Homologous priming in chemotactic peptide-stimulated neutrophils

The kinetics and dose-dependence of activation of human neutrophils exposed to sequential additions of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) have been investigated by multiwell microplate assays. Treatment of neutrophils with medium–high doses (from 10^?8 to 5 × 10^?7 M) of fMLP caused activation of superoxide anion (O) production, but prevented further activation by a subsequent addition of an optimal dose (from 10^?7 M to 5 × 10^?7 M) of fMLP. These findings represent an example of cell desensitization, or adaptation. However, neutrophils treated with low, sub-stimulatory doses (from 10^?10 to 5 × 10^?9 M) of the peptide and then treated with optimal doses of fMLP exhibited an O production that was two to three-fold higher than that induced by the same optimal doses on untreated cells. A similar phenomenon of homologous priming of the oxidative metabolism of neutrophil has not previously been described or characterized. Priming was maximal after about 30 min of incubation with fMLP, which differed from desensitization, which required only a few minutes. Homologous priming was not confined to O production, but was also observed with the release of the granule enzyme, lysozyme. Low doses of fMLP were also capable of triggering an increase of intracellular free Ca²⁺ and of fMLP membrane receptors, which are possible mechanisms responsible for priming.     

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.     

Chemiluminescent assay of various enzyme activites and its application to enzyme immunoassays

A highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O) and hydrogen peroxide (H₂O₂) in the presence of 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS) as electron mediator. The produced O and H₂O₂ can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m-POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10^?9 mol/I to 10^?5 mol/I. This chemiluminescent reaction has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), β-D -galactosidase (β-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, β-Gal and ALP were 10^?18 mol, 10^?20 mol and 10^?18 mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17α-hydroxy-progesterone and DNA hybridization assay using these enzymes as label.     

Conformation of poly(L-homoarginine)

Poly(L -arginine) assumes the α-helix in the presence of the tetrahedral-type anions or some polyanions by forming the “ringed-structure bridge” between guanidinium groups and anions which is stabilized by a pair of hydrogen bonds and electrostatic interaction [Ichimura, S., Mita, K. & Zama, M. (1978) Biopolymers 17 , 2769–2782; Mita, K., Ichimura, S. & Zama, M. (1978) Biopolymers 17 , 2783–2798]. This paper describes the parallel CD studies on the conformational effects on poly (L -homoarginine) of various mono-, di-, polyvalent anions and some polyanions, as well as alcohol and sodium dodecylsulfate. The random coil to α-helix transition of poly(L -homoarginine) occurred only in NaClO₄ solution or in the presence of high content of ethanol or methanol. The divalent and polyvalent anions of the tetrahedral type (SO, HPO, and P₂O), which are strong α-helix-forming agents for poly(L -arginine), failed to induce the α-helical conformation of poly(L -homoarginine). By complexing with poly(L -glutamic acid) or with polyacrylate, which is also a strong α-helix-forming agent for poly(L -arginine), poly(L -homoarginine) only partially formed the α-helical conformation. Monovalent anions (OH^?, Cl^?, F^?, and H₂PO) did not change poly(L -homoarginine) to the α-helix, and in the range of pH 2–11, the polypeptide remained in an unordered conformation. In sodium dodecylsulfate, poly(L -homoarginine) exhibited the remarkably enlarged CD spectrum of an extended conformation, while poly(L -arginine) forms the α-helix by interacting with the agent. Thus poly(L -homoarginine), compared with poly(L -arginine), has a much lower ability to form the α-helical conformation by interacting with anions. The stronger hydrophobicity of homoarginine residue in comparison with the arginine residue would provide unfavorable conditions to maintain the α-helical conformation.     

Nmr studies on complex formation of poly(L-lysine HBr) with carbonate ion in aqueous solution

Diemer formation of poly(L -lysine HBr) in carbonate buffer at pD 10.5 was reported in our previous paper [Biopolymers(1981) 20 , 345–357]. The mechanism of the dimer formation was investigated employing carbon-13 and proton nmr. pD dependence of the ¹³C-nmr spectrum of poly(L -lysine HBr) in the presence of carbonate ion clearly shows that a complex formation between the CO ion and protonated ε-amino group is involved in the stabilization of the dimer form. The lifetime of the complex is longer than 10^?2 s. A stoichiometric evaluation suggests that CO bridges two lysyl side chains. A molecular model of the dimer form designated as a single antiparallel β-ribbon was proposed and discussed in the light of hydrodynamic and ir spectroscopic properties reported earlier. Concentration change experiments indicate that CO binds not only to the dimer formation is inferred as stabilization of the single antiparallel β-ribbon as an intermediate structure in the conversion between the α-helix and β-sheet. The α-CH proton signal of the lysine residue located in the ordered structure (α-helix and β-form) was observed to be separate from that in the random-coil region.     

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.     

On the blue color of triiodide ions in starch and starch fractions. III. Resonance Raman spectra of bluing species in amylose

In order to clarify the characteristics of the basic units responsible for the blue coloring of iodine/iodide in amylose, we made a resonance Raman spectroscopic study at several KI concentrations using excitation by Ar⁺, He-Ne, and Kr⁺ lasers and amyloses with the degrees of polymerization (DP) of 30, 100, 300, and 1000. Similar Raman spectra were observed, regardless of the KI and I₂ concentrations, DP, and excitation wavelengths. Four Raman lines appearing at 159, 111, 55, and 27 cm^?1 were obviously fundamental tones, with a degree of depolarization ρ of ca. 1/3 for every spectrum. However, the internal ratios of the intensities of the 159, 55, and 27 cm^?1 lines to that of the 111-cm^?1 line decreased with increasing KI concentration. Based on the value of ρ, the assignment of the fundamental lines was made by taking a schematic model of the true motions as a projection in separately analyzing the modes of stretching and bending vibrations for a pseudolinear polyiodide chain, which we found to be perturbed by the external forces of the amylose lattice. In accordance with the variation of the force constants from the assignment of the spectra associated with the change in the composition of the bound species, it was concluded that the basic unit changed from I to I through I with decreasing KI concentration.     

Reactivity of Hg(II) with superoxide: Evidence for the catalytic dismutation of superoxide by HG(II)

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H₂O₂) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H₂O₂ production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC₅₀ ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO₂ (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO₂ in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO₂. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H₂O₂ by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

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.     

Optimum Designs for Estimating the Variance Components in a Simple Variance Component Model (Model II), I

The methods of optimal designing of experiments proposed by WALD (1943) are used for determination of an A_σ2-optimal concrete design for estimation of σ^2′ = (σ, σ) in case of one-way analysis of variance. Starting point of definition of the optimality criterion is a quadratic loss matrix.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Conformational analysis of acetyl-L-phenylalanine p-acetyl and p-valeryl anilides

Empirical force-field calculations and ir and ¹H-nmr spectra indicate that five-membered (C₅) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C₅/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.     

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.     

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages

Maximal rates of superoxide (O) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14–18 nmol O/min/10⁷ cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H₂O₂ by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H₂O₂ staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H₂O₂ staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.     

Regulation of the Na+-K+(NH)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH₄Cl, and the variations of the intracellular concentration of calcium ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured using fura-2 and 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH₄Cl significantly increased the [Ca²⁺]_i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH₄Cl, the cells acidified immediately. At 30 mmol/l, NH₄Cl first alkalinized the cells and the pH_i subsequently dropped. This drop reflects the uptake of NH ions that dissociate to NH₃ and H⁺ in the cytosol. These protons are exchanged for extracellular sodium by the Na⁺/H⁺ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH₄Cl. Ouabain partly blocked the uptake of NH. In the combined presence of ouabain and bumetanide (an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter), 1 mmol/l NH₄Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na⁺-K⁺-2Cl⁻ cotransporter in the uptake of NH was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β-adrenergic agonist. ATP increased the release of ³H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na⁺-K⁺-2Cl⁻ cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley-Liss, Inc.     

The influence of environmental conditions on the macromolecular composition of Candida utilis

Glucose-limited chemostat cultures of Candida utilis were cultivated at various pH levels (3.0–7.5), temperatures (15–37.5°C), dilution rates (0.06–0.42 hr^?1), and with different nitrogen sources (NH and NO). The ratio of total nucleic acid to protein increased with increase in dilution rate at constant temperature and decreased with increase in temperature at constant dilution rate. The pattern of these variations is consistent with the hypothesis that the nucleic acid to protein ratio is a function of the ratio of the actual dilution rate to the critical dilution rate corresponding to each one of the cultivation temperatures. This ratio is called “reduced dilution rate.” A basis is proposed on which various microorganisms may be compared with respect to the ratios of cell protein to nucleic acid, RNA, ribosomal RNA, and polysomes.     

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.     

A population genetic study of the Banks and Torres Islands (Vanuatu) and of the Santa Cruz Islands and polynesian outliers (Solomon Islands)

As part of a multidisciplinary survey of populations in the Banks and Torres Islands of Vanuatu and the Southern and Central Districts of the Solomon Islands, nearly 2,400 persons have been tested for ABO blood groups and a number of serum protein and red cell enzyme genetic marker systems. For the ABO system, the populations are characterized in general by high gene O and low gene B frequencies except in two of the Polynesian Outlier Islands, Rennell and Bellona, which have high frequencies of B. Among the serum proteins, several alleles have distributions indicating significant movement of people between islands. These include Albumin ^{New Guinea} and the transferrin alleles Tf, and Tf, and Tf. Similar specific alleles for red cell enzymes also show distributions reflecting interisland population movement as well as contact with persons from outside the southern Pacific region. Examples are ACP in the acid phosphatase system, PGM and PGM, PGM and PGM, PGK⁴ and also HbJ^Tongariki. The data available for 11 polymorphic systems were used to generate genetic distances. Of the four Polynesian Outlier Islands, Anuta is most remote genetically, with Rennell and Bellona also relatively isolated. The fourth Polynesian Outlier, Tikopia, occupies a position genetically close to the Melanesian populations of the Banks and Torres Islands and the southern Solomons. The history of early European contact and voyaging in the Pacific, as well as archaeological and linguistic evidence and local legends, indicate that significant movements of people occurred between islands and provided opportunities for genes to be introduced from Europeans, Africans, and Asians. The genetic marker studies give evidence for genes from all these sources, though at a low level. Despite this admixture, the Polynesian Outlier and Melanesian populations have preserved their own distinctive genetic patterns.     

Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP: Thermal unfolding studies

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41 → Val and Leu-41 → Ala) in the dimeric 4-α-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in α -helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. ΔH⁰ values at the respective transition temperatures, T_1/2 (71.0, 65.3, and 52.9°C at 0.5 mg/ml) decrease from 580 ± 20 to 461 ± 20 kJ/(mol of dimmer) and 335 ± 20 kJ/(mol of dimmer) for wildtype ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L⁴¹V, and L⁴¹A, respectively. The conformational stabilities at 25°C expressed by the standard Gibbs energies of denaturation, ΔG, are 71.7, 61.1, and 46.1 kJ/(mol of dimmer). The corresponding transition enthalpies have been obtained from extrapolation using the c(T)and c(T) functions. Their values at 25°C are 176.3, 101.9, and 141.7 kJ/(mol of dimmer) for wild-type ROP, L⁴¹V, and L⁴¹A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH₂ group, the average ΔΔG value is ?5.0 ± 1 kJ/(mol of CH₂ group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu → Yal and Leu → Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state. © 1995 Wiley-Liss, Inc.