An investigation of calcium ion binding to heparins

Using the line charge model of Manning and, where appropriate, a form of the empirical corrections to his limiting laws proposed by Wells, we have investigated the ion condensation phenomenon for four pure calcium heparin preparations and similar Ca-heparin/CaCl₂ mixtures in aqueous solution. This has been expressed in terms of the single ion activity coefficient of the total calcium counter ion present. The results found for the single counter ion activity coefficient of pure Ca-heparin agree moderately well with the limiting law of Manning for a pure divalent counter ion polyanion salt in dilute solution. The results for Ca-heparin/CaCl₂ mixtures when corrected for small ion-small ion interactions give a surprisingly good fit to the theoretical counter ion activity coefficient for a divalent counter ion/divalent cation simple electrolyte system, for values of X (X = the polyanion/co-ion ratio) up to 4, with slight deviations for values of X > 4. In the mixtures with the largest excess of polyelectrolyte, the single ion activity coefficients of the Ca²⁺ ion are in excess of the value predicted by Manning's theory. Ion exchange of sodium heparins to give the Ca-heparin samples used in the work did not appear to cause regular changes in the anticoagulant activity.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

A comparison of the reactivating efficacy of a novel bispyridinium oxime K203 with currently available oximes in VX agent-poisoned rats

The ability of a novel bispyridinium oxime K203 to reactivate VX agent-inhibited acetylcholinesterase was compared with the reactivating efficacy of four commonly used oximes (obidoxime, trimedoxime, methoxime, HI-6) using in vivo model. Our results showed that the reactivating efficacy of the oxime HI-6 is higher than the reactivating efficacy of the other oximes studied including the oxime K203 although the differrences between the oxime HI-6 and some other oximes are not significant, especially in the blood. Based on the obtained data, we can conclude that the antidotal treatment involving the oxime HI-6 brings the higher benefit for the antidotal treatment of acute poisonings with VX agent than other oximes.     

Effects of cytokinin on photosynthetic gas exchange, chlorophyll fluorescence parameters and antioxidative system in seedlings of eggplant (Solanum melongena L.) under salinity stress

The effects of 6-benzyladenine (6-BA) on plant growth, photosynthetic gas exchange, chlorophyll fluorescence and antioxidant systems of eggplant (Solanum melongena L.) under salt stress were investigated. Eggplant seedlings were exposed to 90?mM NaCl with four levels of 6-BA (5, 10, 20 and 50???M) for 10?days. 6-BA at lower concentrations increased chlorophyll concentration, the net photosynthetic rate (P _N), stomatal conductance (g _s), and transpiration rate (E), intercellular CO₂ concentration (C _i) and water use efficiency (WUE), as well as the quantum efficiency of PSII photochemistry (??PSII), photochemical quenching (q _p), and decreased non-photochemical quenching (NPQ), while higher concentrations reduced the effects or even exacerbated the occurrence of photosynthetic capacity. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) increased significantly during salt treatments, and induced the increase of the activities of these enzymes at certain concentrations of 6-BA. 6-BA also reduced significantly malonaldehyde (MDA) contents and O ₂ ^·? production. It was concluded that 6-BA could alleviate the detrimental effects of salt stress on plant growth by increasing photosynthetic efficiency and enhancing antioxidant enzyme systems in leaves at a proper concentration and of the varying 6-BA concentrations used, the most effective concentration for promoting growth was 10???M under saline conditions.     

Identification of a Novel Selenium-containing Compound,Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N_α,N_α,N_α-trimethyl-l-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H]⁺ ion of the compound was 533.0562 and the molecular formula was C₁₈H₂₉N₆O₄Se₂. Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound “selenoneine.” By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2–4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.     

The Enhancing Effect of Ion-pairing on the Skin Permeation of Glipizide

The purpose of the present study was to investigate the permeation of glipizide (GP) and observe the effect of an interaction with amines as counter ions, including diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, N-(2-hydroxylethyl) piperidine. Permeation experiments were performed in vitro, using rat abdominal skin as a barrier. The lipophilic donor system consisting of isopropyl myristate (IPM) and ethanol (EtOH; EI system, 8:2) produced a marked enhancement of GP flux through rat skin. All the amines investigated in this study had performed an enhancing effect on GP flux, and triethylamine had the most potent enhancing effect on GP in the vehicle IPM:EtOH = 8:2(w/w). In the presence of counter ions, the solubility of GP in the donor solution (IPM:EtOH = 8:2) was increased and the log K_o/w of GP was decreased, which may due to higher solubility of the GP in the IPM:EtOH = 8:2(w/w). ¹³C NMR spectroscopy was used to identify the ion-pairing formation between GP and the respective counter ion. It was surprising that all the four enhancers examined, such as isopropyl myristate, propylene glycol, N-methyl-2-pyrrolidone, azone, and oleic acid, had no enhancing effect on the percutaneous permeation of GP. This study showed that the formation of ion-pairs between GP and counter ions is a useful method to promote the skin permeation of GP.Key words: amine, counter ion, glipizide, ion-pair, percutaneous absorption     

Examination of the Role of Central Cholinergic Mechanisms in the Therapeutic Effects of HI-6 in Organophosphate Poisoning

Abstract: In atropine-pretreated rats, HI-6 (125 mg/kg i.p.) raised the LD₅₀ of Soman (subcutaneous) 5.7 times. Addition of HI-6 (25 μg i.c. v.) failed to enhance this protection further. HI-6 (intraperitoneal) also protected animals from intracerebroventricular Soman. HI-6, administered intracerebroventricularly either alone or in combination with intraperitoneal HI-6, failed to increase protection, nor did it reactivate Soman-inhibited acetylcholinesterase (AChE) in several brain areas. HI-6 (125 or 62.5 mg/kg i.p.) protected rats from Sarin lethality, but only the higher dose significantly altered the brain AChE activity. Furthermore, HI-6 (intraperitoneal) failed to block the Soman-induced increase in acetylcholine (ACh) or choline (Ch) levels in any of the brain areas examined. These data indicate that HI-6 is a very beneficial therapy against Soman, but that no definitive central anticholinergic activity of the compound could be found to explain its protective effects. It is possible that HI-6 acts by noncholinergic central mechanisms, or that it produces its beneficial effects outside the CNS. Furthermore, brain AChE activity does not appear to be indicative of protective effects of this oxime. ACh or Ch levels in this study were not good parameters to predict the outcome of Soman poisoning.     

The enzymatic oxidation of Desferal to a nitroxide free radical

Desferrioxamine mesylate (Desferal), a transition metal ion chelator, has been used to inhibit the in vitro redox cycling of transition metal ions. ESR spectroscopy was utilized to detect and identify Desferal's one-electron oxidation product. We demonstrate that a horseradish peroxidase/H₂O₂ system, a xanthine oxidase/hypoxanthine system, and a hydroxyl radical-generating system are all capable of oxidizing Desferal to a nitroxide free radical. The same 9-line ESR spectrum (g = 2.0065, a^N = 7.85 G, a^H(2) = 6.35 G) was detected in all of the above systems. We, therefore, stress that care must be taken when using Desferal as a transition metal ion chelator to keep its concentration low enough to minimize these reactions, or to use a different metal ion chelator.     

Aminolysis of oxalate esters in toluene: A model for a carboxylate buried in a hydrophobic environment at an active site

We have examined the aminolysis of the mono-o-nitrophenyl ester of oxalic acid by piperidine in toluene with the purpose of determining how a carboxylate “buried” at an active site might affect an enzyme-catalyzed reaction. The oxalate ester and piperidine form an ion pair (R₂NH₂⁺⁻O₂CCO₂Ar) even in extremely dilute toluene solutions. This conclusion is supported by the kinetic effects of acidic and basic additives and by a “concentration inversion” experiment. The oxalate ester was found to react more than three orders of magnitude faster than o-nitrophenyl acetate. The neighboring carboxylate in the ion pair apparently accelerates the decomposition of a tetrahedral intermediate by accepting a proton from the amine nitrogen. The implications of this anchimeric assistance to enzymatic systems is discussed briefly.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

A refined atomic scale model of the Saccharomyces cerevisiae K+-translocation protein Trk1p combined with experimental evidence confirms the role of selectivity filter glycines and other key residues

Potassium ion (K⁺) uptake in yeast is mediated mainly by the Trk1/2 proteins that enable cells to survive on external K⁺ concentration as low as a few μM. Fungal Trks are related to prokaryotic TRK and Ktr and plant HKT K⁺ transport systems. Overall sequence similarity is very low, thus requiring experimental verification of homology models. Here a refined structural model of the Saccharomyces cerevisiae Trk1 is presented that was obtained by combining homology modeling, molecular dynamics simulation and experimental verification through functional analysis of mutants. Structural models and experimental results showed that glycines within the selectivity filter, conserved among the K-channel/transporter family, are not only important for protein function, but are also required for correct folding/membrane targeting.A conserved aspartic acid in the P_A helix (D79) and a lysine in the M2_D helix (K1147) were proposed earlier to interact. Our results suggested individual roles of these residues in folding, structural integrity and function. While mutations of D79 completely abolished protein folding, mutations at position 1147 were tolerated to some extent. Intriguingly, a secondary interaction of D79 with R76 could enhance folding/stability of Trk1 and enable a fraction of Trk1[K1147A] to fold.The part of the ion permeation path containing the selectivity filter is shaped similar to that of ion channels. However below the selectivity filter it is obstructed or regulated by a proline containing loop. The presented model could provide the structural basis for addressing the long standing question if Trk1 is a passive or active ion-translocation system.     

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

α_s1- and β-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (α_s1- and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α_s1- and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.     

Quantification of diadenosine polyphosphates in blood plasma using a tandem boronate affinity–ion exchange chromatography system

Endogenous diadenosine polyphosphates (Ap_nAs) have been associated with a variety of biological effects but quantifying their concentration in blood is difficult. We report on the development of a tandem affinity–ion exchange high-performance liquid chromatography (HPLC) system that employs boronate affinity upstream of ion exchange chromatography for automated rapid (45-min) resolution and extraction of Ap_nAs from human plasma. This system obviates previous requirements for multiple column separations and handling steps, so it is ideally set up for time- and cost-efficient screening of blood samples for Ap_nA pharmacokinetic and biodistribution studies.     

Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.     

PHOSPHATE ION AS A PROMOTER CATALYST OF RESPIRATION

The active component of phosphate solutions, in relation to promoter action on oxidising enzymes, is the PO₄ ^'''''' ion. This is shown by the demonstration of a hyperbolic relationship between per cent production of CO₂ (of Elodea) and pPO₄, the measure of the phosphate ion potential. This is consistent with the rate of respiration as affected by changing pPO₄ through change of total phosphate concentration while pH is kept constant. The equation for this relationship is (CO₂ – a) (pPO₄ – b)ⁿ = K where a, b, n, and K are constants and n = 1. The same relationship to phosphate ion concentration, expressed by the equation (Activity of enzyme) (pPO₄)ⁿ = K, where n and K are constants and n varies from 1 to 6 under different conditions, appears to hold for some other enzyme actions, including those of peroxidase and pancreatic lipase.     

Modelling of a single-staged bioseparation process for simultaneous removal of iron(III) and chromium(VI) by using Chlorella vulgaris

In this study, the competitive biosorption of iron(III) and chromium(VI) to Chlorella vulgaris from a binary metal mixture was investigated in a single-staged batch reactor as a function of V₀/X₀ (volume of solution containing heavy metal mixture/quantity of biosorbent) ratio and second metal ion concentration at pH 2. The obtained results showed that the increase in biomass quantity (or the decrease of V₀/X₀ ratio) with the addition of second metal ion affected the removed quantities of iron(III) or chromium(VI). The sorption phenomenon was expressed by the competitive, multi-component Langmuir adsorption isotherm and this expression was used for calculating each residual or adsorbed metal ion concentration at equilibrium (C_eq,i or C_ad,eq,i) at a constant V₀/X₀ ratio for a given combination of heavy metal ions in a single-staged batch reactor. Experimental C_eq,i and C_ad,eq,i values were compared to those calculated and graphically determined.     

Effects of Replacement of External Sodium Chloride with Sucrose on Membrane Currents of the Squid Giant Axon

It was observed that a reduction of the sodium chloride concentration in the external solution bathing a squid giant axon by replacement with sucrose resulted in marked decreases in the peak inward and steady-state outward currents through the axon membrane following a step decrease in membrane potential. These effects are quantitatively acounted for by the increase in series resistance resulting from the decreased conductivity of the sea water and the assumption that the sodium current obeys a relation of the form I = k₁C₁ - k₂C₂ where C₁, C₂ are internal and external ion activities and k₁, k₂ are independent of concentration. It is concluded that the potassium ion current is independent of the sodium concentration. That the inward current is carried by sodium ions has been confirmed. The electrical potential (or barrier height) profile in the membrane which drives sodium ions appears to be independent of sodium ion concentration or current. A specific effect of the sucrose on hyperpolarizing currents was observed and noted but not investigated in detail.     

The effect of low temperatures on fatty acid biosynthesis in plants

1. Of three systems, bulb tissue, plant leaf tissue and intact green algal (Chlorella vulgaris) cells, only the former shows an increase in rate of formation of unsaturated fatty acids with decrease in temperature. 2. In bulb tissue the oxygen concentration is rate-limiting for synthesis of unsaturated fatty acids at temperatures down to 10°. 3. At elevated oxygen concentrations the formation of unsaturated fatty acids in bulb tissue increases with temperature. 4. The failure of photosynthetic tissues to respond to either lower temperatures or increased oxygen concentrations in the presence of light is attributed to photosynthetic production of excess of oxygen. This is supported by the fact that in the dark a potentiating oxygen effect on the formation of unsaturated fatty acids can be demonstrated. 5. The HCO₃⁻ ion concentration has a small effect on the formation of unsaturated fatty acids. 6. Elevated content of unsaturated acids at lower temperatures in plants is attributed to increases in oxygen concentration in solution.     

Multinuclear NMR studies of intracellular cations in the prehypertensive rat kidney

We previously reported a significant derangement of intracellular free calcium ion concentration in the isolated perfused kidney of adult spontaneously hypertensive rat (SHR) (J. Biol. Chem. 267, 3637–3643, 1992). In order to investigate whether an abnormality in intracellular free calcium or another ion precedes the development of elevated blood pressure in SHR, we have now compared intracellular free Ca²⁺, Na⁺ and pH, using ³¹P, ¹⁹F, and triple quantum-filtered (TQ) ²³Na NMR, in perfused kidneys from prehypertensive young SHR and normotensive young Wistar-Kyoto (WKY) rats (5–6 weeks old) which showed no significant difference in blood pressure B.P.=120±5 mmHg and 115±3 mmHg, for SHR and WKY rats, respectively). Like the adult kidney, no significant differences in intracellular ATP concentration or intracellular pH were found between young prehypertensive SHR and normotensive WKY rat kidneys. The TQ ²³Na NMR signal was 47% higher in the SHR kidney, but, due to biological variability and measurement errors, this difference could not be shown to be statistically significant. However, a significant (40%; P<0.05) increase was found in O₂ consumption rate, a measure of the Na⁺/K⁺-ATPase activity, of the young prehypertensive SHR kidney in comparison to the age-matched WKY rat kidney (7.25±0.75 for SHR vs. 5.17±0.18 μmola O₂/min g for WKY rat, n = 6). Furthermore, a highly significant (92%; P<0.02) increase in intracellular free Ca²⁺ concentration was observed in kidneys from young SHR that had noy yet been developed high blood pressure in comparison to the kidneys from young normotensive WKY rats (648±76 nM vs. 339±39 nM, n = 4, despite the fact that there was no significant difference in blood pressure. Increased intracellular free Ca²⁺ thus appears to be part of a primary defect, in the prehypertesive young SHR kidney, which may, by way of increased release of arachidonic acid, and subsequent increased production of vasoconstricting arachidonic acid metabolites via the cytochrome P450 pathway, induce elevated blood pressure in the adult SHR.     

Variable product specificity of solanesyl pyrophosphate synthetase

The distribution of polyprenyl pyrophosphates synthesized by the action of solanesyl pyrophosphate synthetase from Micrococcus luteus is dramatically changed depending on the Mg⁺⁺ concentration. When the metal ion concentration is higher than 5 mM, octaprenyl and solanesyl (nonaprenyl) pyrophosphate are synthesized predominantly. On the other hand, when the metal ion level is lower than 0.5 mM, a variety of polyprenyl pyrophosphates ranging in carbon chain length from C₁₅ to C₄₀ are formed. Heptaprenyl pyrophosphate is the longest of the products formed at 0.1 mM of Mg⁺⁺.