Native Calf Research Articles

Hyaluronan Decreases Surfactant Inactivation In Vitro

Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.

Atomic force microscopy of DNA self-assembled on a highly oriented pyrolytic graphite electrode surface

Different types of self-assembled DNA networks were obtained and characterized by tapping mode atomic force microscopy (AFM) on highly oriented pyrolytic graphite (HOPG) electrodes. Both native calf thymus DNA and synthetic DNA with specific base sequences were investigated. The freely adsorption of DNA on the surface of HOPG forms two-dimensional networks. The double-stranded DNA molecules may form cross-linking points on double helix chains, forming intermediate attachments. The differences in AFM morphology of these DNA condensations can be related to the intrinsic properties of the DNA, which lead to different interactions with HOPG. Three-dimensional networks were formed on the HOPG under applied electric field. The effects of external electric field on the condensation of calf thymus DNA have been investigated in detail. It led to a proposition that the DNA molecules may be polarized in the electric field, and attached to the HOPG surface via its one end with the phosphate backbone lying or standing on the substrate depending on the applied potential. The AFM morphological changes of DNA in the presence of external electric field provided insight into DNA interactions.

Magnetic relaxation dispersion of lithium ion in solutions of DNA.

The magnetic field dependence of the nuclear spin-lattice relaxation rate constant defines the magnetic relaxation dispersion (MRD) and provides a direct characterization of the molecular dynamics that cause fluctuations in the magnetic couplings in the system and may also indicate the dimensional constraints on the motion. The counterion cloud surrounding a linear polyelectrolyte ion, such as DNA in solution, provides an interesting opportunity for ion confinement that helps in understanding the thermodynamics and the dynamics of the interactions between the polyion and other solutes. The MRD profiles of lithium ion and tetramethylammonium ion were recorded in dilute aqueous solutions of native calf thymus DNA, which provides a long, charged rod that reorients slowly. The 7Li ion relaxes through the nuclear electric quadrupole coupling and the proton-lithium dipole-dipole coupling; the protons of the tetramethylammonium ion relax by dipole-dipole coupling. MRD profiles of the 7Li+ ion are dominated by transient interactions with the DNA that yield a linear dependence of the spin-lattice relaxation rate constant on the logarithm of the Larmor frequency. This magnetic field dependence is consistent with diffusive ion motions that modulate two spatial coordinates that characterize the relaxation couplings in the vicinity of the polyion. Motions around the rod and fluctuations in the ion distance from the rod are consistent with these constraints for lithium. The magnetic field dependence of the tetramethylammonium ion proton relaxation rate constant is weak, but also approximately a linear function of the logarithm of the Larmor frequency, which implies that the field dependence is caused in part by local order in the DNA solution.

The interaction of native DNA with iron(III)- N, N′-ethylene-bis(salicylideneiminato)-chloride

The interaction between native calf thymus deoxyribonucleic acid (DNA) and Fe III- N, N ′-ethylene-bis (salicylideneiminato)-chloride, Fe(Salen)Cl, was investigated in aqueous solutions by UV–visible (UV–vis) absorption, circular dichroism (CD), thermal denaturation and viscosity measurements. The results obtained from CD, UV–vis and viscosity measurements exclude DNA intercalation and can be interpreted in terms of an electrostatic binding between the Fe(Salen) + cation and the phosphate groups of DNA. The trend of the UV–vis absorption band of the Fe(Salen)Cl complex at different ratios [DNA phosphate]/[Fe(Salen)Cl] and the large increase of the melting temperature of DNA in the presence of Fe(Salen)Cl, support the hypothesis of an external electrostatic interaction between the negatively charged DNA double helix and the axially stacked positively charged Fe(Salen) + moieties, analogously to what reported for a number of porphyrazines and metal–porphyrazine complexes interacting with DNA.

Role of nitric oxide modified DNA in the etiopathogenesis of systemic lupus erythematosus.

The role of the nitric oxide (NO) radical in systemic lupus erythematosus (SLE) pathogenesis has been investigated in the present study. The binding characteristics of SLE autoantibodies with native calf thymus DNA, native and NO-modified plasmid DNA were assessed. Binding characteristics and specificity of antibodies were analysed by direct binding and inhibition ELISA, gel retardation assay and quantitative precipitin titration. The data shows preferential binding of SLE autoantibodies to NO-modified plasmid DNA (NO-DNA) in comparison with native plasmid DNA. Inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of NO-DNA by anti-DNA autoantibodies. The binding affinity of modified and native plasmid DNA with one of the SLE IgGs was calculated, using the Langmuir plot. The apparent association constant for NO-plasmid DNA was found to be highest, followed by native calf thymus DNA and native plasmid DNA. The results suggest that the NO radical modification of plasmid DNA causes perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen. The DNA modified with the NO radical may be one of the factors for the induction of circulating SLE anti-DNA autoantibodies.

Studies on the interaction of altromycin B and its platinum(II) and palladium(II) metal complexes with calf thymus DNA and nucleotides

The interaction of the anticancer antibiotic altromycin B and its isostructrural Pt(II) and Pd(II) metal complexes with native calf thymus (CT) DNA was studied using UV–thermal denaturation experiments, circular dichroism spectroscopy and temperature controlled spectrophotometric titrations. Altromycin B stabilizes the double helix by raising the T m, mainly by intercalation of its chromophore between the base pairs and interacting electrostatically via its sugar moieties with the edges of the DNA helix. Moreover, altromycin B induces a B→A structural transition of CT DNA. The effect on DNA stability and conformation depends on the metal ion. Pt(II) and Pd(II) complexes induce the B→A structural transition and stabilize the double helix similarly but they present lower final hyperchromicity due to premelting effects which were caused by intra- and interstrand crosslinking. Thus, a synergic effect of the metal ions to altromycin B–CT DNA interaction is observed in both cases. Altromycin B interacts with 5′-GMP, 5′-AMP and 5′-CMP by electrophilic attack of the opened epoxide ring to the N(7)G, N(1)/N(7)A and N(3)C. Thus, covalent binding between these nucleotides and altromycin B takes place and explain the multiple binding mode suggested by the studies of the interaction of altromycin B and its complexes with DNA. The [Pd(II)–altroB] complex dissociates in the presence of the nucleotides, and various species of Pd(II)–nucleotide complexes, especially with 5′-GMP, are formed. The [Pt(II)–altroB] complex dissociates too, but only one or two species of Pt(II)–nucleotide complexes are formed, and in the case of 5′-AMP interaction the formation of a tertiary altroB–Pt(II)–5′AMP complex is proposed. 5′-TMP reacts very weakly in comparison with the other three nucleotides. These interactions were followed by 1H-NMR.

Microparticles derived from marine sponge collagen (SCMPs): preparation, characterization and suitability for dermal delivery of all- trans retinol

Collagen microparticles were prepared using marine sponge collagen. For this purpose a previous method by Rössler et al. (J. Microencapsul. 12 (1995) 49) of emulsification and cross-linking of native calf collagen was modified. The modified method for sponge collagen microparticles (SCMPs) achieved a yield of 10%. Scanning electromicroscopic photographs showed spherical particles with a diameter of 120–300 nm and photon correlation spectroscopic measurements indicated particle size range from 126 (±2.9) to 2179 (±342) nm. This broad size distribution was caused by some agglomerates that could not be destroyed by ultrasonication. The surface charge was measured as a function of pH. At pH 2.8 the particles were nearly uncharged, at pH 9.0 the particles showed a strong negative charge of about −60 mV. The preformed SCMPs were loaded by adsorption of all- trans retinol. A loading of up to 8% was obtained. Retinol-loaded SCMPs were incorporated into hydrogels and drug stability was investigated. The in vitro penetration of retinol into hairless mice skin in this formulation was compared to retinol formulations without microparticles. The SCMPs had no influence on the chemical stability of retinol in the hydrogel. The dermal penetration of retinol into the skin increased significantly by approximately two-fold.

Reconstitution of human DNA polymerase delta using recombinant baculoviruses: the p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme.

Eukaryotic DNA polymerase delta is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase delta. However, rigorous purification of human or bovine DNA polymerase delta from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase delta, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase delta displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase delta efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase delta.

Dielectric and UV spectrophotometric study of physicochemical effects of ionizing radiation on mammalian macromolecular DNA

The purpose of this work was the comparative investigation of physicochemical effects induced by /spl gamma/ and /spl alpha/ radiation on the thermal denaturation dynamics of native macromolecular calf thymus DNA, combining dielectric relaxation spectroscopy (DRS) and UV thermal transition spectrophotometry (TTS), while the apparent molecular weight (MW) distribution of the DNA macromolecules was measured by pulse field gel electrophoresis (PFGE). A DNA thermal stability enhancement over the non-irradiated samples was observed at radiation doses <10 Gy for /spl gamma/ rays and 32 Gy for /spl alpha/ particles, attributed to the development of intra and/or intermolecular DNA-DNA interactions, whereas at higher irradiation doses, the expected decrease of thermal stability was observed. Moreover, in all studied cases, the recorded conductivity changes were found to precede by 2 to 3/spl deg/C those of UV TTS absorbance changes, a fact indicative of a multi-stage process for DNA thermal transition. Finally, the displacement of the low dose region limit towards higher doses in the case of /spl alpha/ particles, confirm earlier results attesting higher yields of DNA strand breaks induced by /spl gamma/ rays compared to /spl alpha/ particles.

Effects of radical scavengers on radiation-induced DNA double strand breaks

Purpose: To investigate possible effects of Tris and phenol on the dynamic properties of gamma-irradiated DNA molecules in addition to their well known scavenging capacity. Materials and methods: Native and fragmented calf thymus DNA molecules were exposed to various doses of 60 Co gamma-rays at ~4.5Gy/min. Using thermal transition spectrophotometry, pulsed field gel electrophoresis and standard agarose gel electrophoresis, the effects of Tris, phenol and NaCl on the double helix to single coil thermal transition temperature, Tm, and the yield of the double-strand breaks (G dsb) of the irradiated DNA molecules have been studied. Results: DNA molecules exposed to gamma-rays showed a decreased T m and a corresponding increase of the G dsb yield. Tris, as well as phenol, exhibited a strong protection against preventing these radiation-induced alterations. In addition, both substances strongly affected the thermal stability of the non-irradiated DNA samples. These results, compared with data obtained by NaCl and its effects on DNA thermostability and G dsb, revealed that in the presence of both scavengers the observed dsb decrease was correlated to an increased molecular stability of DNA. Conclusions: This work suggests that the total protective effect of Tris and phenol against radiation-induced dsb is mainly attributed to their well-known radical scavenging properties, while relatively minor protective effects arise from their contribution to an increased molecular stability of DNA.

Antigen Binding Characteristics of Experimentally-Induced Antibodies Against Hydroxyl Radical Modified Native DNA

Hydroxyl radical, generated by UV irradiation of hydrogen peroxide caused damage to native calf thymus DNA leading to strand breaks, base modification and decrease in melting temperature. The ROS-DNA was highly immunogenic in goat. The antibody activity was inhibited to the extent of 89% with the immunogen as inhibitor. Antigenic specificity of anti-ROS-DNA antibodies by competition ELISA showed multiple cross-reactivity, recognizing B-, A-and allied conformations. The immune complex formation with native and ROS-DNA was substantiated by band shift assay. A striking observation, is the enhanced recognition of ROS-thymine and ROS-poly(dT) by the induced antibodies. These investigations suggest that ROS might be the plausible candidate for the presentation of unique epitopes on ROS-DNA, in particular of thymine, inducing antibodies cross-reacting with native DNA and play an important role in the pathogenesis of SLE.

SLE autoantibodies binding to native calf thymus DNA brominated in high salt.

Native calf thymus DNA was brominated in high salt to achieve B-->Z conformational transition. Ultraviolet and circular dichroism spectroscopic studies point towards the conformational modification of the native DNA. Specific binding of the monoclonal anti-Z-DNA antibody (Z-22) to the DNA brominated in high salt further confirmed the B-->Z conformational isomerization of native DNA. The role of Z-DNA in the etiopathogenesis of systemic lupus erythematosus has been investigated in the light of the binding of naturally occurring human anti-DNA autoantibodies to the induced Z-DNA.

Binding of naturally occurring anti-DNA antibodies to estradiol.

Binding of naturally occurring human anti-DNA autoantibodies to beta-estradiol and native DNA has been investigated. Sera from seven SLE patients were tested for their reactivity with beta-estradiol and native calf thymus DNA. Solid Phase enzyme immunoassay was performed using both colorigenic and fluorogenic substrates. The results show enhanced binding of human anti-DNA autoantibodies to beta-estradiol as compared to native DNA. This was further substantiated by increased binding of affinity purified SLE IgG to beta-estradiol. These results suggest a role for female sex hormone in the etiopathogenesis of SLE and are significant vis-a-vis female predominance of SLE.

Reactivity of Haloketenes and Halothioketenes with Nucleobases: Reactions in Vitro with DNA

Ketenes are important and highly reactive intermediates. Thioketenes are formed by cysteine conjugate beta-lyase-dependent biotransformation of 1-halovinylcysteine S-conjugates which are metabolites of several halogenated olefins. Nucleic acid constituents react with haloketenes and halothioketenes in vitro. Thioketenes induce DNA strand breaks in incubations of 1,2-dichlorovinyl 2-nitrophenyl disulfide, a thioketene precursor, with pBr 322 plasmid DNA. After treatment of single-stranded or native calf thymus DNA with chlorothioketene generated by the hydrolysis of 1,2-dichlorovinyl 2-nitrophenyl disulfide, the formation of 3,N4-thioacetylcytosine could be demonstrated. N6-(Dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine, however, adducts formed by dichloroketene in vitro, are labile to hydrolysis. Therefore, the binding of this compound to DNA constituents in intact DNA is difficult to demonstrate. Substitution of one chlorine atom by fluorine allowed us to use 19F NMR as a tool to demonstrate the formation of adducts by dihaloketenes in intact DNA. N6-(Chlorofluoroacetyl)adenine and N4-(chlorofluoroacetyl)cytosine were synthesized (yields 77%, 15%, respectively) as references and characterized by LC/MS, 1H, 13C, and 19F NMR, FT-IR, and elemental analysis. To demonstrate the ability of dihaloketenes to bind to DNA, poly-dA (1 mg) and calf thymus DNA (10 mg) were suspended in DMF and treated with different concentrations of chlorofluoroketene (50-200 micromol). Analysis of the polymeric DNA by 19F NMR showed one doublet at -137.2 ppm downfield from the reference (CFCl3). A doublet at -146.9 ppm, characteristic for chlorofluoroacetic acid, an expected product of DNA adduct hydrolysis, was not detected. These results demonstrate the formation of a stable adenine adduct by dihaloketenes in intact calf thymus DNA.

DNA BINDING STUDIES OF MAGNESIUM(II), CALCIUM(II), BARIUM(II) AND ATP COMPLEXES

We have investigated the ways by which magnesium (II), calcium (II) and barium (II) and their ATP complexes bind to B-DNA. Effects on native calf thymus DNA conformation were studied and compared with those involving the uncomplexed metal ions and the polymer. It was found that MgATP−2, CaATP−2 and BaATP−2, interact with the phosphates of the polynucleotide; only BaATP −2 appears to bind also through the bases. ATP complexes cause transitions mainly to the B family. The C form is induced by small amounts of MgATP −2 or CaATP−2. At high complex concentrations (r = 2.5 or 5) the polynucleotide adopts the ψ conformation, while BaATP−2 induces this transition at r = 0.6. A contribution of the A form is observed in the case of CaATP-DNA at r = 0.6 and at the beginning of the reaction in the Ba-DNA system at 22°C, for r = 5. The alkaline earth metal ions induce a stabilization of the B conformation. Structural changes with magnesium (II) at r = 2.5 are interpreted in terms of a B to C transition. Calcium (II) at r = 5 produces the A form. In other cases the alkaline earth metal ions stabilize the B conformation. It was found that they all interact with the phosphates of the polynucleotide. The binding ratio, r, is an important factor in all structural changes.

X-ray small angle scattering study of chromatin as a function of fiber length.

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.

Binding characteristics of SLE anti-DNA autoantibodies to modified DNA analogues.

Native calf thymus DNA was modified with furocoumarins and reactive oxygen species (ROS). The modifications were probed by UV & Fluorescence spectroscopy, thermal denaturation and hydroxyapatite chromatography. In CD spectroscopy changes in the ellipticity of the DNA molecule were observed as a result of modification. The binding specificity of naturally occurring anti-DNA antibodies with modified DNA molecules was assessed by ELISA and quantitative precipitin titration. The affinity of anti-DNA antibodies for modified conformers was found to be quite high.

High salt and solvent induced Z-conformation in native calf thymus DNA.

The Z-DNA forming potentiality of native calf thymus DNA using spectrophotometric measurements and diethyl pyrocarbonate reactivity have been investigated. The Z value was evaluated from absorbance ratio for B- and Z-DNA and compared with the values obtained for native DNA. The results suggest that native DNA undergoes B- to Z-/Z-analogous transition to an extent of 20 per cent under constraint conditions. Possible Z-DNA formation was also characterized by hyper-reactivity of native DNA towards diethyl pyrocarbonate in aqueous solvent. The thermal melting profile of native and modified DNA and nuclease S1 digestibility data, in addition, reflects B- to Z-/Z-analogous transition in native DNA.

SLE autoantibodies recognize spermine induced Z-conformation of native calf thymus DNA.

The binding specificity of naturally occurring anti-DNA antibodies with spermine modified DNA was analyzed by direct binding and competition ELISA. The binding was visualised by band shift assay. The SLE anti-DNA antibodies appreciably recognized the spermine induced Z-conformation of native DNA. The degree of binding of these antibodies to the Z-conformer was almost similar to their binding with native DNA. The spermine-DNA complex was found to be thermodynamically more stable than native DNA.

Nucleic acid-8-methoxypsoralen crosslinks bind monoclonal anti-Z-DNA antibody.

Native calf thymus DNA and poly(dA-dT).poly(dA-dT) were photo-adducted with 8-methoxypsoralen and characterized by thermal denaturation (Tm) and hydroxyapatite column chromatography. The data demonstrated the formation of interstrand photo-crosslinks. It has been shown by competition ELISA and band shift assays that crosslinked species of DNA-8-MOP and poly(dA-dT)-8-MOP photoadducts recognize previously defined monoclonal anti-Z-DNA antibody (Z22). The results indicate the possible presence of Z- or Z-like epitopes on nucleic acid-8-MOP crosslinks as Z22 antibody does not recognize other nucleic acid conformations. These studies also point out that conformational changes in DNA arising from the photo-addition could induce antibodies to DNA or could cause autoimmune disease.