Effect of Mn2+ and Mg2+ ions on DNA conformation

The DNA conformation was studied at different relation between Na+ and Me2+ (Mn2+ or Mg2+) ions in solution at the fixed total ionic strength mu. At low mu the intrinsic viscosity of DNA [eta] decreased to the limited fixed value with the increasing of Mn2+ or Mg2+ concentration (CMe2+). At higher mu greater than or equal to 0.1 M [eta] doesn't depend on CMe2+. The presence of Mn2+ in solution caused a decrease of the optical anisotropy of DNA and the value of epsilon 260 (p) independent on ionic strengths. In contrary, these parameters of DNA didn't change in solution with Mg2+-concentration. The observed differences in the effects of Mn2+ and Mg2+ on the optical properties of the macromolecule suggest that there are different modes of binding of these ions to DNA. It has been concluded, that Mn2+ interacts with bases and phosphate groups of DNA, but Mg2+--only with phosphates. The persistence length of DNA doesn't depend on Me2+ concentration under the conditions of the experiment (mu greater than or equal to 0.005 M).     

Conformation of the denatured DNA molecule in solutions of different ionic strengths]

The conformation of the denatured DNA molecule of different molecular weights in the solutions of various ionic composition was studied by the methods of viscometry, light scattering and flow birefringence. Formaldehyde purified from metallic ions with the help of ionites was used for fixation of the denatured state of the DNA molecule. It has been shown that theories developed for flexible macromolecules are in a sufficient accordance with hydrodynamical and optical data. The unperturbed dimensions, equilibrium rigidity of the macromolecule in solutions of different ionic strengths, mu, were determined. In the range of mu greater than or equal to 0.005 the length of Kuhn's segment (A) is equal to approximately 40 A and its value increases with an increase of mu. At mu 0.001 A approximately 60 A and mu 0.0005 A approximately 85 divided by 100 A. A relation between intrinsic viscosity and molecular weight of the denatured DNA molecule was established. Data on the flow birefringence in the solutions of the denatured DNA have shown that the sigh of optical anisotrophy of the macromolecule depends on the ionic strength. The observed dependency may be explained only by assuming that ionic strength influences the equilibrium orientation of nitrogen base planes with respect to the main chain of the macromolecule.     

Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA

Release of free bases from calf thymus DNA upon irradiation in aerated 0.1 mol dm-3NaClO4 at pH 7 has been measured by HPLC and shown to be markedly influenced by the presence of thiols during irradiation. The ability of thiols to protect DNA was shown to depend upon the net charge (Z) at pH 7 in the order WR 1065 (Z = +2) greater than cysteamine (Z = +1) greater than 2-mercaptoethanol (Z = 0) approximately equal to dithiothreitol (Z = 0) greater than GSH (Z = -1) approximately equal to 2-mercaptoethanesulfonic acid (Z = -1) approximately equal to 2-mercaptosuccinate (Z = -2). A similar dependence of protection upon net charge was found for disulfides: cystamine (Z = +2) greater than 2-mercaptoethyl disulfide (Z = 0) greater than GSSG (Z = -2). Protection by WR 1065, but not by 2-mercaptoethanol or GSH, was found to decrease significantly with increasing ionic strength. Protection by WR 1065 and GSH was not markedly dependent upon pH between pH 6 and 8. The results are explained in terms of electrostatic interaction of the thiols with DNA, leading to high concentrations of cations near DNA, which allow them to scavenge hydroxyl radicals and repair DNA radicals effectively and to low concentrations of anionic thiols near DNA, which limit their effectiveness as protectors. Poly(dG,dC) and calf thymus DNA exhibited comparable release of G and C upon changing from 0.1 to 0.7 mol dm-3 MgSO4. Since this change causes poly(dG,dC), but not calf thymus DNA, to undergo a change from the B-form to the Z-form of DNA, both forms must have a comparable susceptibility to radiation-induced base release.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.     

Genealogy of neutral genes in two partially isolated populations

Gene genealogy in two partially isolated populations which diverged at a given time t in the past and have since been exchanging individuals at a constant rate m is studied based upon an analytic method for large t and a simulation method for any t. Particular attention is paid to the conditions under which neutral genes sampled from populations are mono-, para-, and polyphyletic in terms of coalescence (divergence) times of genes. It is shown tha the probability of monophyly is high if M = 2Nm less than 0.5 and T = t/(2N) greater than 1, where N is the size of ancestral and descendant haploid populations, in which case most gene genealogies are likely to be concordant with the population relatedness. This probbility decreases as the sample size of genes increases. On the other hand, the case where the probability of monophyly is low will be either that of M greater than 1 and any T or that of M less than 1 and T less than 1, but the clear distinction between these conditions appears very difficult to make. These results are also examined if the gene genealogy is reconstructed from nucleotide differences. It is then shown that the results based upon coalescence times remain valid if the number of nucleotide differences between any pair of genes is not much smaller than 10. To observe such large nucleotide differences in small populations and therefore infer a reliable gene genealogy, we must examine a fairly long stretch of DNA sequences.     

Amplitude of the tension developed by glycerinated muscle fibers during rigidity depends on the nature of the structural reorganizations in F-actin induced by formation of the actomyosin complex

Dependence between the amplitude of tension, developed by glycerinated muscle fibers during rigidity, and the character of structural changes in F-actin, induced by the formation of actomyosin complex, was studied by polarized microfluorimetry and tensiometry. It is shown that during rigidity the anisotropy of intrinsic tryptophan residues as well as of rhodamine phalloidin bound to F-actin, and amplitude of tension depend on pH (6-8) and ionic strength (mu = 0.07 M-0.14 M) of solution. Greater changes in polarized fluorescence and in amplitude of tension were registered during rigidity in solutions with low ionic strength (mu = 0.07 M) and pH 8. It suggested that the amplitude of muscle fibre tension depends on the relative quantity of actin monomers, being in the "switched on" state.     

Effect of temperature on the conformation of native DNA in aqueous solutions of various electrolytes

Effect of the temperature on the conformation of the native DNA molecule in solution of different electrolytes (LiCl, NaCl, KCl, CsCl, Gu-HCl) at ionic strengths mu = 5; 0.1; 0.01; 0.005 and temperatures ranging from 10 to 40 degrees C were studied by the methods of flow birefringence and viscometry. The experiments showed that the value of intrinsic viscosity [eta] of DNA increases at increase of temperatures in solutions of all the chlorides studied, excluding guanidine. The effect of temperature on the value of [eta] doesn't depend on the type of the cation at a fixed value of mu and is elevated when mu decreases. The observed alterations of the value of [eta] for DNA in water-salt solutions at different temperatures can be explained by an increase in the hydration of the alkaline ions at temperature increase. The experiments showed the specificity of the effect of different ions on the dimensions of the DNA molecule in solution. The data on optical anisotropy of the DNA molecule testify, that the thermodynamic rigidity of the latter doesn't depend on the temperature of solutions of different electrolytes in the temperature range studied.     

DNA conformation and thermostability of aqueous solutions of beta-alanine and gamma-aminobutyric acid

It has been shown that at low concentrations of rare amino acids (from 10(-3) M to 10(-1) M of the substance) stechiometric complexes amino acid -- DNA are formed, which bring about partial substitution of counterions screening phosphate groups and to a change of spatial structure of DNA water molecules. The DNA-solvent molecular interactions are changed, accompanied by an abrupt decrease of helix-coil enthalpy transition which leads to the unwinding of DNA double helix. In the region of amino acid high concentrations (greater than 1-1,5 M) a rise of thermostability and winding of DNA double helix is observed. It has been established that B----C-like conformational transition stimulated by the rise of DNA thermostability is a result of counterions dehydration and the increase of effective ionic strength of the solution which is due to the rise of amino acid-zwitterions content in it.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

DNA interaction with the bifunctional acridine dye

The comparative studies of the formation of DNA-complexes with the acridines containing one and two chromophores were accomplished. It was shown that both of acridines were bonded with DNA by means of intercalation irrespective of the ionic strength of medium (mu). When mu = 0.1 the diacridine (1,6-bis(9-acridylamino)-hexan) behaves as an mono-intercalator. Under these conditions both of the ligands exert equal influence of the molecular parameters of DNA. When mu = 0.001 the binding mode of the diacridine with DNA depends on its concentration in a complex. If a number of diacridine molecules on a pair of nucleotides (r) falls in a region 0 less than r less than 0.2 its binding with DNA is accomplished via the bis-intercalation mode and accompanied by the structure distortion of the monomer remnant of the macromolecule. As r increases from 0.2 to 0.4 the gradual change of the binding mode of the diacridine with DNA from bis-intercalation to mono-intercalation takes place. Moreover the structure of nucleotides is reduced. When mu = 0.001 the behaviour of DNA complexes with mono-acridine is analogous to the observed one when mu = 0.1.     

Influence of Plant Growth at High CO2 Concentrations on Leaf Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Intracellular Distribution of Soluble Carbohydrates in Tobacco,Snapdragon, and Parsley

We have examined the possible role of leaf cytosolic hexoses and the expression of mannitol metabolism as mechanisms that may affect the repression of photosynthetic capacity when plants are grown at 1000 versus 380 [mu]L L-1 CO2. In plants grown at high CO2, leaf ribulose-1,5-bisphosphate carboxylase/oxygenase content declined by [greater than or equal to]20% in tobacco (Nicotiana sylvestris) but was not affected in the mannitol-producing species snapdragon (Antirrhinum majus) and parsley (Petroselinum hortense). In the three species mesophyll glucose and fructose at midday occurred almost entirely in the vacuole (>99%), irrespective of growth CO2 levels. The estimated cytosolic concentrations of glucose and fructose were [less than or equal to]100 [mu]M. In the three species grown at high CO2, total leaf carbohydrates increased 60 to 100%, but mannitol metabolism did not function as an overflow mechanism for the increased accumulation of carbohydrate. In both snapdragon and parsley grown at ambient or high CO2, mannitol occurred in the chloroplast and cytosol at estimated midday concentrations of 0.1 M or more each. The compartmentation of leaf hexoses and the metabolism of alternate carbohydrates are further considered in relation to photosynthetic acclimation to high levels of CO2.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

Stark effect in wild-type and heterodimer-containing reaction centers from Rhodobacter capsulatus

The effect of an external electric field on the optical absorption spectra of wild-type Rhodobacter capsulatus and two Rb. capsulatus reaction centers that have been genetically modified through site-directed mutagenesis (HisM200----LeuM200 and HisM200----PheM200) was measured at 77 K. The two genetically modified reaction centers replace histidine M200, the axial ligand to the M-side bacteriochlorophyll of the special pair, with either leucine or phenylalanine. These substitutions result in the replacement of the M-side bacteriochlorophyll with bacteriopheophytin, forming a bacteriochlorophyll-bacteriopheophytin heterodimer. The magnitude of the change in dipole moment from the ground to excited state (delta mu app) and the angle delta between the Qy transition moment and the direction of delta mu app were measured for the special pair absorption band for all three reaction centers. The values for delta mu app and delta obtained for wild-type Rb. capsulatus (delta mu app = 6.7 +/- 1.0 D, delta = 38 +/- 3 degrees) were the same within experimental error as those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. The values for delta mu app and delta obtained for the red-most Stark band of both heterodimers were the same, but delta mu was substantially different from that of wild-type reaction centers (HisM200----LeuM200, delta mu app greater than or equal to 14.1 D and delta = 33 +/- 3 degrees; HisM200----PheM200, delta mu app greater than or equal to 15.7 D and delta = 31 +/- 4 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands.

Localization of the parallel elastic components (PECs) in skinned muscle fibers was investigated by analyzing the change of the resting tension, which accompanies the dissociation of the A- and I-bands. The A-band was dissociated from both ends by increasing the concentration of KCl under relaxing conditions (0.09-0.54 M KCl, 4.0 mM MgATP, 1.0 mM Mg2+, 4.0 mM EGTA, pH 6.0-9.0, 20 degrees C). At sarcomere lengths greater than or equal to 3.5 microns, the length of the A-band was estimated by comparing the intensity of the first-order optical diffraction line with the results of model calculations. These results were supported by differential-interference microscopy and sodium dodecyl sulfate gel electrophoresis. It was shown that the resting tension decreased nearly in proportion to the residual length of the A-band. At sarcomere lengths less than or equal to 4.0 microns, the resting tension after the dissociation of the A-band was lowered to less than 10% of the initial value. On the other hand, at sarcomere lengths greater than or equal to 5.0 microns the resting tension after the dissociation of the A-band still showed approximately 35% of the initial value and did not change even after the I-band was dissociated by a solution containing KI. From these results, we propose that most of the PECs contributing to resting tension bind almost uniformly to the A-band and there are also PECs connecting Z-lines.     

Use of high-performance size-exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column

We have conducted a study of the TSK 3000 SW high-performance size-exclusion column to define under what conditions proteins would migrate most consistently with their known hydrodynamic properties. Our findings include the following: 1) the residual negative charge of the column does cause charge-exclusion or charge-retention effects at low ionic strengths; with elution in deionized water several anionic proteins elute approximately in the void volume; 2) at mu greater than or equal to 0.5, protein migration is not only independent of ionic strength, but consistent with protein molecular weight and hydrodynamic volume; 3) small hydrophobic peptides are retarded by the column; and 4) very asymmetric proteins and other hydrodynamic particles are likely to be retarded by an "end-on insertion" mechanisms.     

Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of M?ssbauer radiation

Hydration relationships of the elastic scattering fraction of M?ssbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.     

Correlation between conformation distortion of the DNA sugar-phosphate backbone and high optical activity of its compact form

Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A).Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml.     

Kinetics of the oxidation of Rhus vernicifera stellacyanin by the Co(EDTA)-- ion.

A kinetic study of the oxidation of the copper(I) form of the blue copper protein stellacyanin (St(I) by Co(EDTA)-- has been performed. Observed rate constants approach a saturation limit with increasing [Co(EDTA)--] at pH 7, consistent with a mechanism involving rapid pre-equilibrium oxidant-protein complex formation followed by rate-limiting intramolecular Cu(I) to Co(III) electron transfer: Co(EDTA)-- + St(i Qp in equilibrium Co(EDTA)-- ---St(I) Co(EDTA)-- ---St(I) k2 leads to Co(EDTA)2-- ---St(II) (Qp = 149 M--1, k2 = 0.169 sec--1; 25.1 degrees, pH 7.0 mu 0.5 M (phosphate)). Activation parameters based on k2 (deltaH not equal to = 1.8 kcal/mol, deltaS not equal to = --56 cal/mol-deg) indicate that the electron transfer process is substantially nondiabatic, in marked contrast with results obtained for Co(phen) 3 3+ as the oxidant. Linear kobsd VS. [Co(EDTA)--] plots are reported for the Co(EDTA)-- oxidation of cuprous stellacyanin at pH 10 (k = 8.9 M--1 sec--1; 25.0, pH 10, mu 0.5 M (carbonate); DELTaH not equal to 11.3 kcal/mol, deltaS not equal to = -16 cal/mol-deg) and at pH 7 in the presence of excess EDTA (k = 21.2 M--1 sec--1; 25.1 degree, pH 7.0, mu 0.5 M (phosphate), [EDTA] tot = 5 X 10(--4) M; deltaH not equal to = 5.9 kcal/mol, delta S not equal to = --33 cal/mol-deg). It is concluded that Co(EDTA)-- adopts an electron transfer mechanism similar to that preferred by Co(phen)33+ under conditions where the oxidant is prevented from binding strongly to reduced stellacyanin.     

Uterotropic actions of relaxin in prepubertal gilts

Uterotropic actions of relaxin (RLX) have been demonstrated in rats and mice but not in other species. Experiments were conducted to assess (1) if RLX has uterotropic effects in prepubertal gilts and (2) if RLX-induced imbibition of water by uterine tissues is mediated by histamine. Prepubertal gilts received (i.m.) either 0.5 mg pRLX (greater than or equal to 3000 U/mg; n = 10) in 0.1 M phosphate-buffered saline (PBS) or PBS vehicle (n = 8) every 6 h for 54 h. Additionally, one-half of each group received either (1) the histamine receptor antagonists (300 mg i.m. of each), pyrilamine maleate (H1) and cimetidine (H2), in PBS and corn oil, respectively, or (2) control vehicles every 6 h for 66 h beginning 12 h prior to initiation of RLX treatment. Treatment with RLX stimulated a twofold increase (p less than or equal to 0.05) in uterine weight in prepubertal gilts. Increases in total uterine weight were manifested by greater (p less than or equal to 0.05) uterine water content and dry weight in RLX-treated gilts. Cotreatment with histamine receptor antagonists did not alter RLX-induced changes in uterine water content or dry weight. Uterine contents of soluble protein and DNA were greater (p less than or equal to 0.05) in RLX-treated gilts compared to controls. Treatment with RLX tended (p approximately 0.08) to reduce protein; DNA ratio of uterine tissues and increased (p less than or equal to 0.05) uterine DNA as a percentage of uterine dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of NO3- Influx in Spruce

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.