Conformation of a DNA molecule in water-salt solutions depends on the salt anion type

The methods of viscosimetry and flow birefringence were used to study the effect of anions F-, Cl-, Br-, J-, NO3-, ClO4-, SCN-, CH3COO- on the dimensions and thermodynamic rigidity of the DNA molecule in solutions in a wide range of ionic strengths and at different temperatures. It was shown that the persistent lengtH of DNA is independent of the anion type at all values of ionic strength, and the changes in its dimensions, which are determined from the changes in intrinsic viscosity, are due to the influence of anions and their hydration on long-range interactions in the macromolecule. Possible reasons for this phenomena and the role of structural changes in water upon hydration of anions are discussed.     

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.     

Effect of Single-Charged Anions of Different Nature on the DNA Molecule Conformation in Water–Salt Solutions

Methods of intrinsic viscosity () and beam flow birefringence were used to study the effects of some single-charged ions (F^–, Cl^–, Br^–, I^–, NO^– ₂, NO^– ₃, ClO^– ₄, SCN^–, CH₃COO^–) on the size and thermodynamic rigidity of a DNA molecule in aqueous solutions of sodium salts in a broad interval of ionic strength when temperature T is changed. It has been shown that the close interactions in a macromolecule and the resulting DNA persistent length a are independent of the type of the salt anion over the whole interval of . On the contrary, the specific volume of the DNA molecule in solution, proportional to the value, is quite sensitive to the anionic composition of the solvent, which is due to the effect of anions and their hydration on the long-range interactions in the macromolecule. The presence of polyatomic and halide anions is manifested differently in the value of DNA. Possible factors responsible for the observed effect and the role of structural alterations of water upon anion hydration are discussed.     

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.     

Chlorate as a Transport Analog for Nitrate Absorption by Roots of Tomato

Several studies have indicated that chlorate (ClO3-) and nitrate (NO3-) may share a common transport system in higher plants. Here, we compared the interactions between ClO3- and NO3-uptake by roots of intact tomato (Lycopersicon esculentum cv T5) plants. Exposure to ClO3- for more than 2 h inhibited both net ClO3- and K+ uptake, presumably because of ClO3- toxicity; consequently, subsequent measurements were conducted after short exposures to ClO3-. The apparent affinity and apparent maximum rate of absorption for net ClO3- and NO3- uptake were very similar. Interactions between ClO3- and NO3- transport were complex; 50 [mu]M NO3- acted as a mixed inhibitor of net ClO3- uptake, but 50 [mu]M ClO3- had no significant effect on net NO3- uptake, and 500 [mu]M ClO3- had no significant effect on 15NO3- influx. If the two ions share a single common high-affinity transport system, it is much more selective for NO3- than would be suggested by the similarity of net NO3- and ClO3- uptake kinetics. Our results indicate that, although NO3- may interfere with root ClO3- uptake, ClO3- is not a useful analog for the root high-affinity NO3- transport system.     

Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density

The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.     

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.     

Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.     

Thermodynamics of anion binding to human serum transferrin

The binding of phosphate, bicarbonate, sulfate, and vanadate to human serum transferrin has been evaluated by two difference ultraviolet spectroscopic techniques. Direct titration of apotransferrin with bicarbonate, phosphate, and sulfate produces a strong negative absorbance near 245 nm, while titration with vanadate produces a positive absorbance in this region. Least-squares refinement of the absorbance data indicates that two anions of sulfate, phosphate, and vanadate bind to each transferrin molecule but that there is detectable binding of only a single bicarbonate anion. A second method used to study the thermodynamics of anion binding was competition equilibrium between anions for binding to the transferrin. The equilibrium constant for binding of the first equivalent of vanadate was determined by competition vs. phosphate and sulfate, while the equilibrium constant for binding of the second equivalent of bicarbonate was determined by competition vs. vanadate. Anion binding was described by two equilibrium constants for the successive binding of two anions per transferrin molecule: K1 = [A-Tr]/[A][Tr] and K2 = [A-Tr-A]/[A][A-Tr] where [A] represents the free anion concentration, [Tr] represents apotransferrin concentration, and [A-Tr] and [A-Tr-A] represent the concentrations of 1:1 and 2:1 anion-transferrin complexes, respectively. The results were the following: for phosphate, log K1 = 4.19 +/- 0.03 and log K2 = 3.25 +/- 0.21; for sulfate, log K1 = 3.62 +/- 0.07 and log K2 = 2.79 +/- 0.20; for vanadate, log K1 = 7.45 +/- 0.10 and log K2 = 6.6 +/- 0.30; for bicarbonate, log K1 = 2.66 +/- 0.07 and log K2 = 1.8 +/- 0.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of spermine on acid-base equilibrium in DNA molecule

The influence of spermine (Sp) on the acid-induced predenaturational and denaturational transitions in the DNA molecule structure has been studied by means of circular dichroism, spectrophotometric and viscometric titration at supporting electrolyte concentration 10 mM NaCl. The data available indicate that at [N]/[P] less than or equal to 0.60 (here [N] and [P] are molar concentrations of Sp nitrogen and DNA phosphours, respectively) the cooperative structural B----B(+)----S transitions are accompanied by the DNA double-helice winding. No competition for proton acceptor sites in the DNA molecule between H+ and Sp4+ cations has been observed when binding to neutral macromolecule. At 0.60 less than or equal to [N]/[P] less than or equal to 0.75 the displacement of the B----B(+)----S transitions midpoints to acidic pH region has been established. This is accompanied by DNA condensation and the appearance of differential scattering of circularly polarized light. The calculations carried out in the framework of the two-variable Manning theory have shown that the acid-induced reduction of the effective polyion charge density facilitates the Sp-induced DNA condensation. It has been shown that the acid-base equilibrium in the DNA molecule is determined by local [H+] in the 2-3 A hydrated monolayer of the macromolecule. An adequate estimation of [H+] can be obtained on the basis of the Poisson-Boltzman approach. The data obtained are consistent with recently proposed hypothesis of polyelectrolyte invariance of the acid-base equilibrium in the DNA molecule.     

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.     

Effects of extracellular anions on cell growth of Chinese hamster V-79 cells

The effects of extracellular anions (10-150 mM, added as Na salts to normal growth medium) on the growth of Chinese hamster V-79 cells were examined. Additions of NaCl and NaNO3 at concentrations greater than 60 mM reduced the growth rate dose-dependently. Several other anions also inhibited cell growth in the decreasing order of potency, SCN- greater than NO2- greater than NO3- greater than Br- greater than Cl- greater than gluconate- glutamate- greater than Mes-. When the added anions were removed, the growth rate was restored to the control rate. Cell survival was markedly reduced by the addition of SCN-, but was less affected by other anions (Cl-,NO3- and NO2-) of comparable potency. The respective syntheses of cellular DNA and protein, as estimated from the incorporation of [3H]-thymidine and [14C]leucine, also decreased with the increase in the concentration (60-120 mM) of anions added, the order of potency being SCN- greater than NO2- greater than NO3- greater than Cl-. After anion-treatment, the cellular Na+ concentration increased and the cellular Cl- concentration decreased in the order of SCN- greater than NO2- greater than NO3-, Cl-, but, the cellular K+ concentration did not change significantly. These data suggest that changes in extracellular anions affect cell growth and survival, probably through changes in the intracellular Na+ or Cl- concentration and in the rates of protein and/or DNA synthesis.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

Synthesis,crystal structure,and nuclease activity of planar mono-heterocyclic base copper(II) complexes

A series of mononuclear copper(II) complexes having a 1:1 molar ratio of copper and the planar heterocyclic base like 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) are prepared from a reaction of copper(II) nitrate.trihydrate and the base (L) in ethanol or aqueous ethanol at different temperatures. The complexes [Cu(dpq)(NO(3))(2)] (2), [Cu(dpq)(NO(3))(H(2)O)(2)](NO(3)) (3), [Cu(dpq)(NO(3))(2)(H(2)O)(2)].2H(2)O (4.2H(2)O) and [Cu(dppz)(NO(3))(2)(H(2)O)].H(2)O (5.H(2)O) have been characterized by X-ray crystallography. The crystal structures show the presence of the heterocyclic base in the basal plane. The coordination geometries of the copper(II) centers are axially elongated square-pyramidal (4+1) in 2, 3 and 5, and octahedral (4+2) in 4. The nitrate anion in the coordination sphere displays unidentate and bidentate chelating bonding modes. The axial ligand is either H(2)O or NO(3) in these structures giving a Cu-L(ax) distance of approximately 2.4 A. The one-electron paramagnetic complexes (mu approximately 1.8 mu(B)) exhibit axial EPR spectra in DMF glass at 77 K giving g(parallel)>g( perpendicular ) with an A(parallel) value of approximately 170G indicating a [d(x)2(-y)2](1) ground state. The complexes are redox active and display a quasireversible cyclic voltammetric response for the Cu(II)/Cu(I) couple near 0.0 V vs. SCE giving an order of the E(1/2) values as 5(dppz)>2-4 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). The complexes bind to calf thymus DNA giving an order 5 (dppz)>2 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). An effect of the extended planar ring in dpq and dppz is observed in the DNA binding. The complexes show nuclease activity with pUC19 supercoiled DNA in DMF/Tris-HCl buffer containing NaCl in presence of mercaptopropanoic acid as a reducing agent. The extent of cleavage follows the order: [Cu(phen)(2)(H(2)O)](ClO(4))(2)>5>2 approximately 3 approximately 4>1. The bis-phen complex is a better cleaver of SC DNA than 1-5 having mono-heterocyclic base. Mechanistic investigations using distamycin reveal minor groove biding for the phen, dpq complexes, and a major groove binding for the dppz complex 5. The cleavage reactions are found to be inhibited in the presence of hydroxyl radical scavenger DMSO and the reactions are proposed to proceed via sugar hydrogen abstraction pathway. The ancillary ligand is found to have less effect in DNA binding but are of importance in DNA cleavage reactions.     

Na2CO3 influence on DNA double helix stability: strong anion destabilizing effect

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.     

Isopycnic sedimentation of DNA in metrizamide: the effect of low concentrations of ions on buoyant density and hydration

Metrizamide, an inert, non-ionic organic compound, dissolves in water to give a dense solution in which DNA bands isopycnically at a density corresponding to that of fully hydrated DNA. Density-gradient centrifugation in solutions of metrizamide has been used to determine the effects of very dilute solutions of salts on the buoyant density of native and denatured DNA. It has been shown that the buoyant density of DNA is dependent on both the counter-cation and the anion present. Interpretation of the data in terms of the degree of hydration of the macromolecule indicates that (i), NaDNA is more highly hydrated than CsDNA; and (ii), the hydration of NaDNA varies with anion in the order sulphate< fluoride< chloride< bromide< iodide.     

Induction of Stomatal Closure by Vanadate or a Light/Dark Transition Involves Ca2+-Calmodulin-Dependent Protein Phosphorylations

Previous studies indicate that a continual source of adenosine 5[prime]-triphosphate is required for both opening and closing of stomata. However, vanadate (Na3VO4 at 500 [mu]M) as well as a light/dark transition induced stomatal closing in epidermal peels of Commelina communis L., showing that the stoppage or even the decrease of the activity of the plasma membrane H+-adenosine 5[prime]-triphosphatase is sufficient to induce stomatal closure. Furthermore, stomatal closing in response to Na3VO4 or a light/dark transition was suppressed by inhibitors of metabolism (10 [mu]M carbonyl cyanide m-chlorophenylhydrazone) and of protein kinases (20 [mu]M 1-[5-iodonaphthalene-1-sulfonyl]-1H-hexa-hydro-1,4-diaz-epine), calmodulin antagonists (20 [mu]M N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide), and the anion channel blocker 5-nitro-2,3-phenylpropyllamino benzoic acid (50 [mu]M). These data suggest that the slow, outward rectifying anion channel, whose opening would be related to the membrane potential, and at least one step requiring a protein phosphorylation by a Ca2+-calmodulin-dependent protein kinase of the myosin light chain kinase type might be implicated in the induction of stomatal closing by vanadate or a light/dark transition.     

Decrease in bicarbonate transport activities during epididymal maturation of porcine sperm

Kinetic studies of [14C]HCO3- and [35S]SO4(2-) transport show the existence of a common transport system for the two anions on the plasma membranes of porcine epididymal sperm. Km value for bicarbonate (8.3mM) is within the range of physiological concentration of bicarbonate. The anion transport activity is dependent on the temperature but independent of co-existence of Na+. Its pH dependency and sensitivity to the several anion transport inhibitors are similar to those observed in erythrocyte. Anion transport activity decreases during sperm maturation in epididymis.     

Error-prone translesion synthesis by human DNA polymerase eta on DNA-containing deoxyadenosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.