Dual effects of formylpeptides on the adhesion of endotoxin-primed human neutrophils

Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (> 5 × 10^?8 M ) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10^?10 M to 5 × 10^?9 M ) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (> 10^?7 M ) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (> 10^?9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10^?9 M ) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10^?9 M ) and high (5 × 10^?7 M ) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.     

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).     

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.     

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.     

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.     

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.     

Equi-replicated balanced block designs generated by a balanced matrix

In this paper it is shown that if N= \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} c_ihN_ih, where c_ih are some non-negative integer numbers and N_ih are such incidence matrices that A_h = \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} i N_ih is a balanced matrix defined by SHAH (1959), for h = 1, 2,…, p, then a block design with an incidence matrix Ñ = [N, N,…,N] is an equi-replicated balanced block design. Here the balance of a block design is defined in terms of the matrix M₀ introduced by CALI?SKI (1971).     

Construction of Equi-replicated Balanced Block Designs with Block Sizes Exceeding the Number of Treatments

In this note it is shown that the block design with incidence matrix Ñ = [NN… N], where N = c_1hN_h + c_oh (11′–N_h). c_oh and c_1h are any non-negative integers and N_h,h = 1, 2,…,p, are incidence matrices of balanced incomplete block designs with the same number of treatments t, is a balanced block design with the block sizes exceeding the number of treatments. In derivation the matrix M₀, introduced by CALIński (1971) is utilized.     

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-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.     

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.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Conformation and folding in histones H1 and H5

Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a T_m and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.     

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.     

An Unbalanced Mixed Model with Random Effects Having Unequal Variances

Consider the mixed model where x_ijk's are known constants, β_k are unknown parameters and a_i, e_ij are random variables independently and normally distributed with zero means and variances σd_i and σ² respectively, where it is assumed that the d_i's are known (d_i >0). This paper presents procedures for estimating the variance components σ, σ², for testing the hypothesis σ = 0, and for making transformations to random variables with uncorrelated errors and constant variances in order to estimate as well as to test hypothesis concerning the β_k's in the model.     

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.     

Polyiodine units in starch–iodine complex: INDO CI study of spectra and comparison with experiments

The starch–iodine blue complex formation does not involve negatively charged iodine species like I, I, or I; rather, neutral iodine units are involved. The heat of reaction is determined to be about ?110 kJ for every mole of I-I unit in the amylose helix, which suggests that the dissociation of I₂ (binding energy 149 kJ/mol) does not take place during the complex formation. Quantum mechanical (INDO CI) calculations indicate that the linear as well as nonlinear polyiodine units, I₆, with interiodine distance of 3.0 Å are responsible for characteristic absorbance bands of the starch–iodine complex. Based on our previous article [(1989) J. Polym. Sci. A 27 , 4161] and the present studies we identify (C₆H₁₀O₅)_16.5I₆ to be the polymeric unit responsible for the characteristic blue color of the complex.     

Hydrobiological Studies on Suraj Kund and Chandrama Kund,Hot Springs of Rajgir,Bihar, India

The hot water of Rajgir springs is used for drinking and bathing purposes by tourists. Certain physico-chemical characteristics (temperature, pH, NO, PO, etc.) of the water along with phycological parameters viz. community composition, species diversity, standing crop etc. were measured from June 1986 to April 1987. The water was deficient in Na, NO and PO ions. The hot springs were mainly dominated by algae belonging to Cyanophyceae and Bacillariophyceae. The algal community comprised 18 species, with dominance of Cyanophyceae over Bacillariophyceae. While Mastigocladus laminosus and species of Phormidium were dominant in Suraj Kund, species of Oscillaroria and Synechococcus dominated in Chandrama Kund. Diatoms comprised about 10 % of the algal community. Though there was a considerable seasonal change in species diversity of the algal community the total biomass (chlorophyll a extracted per unit area from the algal mat) remained constant.