Osmotic fragility of the erythrocyte membrane: characterization by modeling of the transmittance curve as a function of the NaCl concentration

The theoretical extinction of blood suspensions submitted to a slow dialysis is analyzed in terms of their NaCl concentration. The model involves two adjustable parameters, chi and K, related to swelling and hemolysis. During swelling, the erythrocyte volume is supposed to vary linearly with the saline concentration. During hemolysis, an exponential decay of the hemoglobin concentration in the erythrocyte is used. The theoretical transmittance curves are consistent with the measurements carried out at a wavelength of 0.808 microm on native and incubated blood samples. Chi and K are relevant parameters to characterize quantitatively the fragility of the erythrocyte membrane. The effect of a non ideal character of the hemoglobin solutions and of normal distributions of chi and K is also discussed.     

A microstructural model of elastostatic properties of articular cartilage in confined compression

A microstructural model of cartilage was developed to investigate the relative contribution of tissue matrix components to its elastostatic properties. Cartilage was depicted as a tensed collagen lattice pressurized by the Donnan osmotic swelling pressure of proteoglycans. As a first step in modeling the collagen lattice, two-dimensional networks of tensed, elastic, interconnected cables were studied as conceptual models. The models were subjected to the boundary conditions of confined compression and stress-strain curves and elastic moduli were obtained as a function of a two-dimensional equivalent of swelling pressure. Model predictions were compared to equilibrium confined compression moduli of calf cartilage obtained at different bath concentrations ranging from 0.01 to 0.50 M NaCl. It was found that a triangular cable network provided the most consistent correspondence to the experimental data. The model showed that the cartilage collagen network remained tensed under large confined compression strains and could therefore support shear stress. The model also predicted that the elastic moduli increased with increasing swelling pressure in a manner qualitatively similar to experimental observations. Although the model did not preclude potential contributions of other tissue components and mechanisms, the consistency of model predictions with experimental observations suggests that the cartilage collagen network, prestressed by proteoglycan swelling pressure, plays an important role in supporting compression.     

The electron-microscopic structure of nucleoprotein fibrils from metaphase chromosomes treated with salt

The diameter of nucleoprotein fibres of metaphase chromosomes is sensitive to salt concentrations. Treatment of human lymphocyte cells in metaphase with a hypotonic medium and spreading them on a water surface causes swelling of the chromosome fibres from 150–180 Å to 230–250 Å. Treatment of the chromosomes with 0.15–0.45 M NaCl, a concentration at which histones are not yet removed from the nucleoprotein complexes, apparently does not affect the chromosome structure. Treatment with NaCl solutions between 0.6 M and 2.0 M NaCl leads to a progressive extraction of the chromosomal proteins and decreases the diameter of the chromosome fibres to 80–100 Å and less.Dedicated to Prof. Dr. A. Butenandt on the occasion of his 70th birthday.     

Structure and dynamics of M13mp19 circular single-strand DNA: effects of ionic strength

Dynamic and static light scattering, CD, and optical melting experiments have been conducted on M13mp19 viral circular single-strand DNA as a function of NaCl concentration. Over the 10,000-fold range in concentration from 100 microM to 1.0 M NaCl, the melting curves and CD spectra indicate an increase in base stacking and stability of stacked regions with increased salt concentration. Analysis of dynamic light scattering measurements of the single-strand DNA solutions as a function of K2 from 1.56 to 20 X 10(10) cm-2 indicates the collected autocorrelation functions are biexponential, thus revealing the presence of two decaying dynamic components. These components are taken to correspond to (1) global translational motions of the molecular center of mass and (2) motions of the internal molecular subunits. From the evaluated relaxation rates of these components, diffusion coefficients D0 and Dplat are determined. The center of mass translational diffusion coefficient D0, varies in a nonmonotonic manner, by 10%, from 3.75 X 10(-8) to 3.39 X 10(-8) cm2/s over the NaCl concentration range from 100 microM to 1.0 M. Likewise, the radius of gyration RG, obtained from static light scattering experiments, varies by 15% from 699 to 830 A over the same NaCl range Dplat, the diffusion coefficient of the internal subunits, displays a different dependence on the NaCl concentration and decreases, by nearly 22% in a titratable fashion, from 12.46 X 10(-8) to 10.26 X 10(-8) cm2/s, when the salt is increased from 100 microM to 1.0 M. A semiquantitative interpretation of these results is provided by analysis of the light scattering data in terms of the circular Rouse-Zimm chain. Rouse-Zimm model parameters are estimated from the experimental results, assuming the circular chains are composed of a fixed number of Gaussian segments, N + 1 = 15. The rms displacement of the internal segments, b, is estimated to be the smallest (442 A) in 100 mM NaCl. Increases of b to 467 A in 100 microM and 524 A in 1.0 M NaCl are observed. Meanwhile, the hypothetical friction factor of the internal subunits, f, progressively increases as the NaCl concentration is raised. It is inferred from the evaluated Rouse-Zimm model parameters that both the static flexibility of the circular chain and diffusive displacements of the internal subunits decrease with increases in NaCl concentration from 100 mM to 1.0 M.(ABSTRACT TRUNCATED AT 400 WORDS)     

Z-DNA is Formed by poly(dC-dG) and poly (dm5C-dG) at Micro or Nanomolar Concentrations of some Zinc(II) and Copper(II) Complexes

Abstract

We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm⁵C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4μM (poly(dC-dG)) and 44μM (poly(dm⁵C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2μM for poly(dm⁵C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.     

The use of the spectrophotometric analysis for the calculation of the thermodynamic parameters in actinocin derivative-DNA systems

The spectral properties of the actinocin derivative ActII in complexes with DNA were studied by UV visible spectrophotometry. Two binding models with one and two binding sites for competitive binding with different values of parameters were considered. To choose an optimal model of complexation, the optimization program of spectrophotometric concentration dependencies DALSMOD was used. Using this program, it was concluded that at least three complexes with different absorption spectra are present in the system ActII-DNA. The logarithms of K2 and K3 for DNA-ActII mixtures, calculated for models I and II at different sodium ion concentrations, were in good agreement with predictions of the counterion condensation theory. The analysis of the absorption spectra of ActII-DNA mixtures at different temperatures made it possible to obtain the values of deltaH and deltaS for each type of complexes. The values of entropy deltaS were positive in the 0.02 M NaCl solution and negative in the 0.15 M NaCI solution.     

Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes.

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes preceding decapsidation of bromegrass mosaic virus under hydrostatic pressure: a small-angle neutron scattering study

The stability of bromegrass mosaic virus (BMV) and empty shells reassembled in vitro from purified BMV coat protein was investigated under hydrostatic pressure, using solution small-angle neutron scattering. This technique allowed us to monitor directly the dissociation of the particles, and to detect conformational changes preceding dissociation. Significant dissociation rates were observed only if virions swelled upon increase of pressure, and pressure effects became irreversible at very high-pressure in such conditions. At pH 5.0, in buffers containing 0.5 M NaCl and 5 mM MgCl(2), BMV remained compact (radius 12.9 nm), dissociation was limited to approximately 10 % at 200 MPa, and pressure effects were totally reversible. At pH 5.9, BMV particles were slightly swollen under normal pressure and swelling increased with pressure. The dissociation was reversible to 90 % for pressures up to 160 MPa, where its rate reached 28 %, but became totally irreversible at 200 MPa. Pressure-induced swelling and dissociation increased further at pH 7.3, but were essentially irreversible. The presence of (2)H(2)O in the buffer strongly stabilized BMV against pressure effects at pH 5.9, but not at pH 7.3. Furthermore, the reversible changes of the scattered intensity observed at pH 5.0 and 5.9 provide evidence that pressure could induce the release of coat protein subunits, or small aggregates of these subunits from the virions, and that the dissociated components reassociated again upon return to low pressure. Empty shells were stable at pH 5.0, at pressures up to 260 MPa. They became ill-shaped at high-pressure, however, and precipitated slowly after return to normal conditions, providing the first example of a pressure-induced conformational drift in an assembled system.     

Complex of repair DNA polymerase β with autonomous 3′→5′ exonuclease shows increased accuracy of DNA synthesis

The complexes of repair DNA polymerase β with 3′-exonuclease and some other proteins were isolated from the chromatin of hepatocytes of normal rats for the first time. Biopolymers were extracted from the chromatin by the solution of NaCl and Triton X-100. The extract was fractionated by gel-filtration on Sephacryl S-300 columns successively in low and high ionic strength solutions, on hydroxyapatite, and on Sephadex G-100 columns. The complexes have molecular weights of 100 and 300 kDa. They dissociate to DNA polymerase and exonuclease in the course of chromatography on a DNA-cellulose column or after gel-filtration in the presence of 1 M NaCl. The co-purification of the polymerase and exonuclease is reconstituted in 0.1 M NaCl. The fidelity of monomeric and composite DNA polymerase β was measured using phase ?X174 amber 3 as a primer/template. The products of the synthesis were transfected into Escherichia coli spheroplasts, and the frequency of reverse mutations was determined. The complex of DNA polymerase β with 3′-exonuclease was shown to be 30 times more accurate than the monomeric polymerase, which can decrease the probability of repair mutagenesis and carcinogenesis.     

Folding of DNA by histones which lack their NH2-terminal regions

HeLa chromatin core particles were digested with trypsin to excise the NH2-terminal histone regions. The resulting nucleoprotein complexes were dissociated in 2.5 M NaCl; the DNA and polypeptides were then allowed to reassemble by lowering the NaCl concentration. Eighty per cent of the DNA reassociated with the polypeptides. The reassembled nucleoprotein complexes sediment at 9.7 S, have a molecular elipticity at 280 nm of 3000 degrees cm2/dmol of PO4, and contain DNase I-susceptible sites at 10 nucleotide intervals. The pattern of products generated by cross-linking the polypeptides with dimethylsuberimidate is very similar to the pattern generated by cross-linking native core particles. The results indicate that histones which lack their HN2-terminal regions retain both the features necessary for correct protein-protein interactions and the ability to fold DNA into a nucleoprotein complex resembling the chromatin core particle.     

The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.     

Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Nonuniform swelling-induced residual strains in articular cartilage

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swelling-induced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 M Pa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.     

Application of high-performance ion-exchange chromatography to the analysis of cytosolic glucocorticoid receptor

The use of high-performance ion-exchange chromatography (HPIEC) on a Mono Q column was investigated for the analysis of glucocorticoid receptor. In the presence of 10 mM sodium molybdate, both liganded and unliganded glucocorticoid receptor were eluted as a single and sharp peak (0.32 M NaCl). In the absence of molybdate and after exposure to heat and salt, another peak of specifically bound radioactivity was eluted with 0.08 M NaCl. When HPIEC was performed in the absence of molybdate, two molecular forms of the liganded receptor were detected which eluted with 0.08 M NaCl (Stokes' radius Rs = 5.1 nm, s20,w = 4.6 S, calculated mol. wt Mr approximately 100,000) and 0.32 M NaCl (Rs = 7.3 nm, S20,w = 9.0 S, calculated Mr approximately 280,000). Analysis of both forms with mini-columns of DNA-Ultrogel, DEAE-Trisacryl and hydroxylapatite (HA-Ultrogel) confirmed the identity of the two peaks with transformed and non-transformed glucocorticoid-receptor complexes. These results suggest that HPIEC may provide a useful tool for the rapid resolution and quantification of receptor molecular forms.     

Effect of osmolarity on the zero-stress state and mechanical properties of aorta

Some pathological conditions may affect osmolarity, which can impact cell, tissue, and organ volume. The hypothesis of this study is that changes in osmolarity affect the zero-stress state and mechanical properties of the aorta. To test this hypothesis, a segment of mouse abdominal aorta was cannulated in vivo and mechanically distended by perfusion of physiological salt (NaCl) solutions with graded osmolarities from 145 to 562 mosM. The mechanical (circumferential stress, strain, and elastic modulus) and morphological (wall thickness and wall area) parameters in the loaded state were determined. To determine the osmolarity-induced changes of zero-stress state, the opening angle was observed by immersion of the sectors of mouse, rat, and pig thoracic aorta in NaCl solution with different osmolarities. Wall volume and tissue water content of the rings were also recorded at different osmolarities. Our results show that acute aortic swelling due to low osmolarity leads to an increase in wall thickness and area, a change in the stress-strain relationship, and an increase in the elastic modulus (stiffness) in mouse aorta. The opening angle, wall volume, and water content decreased significantly with increase in osmolarity. These findings suggest that acute aortic swelling and shrinking result in immediate mechanical changes in the aorta. Osmotic pressure-induced changes in the zero-stress state may serve to regulate mechanical homeostasis.     

A triphasic analysis of corneal swelling and hydration control.

Physiological studies strongly support the view that hydration control in the cornea is dependent on active ion transport at the corneal endothelium. However, the mechanism by which endothelial ion transport regulates corneal thickness has not been elaborated in detail. In this study, the corneal stroma is modeled as a triphasic material under steady-state conditions. An ion flux boundary condition is developed to represent active transport at the endothelium. The equations are solved in cylindrical coordinates for confined compression and in spherical coordinates to represent an intact cornea. The model provides a mechanism by which active ion transport at the endothelium regulates corneal hydration and provides a basis for explaining the origin of the "imbibition pressure" and stromal "swelling pressure." The model encapsulates the Donnan view of corneal swelling as well as the "pump-leak hypothesis."     

B--Z conformational transition in d(CGCGCGTTAATT), d(CGCGCGTTAA) and d(CGCGCGTT)

Conformational studies on three DNA-oligomers (d(CGCGCGTTAATT), d(CGCGTTAA) and d(CGCGCGTT) in solution by circular dichroism spectroscopy are reported. In low salt solution, all three DNA oligomers exhibit a characteristic B-conformation. However, under the influence of high salt concentration i.e. 5M NaCl, the octamer d(CGCGCGTT) exhibits 'A' conformation whereas the decamer and dodecamer retain B-conformation. On addition of millimolar amount of NiCl2 to the 5M NaCl, solution of oligodeoxynucleotides a B-Z transition is observed in octamer, decamer and dodecamer. However, NiCl2 titrations show that mid point of transition for dodecamer is at 2.25 mM, for decamer is at 13 mM NiCl2 and for octamer is 17 mM at NiCl2. In 60% alcohol all three oligonucleotides remain in the B-conformation. The melting temperatures of oligonucleotides at various salt concentration are also reported. Thermodynamic parameters calculated by melting profile using a two state model show that dodecamer and decamer are most stable in their 5M NaCl, B-form. However, octamer is more stable in its Z form than that of its 'A' form.     

The fractionation of calf thymus DNA on histone KAP - Sepharose 4B columns

It was found that fractionation of calf thymus DNA on homologous histtone KAP covalently bound to CNBr activated Sepharose 4B depends on the molecular weight of DNA. The maximum of elution of high molecular DNA (m. wt. above 5 x 10(6)) was observed at 0.56 M NaCl and that of degraded DNA (m. wt. 0.8 x 10(6)) at 0.52 M NaCl. Significant differences in melting temperatures and melting intervals were observed among fractions obtained from low molecular DNA as a result of enrichment of some fractions in satellite DNAs. These differences were very small in DNA of m. wt. above 5 x 10(6). The results are discussed in terms of specific areas which may exist on calf thymus DNA molecules playing the role of loci, where lysine-rich histone KAP is preferentially bound.