Quasielastic light scattering from solutions of filamentous viruses. I. Experimental

Intensity fluctuations of laser light scattered from filamentous viruses Pf1 [length L (Å) × diameter d (Å) = 20,000 × 90], M13 (9000 × 90), potato virus X (5150 × 130), and tobacco mosaic virus (3000 × 180) in sucrose density gradients were measured with a photon correlation spectrometer over a range of scattering angles from 15° to 120°. The experimental data can be approximated by two exponential decays, “slow” and “fast.” The slow decay rate constant t corresponds to the translational diffusion D of the virus, i.e., t = K²D, where K is the magnitude of the scattering vector. The amplitude of the slow component, i.e., translational diffusion, remains greater than that of the fast component, even at high KL. The fast decay rate constant t is also proportional to K² for viruses such as Pf1, M13, and even potato virus X. In the companion paper, we shall attribute the amplitude enhancement of the translational diffusion to the coupling of its anisotropy to the rotational diffusion modes. In order to explain the excessive decay rates in the fast component, we need to consider the bending mode of rodlike viruses, especially in the longer viruses such as M13 and Pf1, in addition to the usually expected rotational diffusion modes.     

Intramolecular interference effects in dynamic light scattering from rigid rings

A formula for the apparent diffusion coefficient [D_app(κ)] of a rigid ring is derived from the exact first cumulant of its dynamic structure factor. D_app(κ) is expressed in terms of the ring radius, the diffusion coefficients (D_zz and D_xx) for translation parallel and perpendicular, respectively, to the symmetry axis, and the diffusion coefficients (D and D) for rotation around the symmetry and transverse axes, respectively. D_app(κ) exhibits oscillations as a function of the scattering vector k , which depend on D and the anisotropy of translational diffusion (D_zz ? D_xx). The maxima in D_app(κ) are associated with minima in the static structure factor S(κ, 0), which are due to destructive intramolecular interference. The oscillations in D_app(κ) result from periodic variations in the relative intensities of scattered light from different orientations of the ring, which manifest the various motions to different extents. The orientations contributing most to the scattered intensity are those that exhibit the least destructive interference and consequently contribute most to the decay of the dynamic structure factor. © 1993 John Wiley & Sons, Inc.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Conformation of poly(L-arginine). I. Effects of anions

The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P₂O, P₃O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH^?, Cl^?, F^?, H₂PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).     

Characterization of the transition state of Lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state

The influence of proline cis-trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two-state transition model well. The temperature dependencies of k_uf and k_f over a wide temperature range showed that ΔC = 0 and ΔC = ?6.7 kJ K^?1 mol^?1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol^?1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol^?1 is primarily required for the disruption of long-range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.     

On the blue color of triiodide ions in starch and starch fractions. I. Characterization and assignment of absorption and circular dichroism spectra of triiodide ions in amylose

Four fundamental Raman lines were observed at 159, 111, 55 and 27 cm^-1 corresponding to the I bound (I) in amyloses with DP from 20 to 100, regardless of the degree of polymerization of I and the excitation wavelength. The spectral resolution was based on the molar extinction coefficient and molar ellipticity spectra of I. Eight bands, named, S₁, S₂, ?, S₈ from long to short wavelength, were isolated. These were found regardless of the DP. By a resonance excitation Raman study, the characteristics of S₃ and S₄, comprising the shoulder around 480 nm, were found to be different from those of S₁ and S₂, comprising the blue band. The assignment of the spectra was based on the electronic states of the monomeric I in the exciton-coupled dimeric unit. It was concluded that the blue band (S₁,S₂) belonged to the long-axis transitions and the shoulder band (S₃,S₄) to the short-axis ones on the monmeric coordinate system.     

Biopolymer–surfactant interaction. I. Kinetics of binding of cetyltrimethyl ammonium bromide with carboxymethyl cellulose (Na Salt)

The kinetics of binding of the cationic surfactant cetyltrimethyl ammonium bromide with the Na salt of carboxymethyl cellulose was studied by the electrometric method using cetyltrimetlyl ammonium⁺ (CTA⁺) ion-selective polyvinyl chloride membrane electrode. The binding process followed the first-order kinetics and occurred in three stages. Its affinity increased with increasing CTA bromide concentration and decreased with ionic strength. The activation process comprised moderate E and ΔH^≠ and negative ΔS^≠ for all three stages with a ΔH^≠ < TδS^≠ trend proving it to be entropy controlled. The ΔG^≠ values followed the trend ΔG < ΔG < ΔG (in accordance with k₁ > k₂ > k₃). The enthalpies (ΔH^≠) and entropies (ΔS^≠) of activation followed a systematic and interdependent trend. The multiple-stage binding kinetics is grossly comparable with the kinetics of binding of proteins to solid surfaces. © 1995 John Wiley & Sons, Inc.     

A stereospecific complex of poly(I) with ammonium ion

We describe conditions which lead to complete helix formation of poly(I) in the presence of NH. Binding of NH is shown to be specific in the presence of Li⁺, which does not by itself support helix formation under these conditions. The NH–poly(I) complex is characterized by uv, CD, and ir spectroscopy. The CD spectrum is strikingly different from those of the Na⁺ or K⁺ complexes, the first extremum being changed from negative for the metal ions to positive for NH. A stereospecific model is proposed for the NH–poly(I) helix in which the N of NH is located on the axis of the four-stranded helix, midway between planar tetramers formed by the bases. The model is consistent with the tetrahedral symmetry of NH, the requirement for four acceptable hydrogen bonds, the observed stability of the helix, and the accepted geometry of the backbone.     

Number of binding sites of DNA and polynucleotides with tris(2,2'-dipyridyl)ruthenium(II) cations

The binding of tris(2,2′-bipyridyl)ruthenium(II) cations [Ru(bpy)] with single- and double-stranded (ss and ds) DNA, and the polynucleotides poly(A), poly(C), poly(G), poly(I), poly(I) · poly(C), and poly(U), was studied in aqueous solution. Steady-state electrical conductivity measurements with the polynucleotides, ssDNA, and dsDNA reveal that approximately three nucleotides offer one binding site. This may be compared with the ratio [nucleotide]/[Mg²⁺] of 2.4 : 1 for dsDNA. After laser excitation (353 nm), the luminescence of Ru(bpy) bound to nucleic acids shows two decay components. The contribution of the fast component, which is interpreted as resulting from quenching processes of the absorbed ruthenium complex, exhibits a maximum with increasing [nucleotide]/[Ru(bpy)] at a ratio of about three to one. Bound Ru(bpy) can be released from the strand by addition of NaClO₄ [half-concentration: 2.5 and ≤ 10 mM for poly(U) and dsDNA, respectively].     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of temperature on microbial ethanol production. II. A thermodynamic interpretation of the temperature profile curve of ethanol production

An attempt is made to give a thermodynamic interpretation of the complete temperature profile curve of ethanol formation. Taking into consideration an enhancing competition between thermal activation and thermal deactivation of ethanol formation at increasing temperatures and supposing that the ethanol production is affected by a reversible and an irreversible term of thermal deactivation of a modified ARRHENIUS equation being current for the total biokinetic sphere may be derived: . The quantities ΔH and ΔH^D_2T are identical with the temperature functions of the change of entropy caused by reversible and irreversible deactivation of ethanol formation, respectively. Accordingly for the yeast strain Saccharomyces cerevisiae Sc 5 the calculated entropy coefficients of reversible and irreversible thermal deactivation of ethanol formation amount to C = (0.245± 0.013) kJ/mol · deg.² and C = (1.657 ± 0.046) kJ/mol · deg.².     

Interchain hydrogen bonds via bound water molecules in the collagen triple helix

If the collagen triple helix is so built as to have one set of NH ? O hydrogen bonds of the type N₃H₃(A) ? O₂(B), then it is possible to have a linkage between N₁H₁(B) and O₁(A) through the intermediary of a water molecule with an oxygen O leading to the formation of the hydrogen bonds N₁(B) ? O and O (A). In the same configuration, another water molecule with an oxygen O can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O O₁(A) and O O₀ (B). The two water oxygens also become receptors at the same time for CH ? O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ? O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.     

Einfluß extracellulärer Kaliuminoenkonzentrationen auf die celluläre Natriumionenkonzentration von Lodderomyces elongisporus D

It was found that the cellular Na⁺-concentration (C) of Lodderomyces elongisporus D is depended on the extracellular K⁺-concentration (C). The relationship can be described by an equation in the form The function of the natrium ion seem to be to support the utilisation rate of potassium ion at lower extracellular K⁺-concentration.     

Studies on ventilation of culture broths. I. Behavior of CO2 in model systems

The rate of dissolution and dehydration of CO₂ in a liquid model system was investigated. Components in the model system established the main conditions which may exist, in the extracellular space of a microbiological culture liquid. The charge in voltage of a glass electrode was measured which indicated the formation of H⁺ ions in the H₂CO₃ ? HCO H⁺ reaction. The rate of CO₂ hydration increased with the increase of temperature from 0 to 40°C. Likewise the equilibrium of the reaction was shifted towards the forward reaction. Similar results were observed when the tip velocity of the impeller was increased. Data suggest that agitation promotes the dissolution of CO₂ in the culture liquid through the reduction of gas-liquid film resistance in the diffusion of this gas. The rate of hydration of CO₂ into the bulk of the liquid was independent of pCO₂ above the surface of the liquid but depended on pCO₂ in the gas bubble within the liquid. The concentration of HCO was, furthermore, influenced by the buffer components, buffer capacity, and the viscosity of the system. Since pCO₂ and the HCO concentration in the extracellular space depend on both physical and chemical factors, the ventilation of a culture liquid necessitates both exhaust of CO₂ from the gas bubbles of the culture broth and shift of the H₂CO₃ ? HCO + H⁺ reaction towards the backward direction.     

Collapse of DNA caused by trivalent cations: pH and ionic specificity effects

A comparison of the condensation of T4 phage DNA by spermidine and Co(NH₃) at pH values between 5.1 and 10.2 has been made using quasielastic light scattering to determine translational diffusion coefficients and Stokes radii. Co(NH₃) is more effective than spermidine in causing condensation at all pH, indicating that the differences observed in previous work were not due to pH effects, as might have been inferred from recent theories of intermolecular forces. The DNA particles collapsed with Co(NH₃) are smaller than those obtained with spermidine. The hydrodynamic radius of spermide-collapsed structures decreases slightly with increasing pH, while the size of the Co(NH₃)collapsed structures is almost independent of pH. These results confirm that there are specific ion effects in DNA condensation by oligocations, in addition to the dominant general polyelectrolyte effects.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Phenotypic characterization of transgenic mice harboring Nf1+/- or Nf1-/- osteoclasts in otherwise Nf1+/+ background

Skeletal abnormalities in neurofibromatosis type 1 syndrome (NF1) are observed in ～50% of patients. Here, we describe the phenotype of Nf1_Ocl mouse model with Nf1‐deficient osteoclasts. Nf1_Ocl mice with Nf1^+/? or Nf1^?/? osteoclasts in otherwise Nf1^+/+ background were successfully generated by mating parental Nf1flox/flox and TRAP‐Cre mice. Contrary to our original hypothesis, osteoporotic or fragile bone phenotype was not observed. The µCT analysis revealed that tibial bone marrow cavity, trabecular tissue volume, and the perimeter of cortical bone were smaller in Nf1 mice compared to Nf1 control mice. Nf1 mice also a displayed narrowed growth plate in the proximal tibia. In vitro analysis showed increased bone resorption capacity and cytoskeletal changes including irregular cell shape and abnormal actin ring formation in Nf1^?/? osteoclasts. Surprisingly, the size of spleen in Nf1 mice was two times larger than in controls and histomorphometric analysis showed splenic megakaryocytosis. In summary, Nf1Ocl mouse model presented with a mild but specific bone phenotype. This study shows that NF1‐deficiency in osteoclasts may have a role in the development of NF1‐related skeletal abnormalities, but Nf1‐deficiency in osteoclasts in Nf1^+/+ background is not sufficient to induce skeletal abnormalities analogous to those observed in patients with NF1. J. Cell. Biochem. 113: 2136–2146, 2012. © 2012 Wiley Periodicals, Inc.     

Enantioselective interactions of inversion-labile trigonal iron(II) complexes upon binding to DNA

We studied the interactions of the substitution-inert inversion-labile complexes Fe(bipy) and Fe(phen) [and the inversion-stable complex Ru(bipy)] with DNA. The association of these complexes to DNA is mainly electrostatic, and Fe(phen) shows a more effective binding to DNA than the two bipyridyl complexes, possibly owing to a different binding mode. The interactions are enantioselective, leading to a Pfeiffer shift in the diastereomeric inversion equilibria and an excess of the Δ-enantiomer of Fe(phen) and Fe(bipy), which is directly monitorable through CD. The partition constants for the inversion equilibrium range from 1.3 to 2.0 for Fe(bipy) and Fe(phen), depending on ionic conditions. From flow LD information about the orientation of the complexes on DNA was obtained: it is consistent with a fit of the Δ-enantiomer in the major groove of the right-handed DNA helix. The mechanisms of interaction are discussed against equilibrium, spectroscopic, and kinetic data.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.