Efficient Synthesis of Macromolecular DO3A@Gn Derivatives for Potential Application in MRI Diagnostics: From Polymer Conjugates to Polymer Nanoparticles

Herein, the synthesis of three different macromolecular DO3A@Gn conjugates based on poly(2-oxazoline)s is presented. Therefore, poly(2-methyl-2-oxazoline) is synthesized by a ring-opening, cationic polymerization and the polymerization is terminated with DO3A(tBu)₃ . The best results are obtained after 48 h at 120 °C with degree of termination of 86%. After deprotection of the DO3A ligand and complexation with Gn³⁺, relaxivity as measured with a magnetic field strength of 9.4 T (400 MHz) reveals values for r₁ of up to 2.32 mm ⁻¹ s⁻¹. The concept is extended to a block copolymer based on 2-heptyl-2-oxazoline and 2-methyl-2-oxazoline that is again terminated with DO3A(tBu)₃ to form micelles with a size of 12.6 ± 0.7 nm after DO3A(tBu)₃ termination and deprotection of the 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid ligand. After complexation with Gn³⁺, relaxivity r₁ is 10.1 mm ⁻¹ s⁻¹ as determined from the slope of the plot of 1/T₁ against the gadolinium(III) concentration at 9.4 T. Finally, crosslinked nanoparticles are prepared from amphiphilic macro-monomers that form micelles in water and are crosslinked throughout the core in the presence of azoisobutyronitrile (AIBN). The nanoparticle is 32.9 ± 7.8 nm in size after Gn³⁺ complexation and reveals a relaxivity r₁ of 6.77 mm ⁻¹ s⁻¹.     

Relationship between blood and myocardium manganese levels during manganese‐enhanced MRI (MEMRI) with T1 mapping in rats

Manganese ions (Mn²⁺) enter viable myocardial cells via voltage‐gated calcium channels. Because of its shortening of T₁ and its relatively long half‐life in cells, Mn²⁺ can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn²⁺ is its sensitivity over a limited range of concentrations employing T₁‐weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T₁ mapping method for cardiac manganese‐enhanced MRI to enable a quantitative estimate of the influx of Mn²⁺ over a wide range of concentrations in male Sprague‐Dawley rats. This MRI method was used to compare the relationship between T₁ changes in the heart as a function of myocardium and blood Mn²⁺ levels. Results showed a biphasic relationship between ΔR₁ and the total Mn²⁺ infusion dose. Nonlinear relationships were observed between the total Mn²⁺ infusion dose versus blood levels and left ventricular free wall ΔR₁. At low blood levels of Mn²⁺, there was proportionally less cardiac enhancement seen than at higher levels of blood Mn²⁺. We hypothesize that Mn²⁺ blood levels increase as a result of rate‐limiting excretion by the liver and kidneys at these higher Mn²⁺ doses. Copyright © 2010 John Wiley & Sons, Ltd.     

Monitoring dynamic alterations in calcium homeostasis by T1‐mapping manganese‐enhanced MRI (MEMRI) in the early stage of small intestinal ischemia–reperfusion injury

Manganese‐enhanced MRI studies have proven to be useful in monitoring physiological activities associated with calcium ions (Ca²⁺) due to the paramagnetic property of the manganese ion (Mn²⁺), which makes it an excellent probe of Ca²⁺. In this study, we developed a method in which a Mn²⁺‐enhanced T₁‐map MRI could enable the monitoring of Ca²⁺ influx during the early stages of intestinal ischemia–reperfusion (I/R) injury. The Mn²⁺ infusion protocol was optimized by obtaining dose‐dependent and time‐course wash‐out curves using a Mn²⁺‐enhanced T₁‐map MRI of rabbit abdomens following an intravenous infusion of 50 mmol/l MnCl₂ (5–10 nmol/g body weight (BW)). In the rabbit model of intestinal I/R injury, T₁ values were derived from the T₁ maps in the intestinal wall region and revealed a relationship between the dose of the infused MnCl₂ and the intestinal wall relaxation time. Significant Mn²⁺ clearance was also observed over time in control animals after the infusion of Mn²⁺ at a dose of 10 nmol/g BW. This technique was also shown to be sensitive enough to monitor variations in calcium ion homeostasis in vivo after small intestinal I/R injury. The T₁ values of the intestinal I/R group were significantly lower (P < 0.05) than that of the control group at 5, 10, and 15 min after Mn²⁺ infusion. Our data suggest that MnCl₂ has the potential to be an MRI contrast agent that can be effectively used to monitor changes in intracellular Ca²⁺ homeostasis during the early stages of intestinal I/R injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Alginate‐coated magnetic nanoparticles for noninvasive MRI of extracellular calcium

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca²⁺), are essential ingredients for life. Despite the tremendous importance of Ca²⁺ for the well‐being of living systems, the noninvasive determination of the changes in Ca²⁺ levels in general, and extracellular Ca²⁺ levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca²⁺ levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca²⁺ concentrations in the range of 250 µm to 2.5 mm , even in the presence of competitive ions. The alginate‐coated magnetic NPs (MNPs) aggregate in the presence of Ca²⁺, which, in turn, affects the T₂ relaxation of the water protons in their vicinity. The new alginate‐coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca²⁺ levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca²⁺ levels in vivo. These results demonstrate that alginate‐coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca²⁺ levels in myriad in vitro and in vivo biomedical applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Inhibition of the sodium–calcium exchanger via SEA0400 altered manganese‐induced T1 changes in isolated perfused rat hearts

Manganese (Mn²⁺)‐enhanced MRI (MEMRI) provides the potential for the in vivo evaluation of calcium (Ca²⁺) uptake in the heart. Recent studies have also suggested the role of the sodium–calcium (Na⁺–Ca²⁺) exchanger (NCX) in Mn²⁺ retention, which may have an impact on MEMRI signals. In this study, we investigated whether MEMRI with fast T₁ mapping allowed the sensitive detection of changes in NCX activity. We quantified the dynamics of the Mn²⁺‐induced T₁ changes in isolated perfused rat hearts in response to SEA0400, an NCX inhibitor. The experimental protocol comprised 30 min of Mn²⁺ perfusion (wash‐in), followed by a 30‐min wash‐out period. There were three experimental groups: 1, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐in only (SEAin, n = 6); 2, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐out only (SEAout, n = 6); 3, no NCX inhibition during both wash‐in and wash‐out to serve as the control group (CNTL, n = 5). Rapid T₁ mapping at a temporal resolution of 3 min was performed throughout the perfusion protocol using a triggered saturation–recovery Look–Locker sequence. Our results showed that NCX inhibition during Mn²⁺ wash‐in caused a significant increase in relaxation rate (R₁) at the end of Mn²⁺ perfusion. During the wash‐out period, NCX inhibition led to less reduction in R₁. Further analysis of Mn²⁺ content in myocardium with flame atomic absorption spectroscopy was consistent with the MRI findings. These results suggest that Mn²⁺ accumulation and retention in rat hearts are, in part, dependent on NCX activity. Hence, MEMRI may provide an imaging method that is also sensitive to changes in NCX activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Interaction of divalent metal ions with poly(riboinosinic acid)

Methods of differential UV and visible spectroscopy are used to study the interaction of Mg²⁺, Ca²⁺, Mn²⁺ and Cu²⁺ ions with four (4×I)‐ and single‐stranded poly I in solutions with 1 mol·l^–1 Na⁺. Up to concentrations of about 0.1 mol·l^–1 Mt²⁺, Mg²⁺ and Ca²⁺ ions do not bind to heteroatoms of hypoxanthine of 4×I. Cu²⁺ ions interact with N7 and/or N1 and possibly with O6 through the water molecule of the hydrate shell of the ion. It is likely that the interaction with O6 causes enolization of the hypoxanthine, N1 deprotonation and, as a result, this leads to the melting of the four‐stranded helix. In single‐stranded chains, Cu²⁺ ions induce the formation of compact particles which have the effective radii r_e ≈ 100 Å. The Mn²⁺‐induced differential spectra are similar to those observed in the presence of Cu²⁺ ions but in the case of Mn²⁺ they occur at concentrations about two orders of magnitude higher. In contrast to the positive cooperativity of the Cu²⁺ binding to bases of single‐stranded poly I, their interaction with 4×I has the negative cooperativity.     

Manganese and the heart: acute cardiodepression and myocardial accumulation of manganese

The aim of study was to assess acute effects of the divalent manganese ion (Mn²⁺) in an intact but isolated heart preparation. Rat hearts were perfused in the Langendorff mode at constant flow rate. Left ventricular (LV) developed pressure (LVDP), LV pressure first derivatives (LVdp/dt max and min), heart rate (HR) and aortic pressure (AoP) were recorded. Ventricular contents of high energy phosphate compounds (HEP) and Mn metal were measured at the end of experiment. Infusion of MnCl₂ for 5 min with perfusate concentrations 1–3000 μM induced an immediate depression of contractile function at and above 30 μM and negative chronotropy at and above 300 μM . These EC₅₀ values were found (μM ): LVDP 250; LVdp/dt max 160; LVdp/dp min 120; HR 1000; and increase in AoP 80. Recovery of function during a 14 min washout period was rapid and extensive, except for Mn²⁺ 3000 μM . Somewhat unexpected, Mn²⁺ 30–1000 μM raised coronary vascular resistance up to about twice the control level, whereas the vasoconstrictory response was overcome at 3000 μM . Mn²⁺ 3000 μM reduced tissue HEP. Ventricular Mn content rose stepwise for perfusate Mn²⁺ above 1 μM up to about 55 times the control level for perfusate Mn²⁺ 3000 μM . It is concluded that: acute effects of Mn²⁺ like depression of contractility and rate is rapidly reversible; and rat hearts accumulate and buffer large amounts of Mn²⁺ without affecting cardiac function or energy metabolism in the acute stage.     

Modulation of manganese currents by 1, 4-dihydropyridines,isoproterenol and forskolin in rabbit ventricular cells

Although often used as a Ca²⁺ channel blocker, Mn²⁺, in fact, permeates through Ca²⁺ channels. Under Na⁺-free conditions, depolarizing pulses evoked slowly-decaying Mn²⁺ currents (I_Mn). Maximal I_Mn densities in the presence of 5 and 20 mM Mn²⁺ were 0.42±0.12 pA/pF (mean±SEM, n=17) and 1.23±0.10 pA/pF (n=40), respectively. At 5 mM, the ratio of maximal amplitude of I_Mn to that of the Ca²⁺ current (I_Ca) was 0.079±0.009 (n=8). I_Mn elicited from a holding potential of –50 mV was depressed by nitrendipine (1 µM) by ~70%. Nitrendipine (0.3 µM) shifted the steady-state inactivation curve to more negative potentials and shifted the potential for half-maximal inactivation (E_0.5) from 1.3 to –8.8 mV and also decreased the time constant of decay of I_Mn at 20 mV from 986.2 to 167.9 ms. BAY K 8644 (1 µM), isoproterenol (10 µM) and forskolin (10 µM) all increased I_Mn and shifted the current/voltage (I/V) relationship to more negative potentials. The small, slowly-inactivating I_Mn is thus modulated by dihydropyridine Ca²⁺ channel modulators and cyclic AMP-mediated phosphorylation in a manner similar to other L-type Ca²⁺ channel currents. L-type Ca²⁺ channels are involved in the regulation of intracellular [Mn] in ventricular myocytes.     

Characterization of an extracellular alkaline lipase from Pseudomonas mendocina M‐37

A strain of Pseudomonas mendocina producing extracellular lipase was isolated from soil. The bacterium accumulates lipase in culture fluid when grown aerobically at 30 °C for 24 h in a medium composed of olive oil (1%) as substrate. Pseudomonas mendocina lipase was optimally active at pH 9.0, temperature of 50 °C and was found to be stable between pH 7.0–11.0. The lipase was inhibited by detergents such as SDS and Tween‐80. The enzyme was stable in various organic solvents tested with maximum stability in chloroform followed by toluene and exhibited 1–3 regiospecificity for hydrolytic reaction. This lipase was capable of hydrolyzing a variety of lipidic substrates and is mainly active towards synthetic triglycerides and fatty acid esters that possess a butyryl group. Metal ions like Mg²⁺, Ca²⁺ and Na⁺ stimulated lipase activity, whereas, Cu²⁺, Mn²⁺ and Hg²⁺ ions caused inhibition. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells

Changes of intracellular calcium activity (Ca _i ²⁺ ) in mouse mammary epithelial cells in primary culture (normal) and in an established cell line (MMT060562, cancerous) were investigated by microfluorometry and image analysis of fura-2 fluorescence. In both types of cells, some populations exhibited occasional Ca _i ²⁺ oscillations with a period of 50–160 s. Slight mechanical stimulation of a cell with a fine glass pipette induced a Ca _i ²⁺ increase, which spread from the stimulated cell to the surrounding cells with a speed of 7–12 m/s. ATP (>1 mol/l) and ADP, but not AMP induced a Ca _i ²⁺ increase in both cell types. Bradykinin was highly effective (> 10 nmol/l) only in the cancerous mammary epithelial cells. In Ca²⁺-free solution, all these Ca _i ²⁺ responses remained unchanged at the first application, and decreased abruptly at the second trial. La³⁺ (>0.5 mmol/l) suppressed the response to ATP but not the response to bradykinin. Addition of extracellular Mn²⁺ rapidly quenched the fura-2 fluorescence in the cell even in a non-stimulated state. Influx of Mn²⁺ did not increase during Ca _i ²⁺ responses. These results indicate that the sources of Ca _i ²⁺ responses in mammary epithelial cells are intracellular stores, which exchange Ca²⁺ with the extracellular medium.     

Effect of α1‐adrenergic stimulation of Cl− secretion and signal transduction in exocrine glands (Rana esculenta)

In the present work, the effect of stimulation of α‐adrenergic receptors on Cl^? secretion via exocrine frog skin glands was investigated. The α‐adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the β‐adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl^? secretion was measured as the amiloride‐insensitive short circuit current (I_SC). Addition of noradrenaline induced a biphasic increase in the I_SC. The increase in I_SC coincided with an increase in the net ³⁶Cl^? secretion. The noradrenaline‐induced increase in I_SC was dose‐dependent with an EC₅₀ of 13 ± 0.3 μM . Epifluorescence microscopic measurements of isolated, fura‐2‐loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca²⁺]_i) response. Application of noradrenaline induced a biphasic [Ca²⁺]_i response, which was dose‐dependent with an EC₅₀ of 11 ± 6 μM . The Ca²⁺ plateau unlike the peak‐response was sensitive to removal of Ca²⁺ from the extracellular medium. The noradrenaline‐induced increase in the Cl^? secretion as well as in [Ca²⁺]_i was sensitive to the α₁‐adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca²⁺]_i indicating that the release was independent of ryanodine‐sensitive Ca²⁺ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5‐trisphosphate (IP₃) content suggesting that the signal transduction pathway leading to the noradrenaline‐induced increase in Ca²⁺ involved IP₃ and a release of Ca²⁺ from IP₃‐sensitive stores.     

Mutants resistant to manganese in Saccharomyces cerevisiae

Summary Several mutants resistant to Mn²⁺ have been isolated and characterized in Saccharomyces cerevisiae. All the mutations are semidominant and allelic to a single nuclear gene (MNRI). Mg²⁺ in the growth medium reverses the inhibitory effect of Mn²⁺ in a competitive way. This appears to be due to the inhibition of the uptake of Mn²⁺ by the cells, not to an increase of the amount of Mg²⁺ inside the cells.The analysis of the distribution of Mn²⁺ taken up by growing cells shows that the amount of the ion present in insoluble form is far higher in resistant than in sensitive cells. We therefore believe that yeast cells have a sequestering system for Mn²⁺ and that the major difference between mutants and wild-type strains lies in the much higher efficiency of this system.     

Identification of the Ca2+ current activated by vasoconstrictors in vascular smooth muscle cells

The noncontractile aortic cell line A7r5 was chosen to study the effect of the vasoconstrictor peptide vasopressin on transmembrane Ca²⁺ movements, using conventional whole-cell patch recording techniques. Conditions in which previously characterised vasoconstrictor-modulated currents were suppressed revealed a tiny inward current component (−18±2 pA,n=50, at -61 mV in 110 mM CaCl₂). The vasopressin-activated inward current was absent when Ca²⁺ was absent from the extracellular solution, and the current amplitude increased with [Ca²⁺] (0.01–110 mM), with an apparent dissociation constant for Ca²⁺ of 9.7 mM. It was highly selective for Ca²⁺ over monovalent cations (permeability ratio Ca/Cs greater than 17). It was not voltage gated, except that the current/potential characteristic showed some inwards rectification. Amplitudes of the evoked inward currents had the same order of magnitude in Sr²⁺ and Ca²⁺, whereas they were much smaller in Mn²⁺, suggesting that this pathway is highly permeable to Sr²⁺ but poorly permeable to Mn²⁺. Inward currents evoked in Ca²⁺ were inhibited by other cations with the following order of potency: La³⁺>Cd²⁺>Co²⁺∼Ni²⁺∼Mn²⁺. The channel producing this current corresponds most probably to the ionic pathway originally called the receptor-operated calcium channel, which produces a long-lasting, constrictor-induced plateau of increased intracellular free calcium concentration in smooth muscle. Dedicated by Catherine Van Renterghem to her father, Jacques Van Renterghem     

Assessing manganese efflux using SEA0400 and cardiac T1‐mapping manganese‐enhanced MRI in a murine model

The sodium–calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca²⁺) concentration by normally mediating net Ca²⁺ efflux. However, the reverse mode of the NCX can cause intracellular Ca²⁺ concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca²⁺ fluctuations produced by this drug. Cardiac manganese‐enhanced MRI (MEMRI) may indirectly assess Ca²⁺ efflux by estimating changes in manganese (Mn²⁺) content in vivo, since Mn²⁺ has been suggested as a surrogate marker for Ca²⁺. This study used the MEMRI technique to examine the temporal features of cardiac Mn²⁺ efflux by implementing a T₁‐mapping method and inhibiting the NCX with SEA0400. The change in ¹H₂O longitudinal relaxation rate, ΔR₁, in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl₂) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 ± 0.6 h post‐MnCl₂ infusion without drug intervention. Furthermore, treatment with 50 ± 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in ΔR₁. At 4.9–5.9 h post‐MnCl₂ infusion, the average ΔR₁ values for the two groups treated with SEA0400 were 2.46 ± 0.29 and 1.72 ± 0.24 s^?1 for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 ± 0.28 s^?1 for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T₁‐mapping technique was shown to effectively quantify Mn²⁺ efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn²⁺ transport mechanisms and rates in vivo, which may reflect changes in Ca²⁺ transport. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of manganese‐enhanced magnetic resonance imaging of the rostral ventrolateral medulla of conscious rats: Importance of normalization and comparison with other regions of interest

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are believed to contribute to pathophysiological alterations in sympathetic nerve activity and the development of cardiovascular disease. The ability to identify changes in the activity of RVLM neurons in conscious animals and humans, especially longitudinally, would represent a clinically important advancement in our understanding of the contribution of the RVLM to cardiovascular disease. To this end, we describe the initial development of manganese‐enhanced magnetic resonance imaging (MEMRI) for the rat RVLM. Manganese (Mn²⁺) has been used to estimate in vivo neuronal activity in other brain regions because of both its paramagnetic properties and its entry into and accumulation in active neurons. In this initial study, our three goals were as follows: (1) to validate that Mn²⁺ enhancement occurs in functionally and anatomically localized images of the rat RVLM; (2) to quantify the dose and time course dependence of Mn²⁺ enhancement in the RVLM after one systemic injection in conscious rats (66 or 33 mg/kg, intraperitoneally); and (3) to compare Mn²⁺ enhancement in the RVLM with other regions to determine an appropriate method of normalization of T₁‐weighted images. In our proof‐of‐concept and proof‐of‐principle studies, Mn²⁺ was identified by MRI in the rat RVLM after direct microinjection or via retrograde transport following spinal cord injections, respectively. Systemic injections in conscious rats produced significant Mn²⁺ enhancement at 24 h (p < 0.05). Injections of 66 mg/kg produced greater enhancement than 33 mg/kg in the RVLM and paraventricular nucleus of the hypothalamus (p < 0.05 for both), but only when normalized to baseline scans without Mn²⁺ injection. Consistent with findings from our previous functional and anatomical studies demonstrating subregional neuroplasticity, Mn²⁺ enhancement was higher in the rostral regions of the RVLM (p < 0.05). Together with important technical considerations, our studies support the development of MEMRI as a potential method to examine RVLM activity over time in conscious animal subjects.     

Effects of divalent cations on chloride movement across amphibian skin

Effects of the divalent heavy metal ions Cd²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ on pathways for sodium and chloride were assessed on isolated amphibian skin (Rana temporaria andesculenta, Bufo marinus andviridis). It was observed that these agents, in addition to the previously reported stimulation of sodium transport, inhibit chloride-related tissue conductance (g _Cl) in frog skin with spontaneously highg _Cl when added to the external incubation medium. Serosal application was ineffective. Half-maximal inhibition ofg _t occurred at approximately 0.2 mmol/l Ni²⁺ and Zn²⁺, 0.5 mmol/l Co²⁺ and Cd²⁺, and more than 3 mmol/l Mn²⁺. The onset of inhibition was rapid, steady state values being reached within 3 min; reversibility was complete with approximately similar time course. Cu²⁺, which could not be tested at concentrations above 0.1 mmol/l, had only minimal and poorly reversible effect ong _Cl. Skin ofBufo was virtually insensitive to these metal ions. Microelectrode determinations demonstrate that the decrease of conductance was restricted to a pathway distinct from the principal cells which show, on the contrary, increase of apical membrane conductance originating from stimulation of sodium permeability. The metal ions might be valuable for characterization of the pathway and the mechanism of transepithelial conductive chloride transport.     

Chelation ion-exchange properties of salicylic acid/thiourea/trioxane terpolymers

Terpolymers prepared by condensation of salicylic acid and thiourea with trioxane in presence of acid catalysts, proved to be selective chelating ion-exchange resins for certain metals. Chelation ion-exchange properties of these terpolymers were studied for Cu²⁺, Ni²⁺, Fe³⁺, Mn²⁺, Co²⁺, Zn²⁺, and UO₂²⁺ ions. A batch equilibration method was employed in the study of the selectivity of metal ion uptake involving the measurement of distribution of a given metal ion between the polymer sample and a solution containing the metal ion. The study was carried out over a wide pH range and in media of various ionic strengths. The terpolymers showed a higher selectivity for Cu²⁺, Fe³⁺, and UO₂²⁺ ions than for Mn²⁺, Co²⁺, and Zn²⁺ ions.     

Blockade of the delayed rectifier K+ currents, I Kr, in rabbit sinoatrial node cells by external divalent cations

We investigated the actions of various divalent cations on the delayed rectifier K⁺ currents (I _Kr) in rabbit sinoatrial node cells using the whole-cell voltage-clamp technique in isotonic K⁺ solutions. External divalent cations decreased the amplitude of currents, accelerated the time course of deactivation and shifted the activation to positive potentials in a dose-dependent manner. The concentrations for half-maximum inhibition of the steady-state currents (K _M) obtained at 0 mV were 0.63, 1.36, 1.65 and 2.16 mM for Ni²⁺, Co²⁺, Mn²⁺ and Ba²⁺, respectively. The effect was voltage dependent (K _M decreased e-fold for 12.2–16.8 mV hyperpolarization), but the dependence did not vary significantly among different cations. Acceleration of the time course of current deactivation by the increase of divalent cation concentration was well fitted by the voltage-dependent block model, and the binding rate constant (k ₁) was obtained. The binding rates for the ions took the following order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. The degree of the shift of activation occurred in the same order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. From these results, we conclude that I _Kr channels are non-selectively blocked by most divalent cations from the external side and that the binding site is located deep inside the channel, resulting in a steep voltage dependence of the blockade. Received: 26 January 1999 / Received after revision: 16 March 1999 / Accepted: 18 March 1999     

Ionophore A23187 can mimick the changes in membrane permeability that occur during acetylcholine-stimulation of pancreatic acinar secretion

Acetylcholine (ACh) released from vagal terminals increases the permeability of the pancreatic acinar membrane to Na⁺ and Ca²⁺ ions. In this report, we compare the induced changes in intracellular Na⁺ and Ca²⁺ electrode potentials (E_Na and E_Ca) due to ACh-stimulation of acini with those observed during stimulation with the calcium ionophore, A23187, which mimicks the action of ACh on pancreatic secretion. Stimulation with ACh concentrations varying from 10^–8 to 10^–5 M and with A23187 concentrations of 10^–6 and 10^–5 M caused parallel increases in cytosolic Ca²⁺ and Na⁺ ([Ca]_i, [Na]_i). The magnitude of the increases in [Ca]_i and [Na]_i due to A23187-stimulation further indicate that when presented with a calcium challenge the acinar cells continue to regulate [Ca]_i close to physiological levels and suggest that the observed increases in ionized calcium could reflect much larger increases in complexed Ca²⁺. ACh-stimulation following removal of either extracellular Na⁺ or Ca²⁺ ions, eliminated the intracellular increases found when the removed ions is present, but did not affect the increases usually found with the other ion. The independence of the permeability changes to either the presence of Ca²⁺ or Na⁺ indicates the ACh-induced currents carried by Na⁺ and Ca²⁺ are also independent. The selective translocation of Na⁺ and Ca²⁺ during acetylcholine-stimulation in a manner analogous to the changes observed when ionophore A23187 was used as stimulus, indicates the ability of the activated acinar membrane to function as an ionophore.     

Purification,characterization, and biological cytotoxic activity of the extracellular cholesterol oxidase produced by Castellaniella sp. COX

A new bacterial strain producing extracellular cholesterol oxidase (ChOx) was isolated and identified as Castellaniella sp. COX. The ChOx was purified by salting-out and ion-exchange chromatography up to 10.4-fold, with a specific activity of 15 U/mg with a molecular mass of 59 kDa. The purified ChOx exhibited pH 8.0 and temperature 40°C for its optimum activity. The enzyme showed stability over a wide pH range and was most stable at pH value 7.0, and at pH 8.0, it retained almost 86% of its initial activity after 3 h of incubation at 37°C. The enzyme possessed a half-life of 8 h at 37°C, 7 h at 40°C, and 3 h at 50°C. A Lineweaver–Burk plot was calibrated to determine its K_m (0.16 mM) and V_max (18.7 μmol·mg⁻¹·min⁻¹). The ChOx activity was enhanced with Ca²⁺, Mg²⁺, and Mn²⁺ while it was inhibited by Hg²⁺, Ba²⁺, Fe²⁺, Cu²⁺, and Zn²⁺ ions. Organic solvents like acetone, n-butanol, toluene, dimethyl sulfoxide, chloroform, benzene, and methanol were well tolerated by the enzyme while iso-propanol and ethanol were found to enhance the activity of purified ChOx. ChOx induced cytotoxicity with an IC₅₀ value of 1.78 and 1.88 U/ml against human RD and U87MG established cell lines, respectively, while broadly sparing the normal human cells.