Evidence for an increase of DNA contour length at low ionic strength.

The polyion chain expansion of DNA was studied by viscometry within the Na+ concentration range c5 = 0.002 M to 0.4 M. The DNA molecular weights M were between 0.5 x 10(6) and 13 x 10(6). The relative change of intrinsic viscosity [eta] is linearly correlated to c5(-1/2) with a slope that increases with increasing M. This behaviour reflects the predominance of helix stiffening in chain expansion. At c5(112) > 0.01(-1/2 M-1/2 (Debye-Hückel screening radius 1/chi > (1/chi)*=3nm) the relative change of [eta] rises with a steeper slope. This effect increases with decreasing M suggesting that helix lengthening contributes to the chain expansion. Our model enables us to interpret other ionic-strength dependent effects known from literature. The start of the significant duplex elongation at (1/chi)* can be correlated to the polyion-charge arrangement. In accordance with our interpretation (1/chi)* is found to be greater for DNA-intercalator complexes.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Removing an interhelical salt bridge abolishes coiled-coil formation in a de novo designed peptide

Alpha-helical coiled coils represent a common protein oligomerization motif that are mainly stabilized by hydrophobic interactions occurring along their coiled-coil interface, the so-called hydrophobic seam. We have recently de novo designed and optimized a series of two-heptad repeat long coiled-coil peptides which are further stabilized by a complex network of inter- and intrahelical salt bridges. Here we have extended the de novo design of such two heptad-repeat long peptides by removing the central and most important g-e' Arg to Glu (g-e'RE) ionic interhelical interaction and replacing these residues by alanine residues. The effect of the missing interhelical ionic interaction on coiled-coil formation and stability has been analyzed by CD spectroscopy, analytical ultracentrifugation, and X-ray crystallography. We show that the peptide, while being highly alpha-helical, is no longer able to form a parallel coiled-coil structure but rather assumes an octameric globular helical assembly devoid of any coiled-coil interactions.     

Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii

Glycolipids are important components of cellular membranes involved in various biological functions. In this report, we describe the identification of the de novo synthesis of glycosphingolipids by Toxoplasma gondii tachyzoites. Parasite-specific glycolipids were identified by metabolic labelling of parasites with tritiated serine and galactose. These glycolipids were characterised as sphingolipids based on the labelling protocol and their insensitivity towards alkaline treatment. Synthesis of parasite glycosphingolipids were inhibited by threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol and L-cycloserine, two well-established inhibitors of de novo sphingolipid biosynthesis. The identified glycolipids were insensitive towards treatment with endoglycoceramidase II indicating that they might belong to globo-type glycosphingolipids. Taken together, we provide evidence for the first time that T. gondii is capable of synthesising glycosphingolipids de novo.     

Effect of purification procedures on the self-association of myosin at high ionic strength

The effects of purification procedures on the self-association of myosin at high ionic strength have been investigated by high-speed sedimentation equilibrium. The method of purification profoundly affects the self-association characteristics of the myosin molecule. Myosin purified by chromatography on Sephadex G-200 (S-myosin) behaves (in the limit) as a single nonideal thermodynamic component with little (if any) reversible dimerization (K₂₁ = 1.05 dl/g) and with a second viral coefficient, B₁ = 0.064 dl/g, compatible with excluded volume effects alone. Conversely, myosin which had been subjected to (NH₄)₂SO₄ precipitation followed by chromatography on DEAE-Sephadex (DEAE-myosin) dimerizes reversibly to a significant extent judged by its apparent association constant, K₂₁ ? 9 dl/g. In addition, higher N-mers appear to partake in the equilibria. The nonideality of this system, B₁ = 2.2 ± 0.1 dl/g, is also greater than that of S-myosin. Two possible explanations to account for these findings are offered: (1) Myosin may undergo a change in conformation (denaturation?) during the preparation steps which lead to DEAE-myosin; (2) another component (C-protein?) which would modulate the self-association properties of myosin may be involved.     

The effect of low ionic strength on the circular dichroic spectrum of chromatin and nucleosomal subunits

Circular dichroism has been used to measure the conformation changes in the DNA of chromatin and chromatin subunits as a function of ionic strength. Transfer of chromatin from 0.15 M to 0.25 mM salt led to an enhancement of the circular dichroic bands at 275 and 285 nm. Removal of histone H1 did not appreciably affect the circular dichroic spectrum when measured in 0.15 M salt, but in 0.25 mM salt H1 depletion led to a marked increase in the ellipticity. Conformation changes due to low ionic strength were also observed with a 145- and a 172-bp chromatin subunit. A linear combination of the ellipticities of the DNA of the two domains in chromatin, namely core and linker, was successful for measurements at 0.15 M salt, but large unexplained discrepancies appeared with the data from measurements in 0.25 mM salt.     

Evidence in support of biosynthesis de novo of free cytokinins

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, ( ²-isopentenyl)adenosine and 2-methylthio-N⁶-( ²-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N⁶-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.     

Caged DNA does not aggregate in high ionic strength solutions.

The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.     

Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength.

Hyaluronidase and high levels of hyaluronan are found together in tumours. It is highly likely that hyaluronidase activity controls the balance between high molecular mass hyaluronan and oligosaccharides, and thus plays an important role in cancer development. The hyaluronan hydrolysis catalysed by bovine testicular hyaluronidase was studied as a model. The kinetics was investigated at pH 5 and 37 degrees C using the colorimetric N-acetyl-d-glucosamine reducing end assay method. While the substrate dependence obtained in the presence of 0.15 mol L(-1) ionic strength exhibited a Michaelis-Menten behaviour, an atypical behaviour was observed under low ionic strength: for increasing hyaluronan concentrations, the initial reaction rate increased, reached a maximum and then decreased to a very low level, close to zero at high substrate concentrations. One of the various hypotheses examined to explain this atypical behaviour is the formation of non-specific complexes between hyaluronan and hyaluronidase based on electrostatic interactions. This hypothesis is the only one that can explain all the experimental results including the variation of the reaction medium turbidity as a function of time and the influence on the initial reaction rate of the hyaluronan concentration over hyaluronidase concentration. However, phenomena such as the high viscosity of highly concentrated hyaluronan solutions or the steric exclusion of hyaluronidase from hyaluronan solutions may contribute to the atypical behaviour. Finally, the biological implications of the non-linear and non-monotonous shape of the hyaluronan-hyaluronidase substrate dependence in the regulation of the hyaluronan chain molecular mass are discussed, in particular in the case of cancer development.     

Efficiency of de novo centromere formation in human artificial chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on alpha-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17alpha, Yalpha) and two with 17alpha or Yalpha and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17alpha HAC vectors generated artificial minichromosomes in 32-79% of the HT1080 clones screened, compared with only approximately 4% for the Yalpha HAC vectors, indicating that Yalpha is inefficient at forming a de novo centromere. The 17alpha HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60-250 kb) interspersed with either vector or HPRT1 DNA.The 17alpha-HPRT1 HACs were less stable than those with 17alpha only, and these results may influence the design of new HAC gene transfer vectors.     

Thermodynamics and dissociation constants of carboxylic acids at high ionic strength and temperature

Dissociation constants (pK_a) of oxalic, iminodiacetic, citric, nitrilotriacetic, ethylenediaminetetraacetic, trans-1,2 diaminocyclohexanetetraacetic acid and diethylenetriaminepentaacetic acid have been determined potentiometrically using a glass electrode at an ionic strength of 6.60 m (NaClO₄) and temperatures of 0-60 °C. The constants of iminodiacetic, nitrilotriacetic and diethylenetriaminepentaacetic acid were measured at 25 °C at ionic strengths from 0.30 to 6.60 m (NaClO₄). The thermodynamic parameters for the dissociation of these carboxylic acids were derived from the temperature dependence of the dissociation constants. The Specific Ion Interaction Theory (SIT) and the Parabolic model successfully described the ionic strength dependencies of the pK_a values. The variation of the pK_a values at high ionic strengths as a function of the type and concentration of supporting electrolyte is discussed and compared with literature data.