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).     

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.     

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.     

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.     

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.     

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.     

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.     

Crystallographic analysis of Thr-200 → His human carbonic anhydrase II and its complex with the substrate,HCO

A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 Å resolution. © 1993 Wiley-Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

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.     

How Large is the Probability for the Estimate of a Variance Component to be Negative?

For a balanced one-way classification, where the normally distributed observations obey a random model y_ij=μ+b_i+c_ij with two variance components var (b_i) = δ and var (c_ij) = δ, the probability is given that the analysis of variance estimate of δ will be negative. This probability depends on δ/δ and the degrees of freedom in the ANOVA table. Tables for this probability are given. If the normally distributed observations obey an intra-class correlation model, the probability that the Mean Square between groups is smaller than the Mean Square within groups can also be evaluated from the given tables.     

High-resolution nmr study of yeast tRNA and the native and denatured conformers of yeast tRNA

The high-resolution nmr spectrum of baker's yeast tRNA, a recently sequenced non-denaturable tRNA, has been compared with the spectra of the native and denatured conformers of the closely related species tRNA. Because of the presence of many common base pairs in the different tRNA's, it is possible to assign most of the low-field resonances to specific secondary-structure base pairs. A comparison of the observed positions of the various resonances with those predicted by a semiempirical ring-current shift theory shows a root-mean-square deviation of 0.14, 0.11, and 0.12 ppm for tRNA (native), and tRNA (denatured), respectively. These results support the ring-current shift theory currently used to interpret the low-field nmr spectra of the tRNA molecules. Differences between the predicted and observed positions of some resonances provide new evidence for higher order effects such as shifts from second nearest neighbors, anomalous shifts exerted by G·U base pairs, and tertiary-structure effects. A model that was previously proposed for the denatured conformer of tRNA is also supported.     

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.     

Effects of hydrophobicity and anions on self‐assembly of the peptide EMK16‐II

Effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptide EMK16‐II are investigated by atomic force microscopy imaging, circular dichroism spectra, light scattering, and chromatography. It is found that the hydrophobicity of the peptide promotes the aggregation in pure water even at a very low concentration, resulting in a much lower critical aggregation concentration than that of another peptide, EAK16‐II. The effect of anions in solution with different valences on electrostatic interactions is also important. Monovalent anions (Cl^? and Ac^?) with a proper concentration can facilitate the formation of peptide fibrils, with Cl^? of smaller size being more effective than Ac^? of larger size. However, only small amounts of fibrils, but plenty of large amorphous aggregates, are found when the peptide solution is incubated with multivalent anions, such as SO, C₆H₅O, and HPO. More importantly, by gel filtration chromatography, the citrate anion, which induces a similar effect on the self‐assembling process of EMK16‐II as that of SO and HPO, can interact with two or more positively charged residues of the peptide and reside in the amorphous aggregates. This implies a “salt bridge” effect of multivalent anions on the peptide self‐assembling process, which can interpret a previous puzzle why divalent cations inhibit the formation of ordered nanofibrils of the ionic‐complementary peptides. Thus, our results clarify the important effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptides. These are greatly helpful for us to understand the mechanism of peptides' self‐assembling process and protein folding and aggregation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 318–329, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprintversion. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com