labeled segments are no longer complexed, for the ESR spectrum at T_m^spcan be simulated with equal contributions from the line shapes of ESR spectra taken before and after the transition. Arrhenius plots obtained by using two different approaches for computing correlation times were qualitatively the same. Computer analysis also revealed that the formation of a (RUGT,U)_n·(A)_n complex can be described by a two-state model, in contrast to results obtained with chemically spin-labeled (U)_n. Thus, using (RUGT,U)_n over chemically spin-labeled (U)_n can offer distinct advantages.     

Counterion binding in partially neutralized poly(D-glutamic acid) and poly(DL-glutamic acid)

Sodium counterion association with partially neutralized poly(D -glutamic acid) or poly(DL -glutamic acid) was measured by use of Wall's transference method with radioactive sodium. In the region where both polyacids are in completely random coil form, fractions of association were considerably less than that with poly(acrylic acid) in the same region of degree of neutralization. Even in the region where poly (D -glutamic acid) is in the helical form, the fraction of association was less than that with poly(acrylic acid) in the same region. No pronounced characteristics attributable to counterion association corresponding to the helix–coil transition could be found. The association phenomena were discussed on the basis of a rodlike model of polyelectrolyte.     

Solution conformation of poly(L-lysyl-L-glutamic acid) and poly(L-lysyl-L-glutamine)

Solution conformation of poly(L-lysyl-L-glutamic acid) (PLGU) and poly(L-lysyl-L-glutamine) (PLGN) was studied in water as a function of pH, added salt, detergents, methanol and trifluoroethanol (TFE). Both the polypeptides exhibit no ordered conformation in the pH range 1.5-12.5; salts and detergents did not have any marked effect. Replacement of side chain carboxyl by an amide group did not help in inducing PLGN to adopt a helical conformation even at pH as high as 12.0, unlike poly(L-lysine). The helicogenic solvents, methanol and TFE, induce formation of weak helices in PLGU as well as in PLGN. It is not unlikely that H-bonding between the side chains leads to stabilizing an unordered conformations.     

Uniaxial drawing and mechanical properties of poly[(R)-3-hydroxybutyrate]/poly(L-lactic acid) blends

Blends of poly(L-lactic acid) (PLLA) with two kinds of poly[(R)-3-hydroxybutyrate] (PHB) having different molecular weights, commercial-grade bacterial PHB (bacterial-PHB) and ultrahigh molecular weight PHB (UHMW-PHB), were prepared by the solvent-casting method and uniaxially drawn at two drawing temperatures, around PHB's T(g) (2 degrees C) for PHB-rich blends and around PLLA's T(g) (60 degrees C) for PLLA-rich blends. Differential scanning calorimetry analysis showed that this system was immiscible over the entire composition range. Mechanical properties of all of the samples were improved in proportion to the draw ratio. Although PLLA domains in bacterial-PHB-rich blends remained almost unstretched during cold drawing, a good interfacial adhesion between two polymers and the reinforcing role of PLLA components led to enhanced mechanical properties proportionally to the PLLA content at the same draw ratio. On the contrary, in the case of UHMW-PHB-rich blends, the minor component PLLA was found to be also oriented by cold drawing in ice water due to an increase in the interfacial entanglements caused by the very long chain length of the matrix polymer. As a result, their mechanical properties were considerably improved with increasing PLLA content compared with the bacterial-PHB system. Scanning electron microscopy observations on the surface and cross-section revealed that a layered structure with uniformly oriented microporous in the interior was obtained by selectively removal of PLLA component after simple alkaline treatment.     

Interferon induction by poly(inosinic acid).poly(cytidylic acid) segmented by spin-labels

Poly(inosinic acid).poly(cytidylic acid) [(I)n.(C)n] duplexes of which the (C)n strand was modified to various degrees chemically or enzymatically with nitroxide radicals (spin-labels) were evaluated for interferon-inducing activity. Upon annealing of the chemically modified (C)n, (1C,Cx)n (X = 1000 or 16), with (I)n, the interferon-inducing activity was similar to that of (I)n.(C)n in PRK cell cultures. However, to overcome hydrolysis of the spin-label linkage in (1C,Cx)n, and enzymatic approach was taken to synthesize (1S(4)U,Cx)n copolymers with x = 100, 38, 16, and 8. The (1s(4)U,Cx)n copolymers were chemically stable, and upon annealing with (I)n the correlation time of the nitroxide moiety in (I)n. (1s(4)U,Cx)n was determined. A comparison of this correlation time with that measured for (RUGT,U100)n.(A)n, which contains the nitroxide moiety in position 5 of the U moiety, suggests that the 1s(4)U residue is in a nonintrahelical conformation and partitions the duplex into double-helical segments of varying size. The interferon-inducing activity of (I)n. (1s(4)U,Cx)n was evaluated in primary rabbit kidney, human skin fibroblast (strain VGS), and mouse L-929 cell cultures as well as in rabbits. The 1s(4)U residue did not cause a significant change in the interferon induction as compared to (I)n.(C)n in most systems tested unless x less than 16. These findings indicate that double-helical segments of approximately 16 base pairs partitioned by nonintrahelical 1s(4)U residues suffice to trigger the interferon response in all systems studied.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Antibody-nucleic acid complexes. Oligo(dG)n and -(dT)n specificities associated with anti-DNA antibodies from autoimmune MRL mice

The specificity of anti-DNA antibodies in the sera of unimmunized autoimmune MRL mice was initially assessed via an enzyme-linked immunosorbent assay (ELISA). Antibody binding profiles to a panel of immobilized antigens (AMP-, GMP-, CMP-, UMP-, and TMP-BSA, ss- and dsDNA) demonstrated high levels of immunoglobulins reacting with GMP and ssDNA and intermediate levels with AMP, TMP, and dsDNA. Fractionation of serum anti-DNA antibodies into subsets on the basis of their binding to GMP- and TMP-agarose indicated that the resulting GMP- or TMP-reactive antibodies bound to their homologous nucleotides and ssDNA. Competition-inhibition studies with soluble mono-, oligo-, and polynucleotides revealed that GMP- and TMP-reactive antibodies were highly specific for oligo(dG)n and -(dT)n sequences, respectively. Whereas the relative affinity of TMP-reactive autoantibodies to oligo(dT)n increased with oligonucleotide length (n = 2, 4, 6, 8, 10, 15), GMP-reactive antibodies preferentially recognized oligo(dG)10 (Ka congruent to 1 x 10(7) M-1). While neither antibody recognized oligo(dA)8 and -(dC)8 competitors, mixed-base oligonucleotides were inhibitory at concentrations approximately 10-fold greater than similarly sized oligo(dG)n and -(dT)n sequences. Similar characterizations of both pooled and individual MRL sera indicated that anti-DNA antibodies represent 8-10% of the total serum IgG. More importantly, GMP-reactive autoantibodies predominated and accounted for 60-70% of the entire unbound anti-DNA antibody population.     

Calorimetric studies of the polynucleotide analog poly(-)2-[2-(thymin-1-yl)propanamido]propenoic acid

The thermally elicited structural and conformational transitions of the polynucleotide analog, poly(-)-2-[2-(thymin-1-yl)propanamido]propenoic acid, P(-)TDHA, have been studied using differential scanning calorimetry (DSC), differential thermal analysis (DTA), and thermogravmetric analysis (TGA). The differential scanning calorimetry curves obtained on solid P(-)TDHA samples exhibited five distinct transitions. The transition occurring at 50°C is attributed to a disruption of interactions involving thymine–thymine stacking. In contrast the transition observed at 83°C is attributed to a hydrogen-bonding interaction involving the thymine residues, whereas the transition occurring at 110°C is assigned to hydrogen bonding of the carboxylic acid side-chain groups. The transitions observed at 50, 83, and 110°C are reversible if the heated and quenched sample is allowed to equilibrate in an atmosphere of high humidity. The transition occurring at 127°C is viewed as a structural rearrangement of the polymer backbone that does not involve the participation of water molecules. The transition observed at a temperature above 197°C is attributed to a structural modification of the polymer resulting from decomposition. Solutions of P(-)TDHA in 0.1M phosphate buffer at pH 7.05 showed only a single transition at 50°C, which is in accord with an observed transition in the solid state assigned to the disruption of base-stacking interactions. The average enthalpy for the transition at 50°C was 0.92 cal/g in the solid state and 1.08 cal/g for the solution, which provides additional support for the assignment.     

Electric dichroism of poly(riboadenylic acid)

Electric dichroism measurements on poly(A) in low-ionic-strength solution demonstrate that below a molecular weight of 130,000 the double-stranded polymer is hydrodynamically rigid and above that molecular weight becomes increasingly flexible. At 500,000 it is considerably more flexible than DNA of the same molecular weight, with a mean end-to-end distance of about 1150 Å compared to approximately 1600 Å for DNA. The fully extended length for both DNA and poly(A) of this molecular weight is about 2750 Å. It is further shown that the orientation of these polyelectrolytes in an electric field is consistent with theoretical treatments of the counter-ion distribution and a preliminary model based on the additivity of classical valence charge anisotropy and counter-ion polarization is postulated for the orientation mechanism. Single-stranded pol (A) is shown not only to retain its base stacking in the presence of the electric field but to extend the persistent regions of stacked bases so that it attains a rodlike structure very similar to the one in the double-stranded polymer is found to be less than that expected from consideration of the x-ray structure. An explanation for this result is sought in the electric asymmetry of the helical polymer.     

Single crystals of poly(L-glutamic acid)

Lamellar single crystals of alkaline earth salts of poly(L -glutamic acid) have been grown by precipitation from dilute aqueous solution and studied by optical and electron microscopy and by x-ray and electron diffraction. The calcium, strontium and barium salts were crystallized in the β form above room temperature and could be converted to crystals of β-poly(L -glutamic acid) by washing in dilute hydrochloric acid. The magnesium salt, on the other hand, was crystallized in the α form at or below room temperature but could not be converted into crystals of α-poly(L -glutamic acid) by washing in hydrochloric acid. The crystalline lamellae are very thin (thicknesses range from 25 to 60 Å in β crystals and are about 100 Å in α crystals) and the polypeptide chains are oriented normal to the planes of the lamellae. It is clear from the disparity between crystal thickness and molecular length that the molecules crystallize by folding at the upper and lower surfaces of the crystals. Conformations of the molecules at these folds are discussed briefly.     

Metalloinitiation routes to biocompatible poly(lactic acid) and poly(acrylic acid) stars with luminescent ruthenium tris(bipyridine) cores

Poly(lactic acid) (PLA) and poly(acrylic acid) (PAA) biomaterials with luminescent ruthenium tris(bipyridine) centers couple drug delivery and imaging functions. Hydrophobic [Ru(bpyPLA2)3](PF6)2 (1) was generated from [Ru[bpy(CH2OH)2]3](PF6)2 in bulk monomer using 4-(dimethylamino)pyridine as the catalyst. The bromoesters, [Ru[bpy(CH2OR)2]3](PF6)2, [Ru[bpy(C13H27)2][bpy(CH2OR]2](PF6)2 (4), and [Ru[bpy(PLAOR)2]3]2+ (9) (R=COCBr(CH3)2), served as initiators for tert-butyl acrylate (tBA) polymerization. Conversion of PtBA to PAA via hydrolysis affords water soluble materials, [Ru(bpyPAA2)3]2+ (7) and [Ru[bpy(C13H27)2](bpyPAA2)2]2+ (8) and the amphiphilic star polymer [Ru[bpy(PLA-PAA)2]3)](PF6)2 (11), which is soluble in a H2O/CH3CN (1:1) mixture. Luminescence excitation and emission spectra of the Ru polymers were in agreement with the parent [Ru(bpy)3]2+ chromophore (lambdaex=468, lambdaem=621 nm). Lifetimes of tau approximately 700 ns in both air and nitrogen atmospheres are typical for most materials; however, the amphiphilic star block copolymer 11 is quenched by oxygen to some degree. Thermal analysis shows the expected glass transitions for the polymeric ruthenium complex materials.