Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca²⁺-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca²⁺-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr²⁺ and Ba²⁺, with ionic radii larger than the ionic radius of Ca²⁺, can bind to Ca²⁺-free ACF I (apo-ACF I), while Mg²⁺, with an ionic radius smaller than that of Ca²⁺, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr²⁺-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca²⁺–ACF I, while Ba²⁺-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca²⁺–ACF I. Mg²⁺ and Sr²⁺, with ionic radii close to that of Ca²⁺, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba²⁺, with a much larger ionic radius than Ca²⁺, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg²⁺, Ca²⁺, and Sr²⁺ ions to FXa may be essential for the recognition between FXa and ACF I.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

Ionic interactions of Ba2+ blockades in the MthK K+ channel

The movement and interaction of multiple ions passing through in single file underlie various fundamental K⁺ channel properties, from the effective conduction of K⁺ ions to channel blockade by Ba²⁺ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba²⁺ with permeant ions within the ion conduction pathway of the MthK K⁺ channel. We found that, as typical of K⁺ channels, the MthK channel was blocked by Ba²⁺ at the internal side, and the Ba²⁺-blocking effect was enhanced by external K⁺. We also obtained crystal structures of the MthK K⁺ channel pore in both Ba²⁺–Na⁺ and Ba²⁺–K⁺ environments. In the Ba²⁺–Na⁺ environment, we found that a single Ba²⁺ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba²⁺–K⁺ environment, Ba²⁺ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba²⁺ blockade of K⁺ channels.     

Net Mg2+ uptake in relation to the amount of exchangeable Mg2+ in the Donnan free space of ryegrass roots

Ammonium acetate and BaCl₂-triethanolamine were used to desorb Mg²⁺ from the root Donnan free space (DFS) of 23-d-old ryegrass (Lolium multiflorum Lam. cvs. Gulf and Wilo). Amounts of desorbed Mg²⁺ increased with the increase in Mg²⁺ activity of the nutrient solution. Slightly less Mg²⁺ was desorbed by Ba²⁺ than by NH₄ ⁺. Previously published data on short-term net Mg²⁺ uptake by intact 23-d-old ryegrass plants of the two cultivars were linearly related to the amount of exchangeable Mg⁺ desorbed from the root DFS (r²=0.90 and 0.81 for the desorption by NH₄ ⁺ and Ba²⁺, respectively). A sward of Mg²⁺ ions attracted to the negative charges of the cell surface is suggested to represent a part of a pool of Mg²⁺ available for active transport through the plasmalemma.     

Molecular dynamics simulation study for ion mobility of alkali earth metal cations in water at 25°C

We present the results of molecular dynamics simulations for alkali earth metal cations (Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺) in an aqueous solution at 25°C using the extended simple point charge water potential with Ewald summation. The ion mobilities (defined by u_i = D_iz_iF/RT) obtained from the simulations are in good accord with the experimental measures. The strong divalent cation–water interactions explain well the static and dynamic properties of the alkali earth metal cations. The classical solvent-berg picture describes the ion mobilities of these cations in water adequately.     

Chemiluminescence behaviour of alkaline earth metal ions in the potassium permanganate–fluorescein reaction

A chemiluminescence (CL) signal was observed when alkaline earth metal ion solution, Mg²⁺ or Ca²⁺ or Ba²⁺, was injected into a reaction mixture of fluorescein and potassium permanganate. A possible CL mechanism is proposed based upon the CL, fluorescence and UV‐visible spectra. Furthermore, the feasibility of the application of these reactions to the analysis of these alkaline earth metal ions was evaluated and the analytical parameters of the methods were determined. Copyright © 2003 John Wiley & Sons, Ltd.     

A highly selective fluorescence and absorption sensor for rapid recognition and detection of Cu2+ ions in aqueous solution and film

A fluorescence and absorption chemosensor (SAAT) based on 5-(hydroxymethyl)-salicylaldehyde (SA) and o-aminothiophenol (AT) was designed and synthesized. SAAT in DMSO–HEPES (20.0 mM, v/v, 1:99, pH = 7.0) solution shows a highly selective and sensitive absorption and an ‘on–off’ fluorescence response to Cu²⁺ ions in aqueous solutions over all other competitive metal ions including Na⁺, Ag⁺, Ba²⁺, Ca²⁺, Cd²⁺, Mg²⁺, Zn²⁺, Cr³⁺, Al³⁺, Hg²⁺, K⁺, Mn²⁺, Ni²⁺, Sr²⁺, Tb³⁺ and Co²⁺. SAAT exhibits ratiometric absorption sensing ability for Cu²⁺ ions. Importantly, SAAT also can sense Cu²⁺ ions using fluorescence quenching, the fluorescence intensity of SAAT showed a good linear relationship with Cu²⁺ concentration, and the detection limit of Cu²⁺ was 0.34 μM. The results of Job's plot, Benesi–Hildebrand plot, mass spectra, and density functional theory calculations confirmed that the selective absorption and fluorescence response were attributed to the formation of a 1:1 complex between SAAT and Cu²⁺. SAAT in test film could identify Cu²⁺ in water samples using the intuitive fluorescence colour change under a UV lamp. SAAT has great application value as a selective and sensitive chemosensor to discriminate and detect Cu²⁺ ions.     

Effects of Ketamine on the Balance of Ions Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>, Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> in the Ischemia-reperfusion Affected Spinal Cord Tissues in Rabbits

To test the effects of ketamine on metal ion balance in the spinal cord tissues after ischemic reperfusion (I/R), 24 white adult Japanese rabbits were randomly assigned to sham operation group, I/R group or ketamine-treated I/R group. Spinal cord injuries in I/R group and ketamine-treated I/R group were induced by aortic occlusions. Rabbits in ketamine-treated I/R group were intravenously infused 10 mg/kg ketamine twice: once at 10 min before aortic clamping and once at the onset of reperfusion. Post-operative neurological functions and concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ in the spinal cord were assessed. Compared with the sham operation group, rabbits in the I/R group showed significantly worsened neurological functions as scored with the modified Tarlov criteria and altered concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺. These unfavorable changes were significantly reversed in the ketamine-treated I/R group, suggesting that the potent protective effects of ketamine against the I/R-induced spinal cord injuries may be due to its ability to maintain ion balance in the I/R affected tissues.     

Types of interaction of meso‐tetraphenylporphyrin with alkali and alkaline‐earth metal ions

The cavity in a porphyrin can accommodate metal ions through electron donor–acceptor (EDA) interaction in acetonitrile media without any specially designed fabrication with the porphyrin subunit. Alkali metal ion forms a complex with meso‐tetraphenylporphyrin (TP) in 2:1 stoichiometry, while the bivalent Mg²⁺ ion follows a 1:1 stoichiometry. A fluorescence interaction study indicated that TP can behave like a chemosensor for these ions present in the blood electrolytes. Specifically, for the alkali metal ions intensity‐based sensing was observed, due to inhibition of photoinduced electron transfer (PET), entailing enhancement of fluorescence intensity, and for the alkaline‐earth Mg²⁺ a mixed quenching was observed. Na⁺ and K⁺ ions can be differentiated depending upon the extent of fluorescence enhancement. Copyright © 2011 John Wiley & Sons, Ltd.     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Competitive Binding of Mg2+ and Na+ Ions to Nucleic Acids: From Helices to Tertiary Structures

Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg²⁺/Na⁺ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg²⁺/Na⁺ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg²⁺/Na⁺ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg²⁺ over Na⁺ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg²⁺/Na⁺ concentrations. Furthermore, the local binding of Mg²⁺/Na⁺ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg²⁺/Na⁺ concentrations.     

Ionic selectivity in L-type calcium channels by electrostatics and hard-core repulsion

A physical model of selective “ion binding” in the L-type calcium channel is constructed, and consequences of the model are compared with experimental data. This reduced model treats only ions and the carboxylate oxygens of the EEEE locus explicitly and restricts interactions to hard-core repulsion and ion–ion and ion–dielectric electrostatic forces. The structural atoms provide a flexible environment for passing cations, thus resulting in a self-organized induced-fit model of the selectivity filter. Experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed using equilibrium Monte Carlo simulations in the grand canonical ensemble. The model pore rejects alkali metal ions in the presence of biological concentrations of Ca²⁺ and predicts the blockade of alkali metal ion currents by micromolar Ca²⁺. Conductance patterns observed in varied mixtures containing Na⁺ and Li⁺, or Ba²⁺ and Ca²⁺, are predicted. Ca²⁺ is substantially more potent in blocking Na⁺ current than Ba²⁺. In apparent contrast to experiments using buffered Ca²⁺ solutions, the predicted potency of Ca²⁺ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with Ca²⁺ for the pore. These experiments depend on the problematic estimation of Ca²⁺ activity in solutions buffered for Ca²⁺ and pH in a varying background of bulk salt. Simulations of Ca²⁺ distribution with the model pore bathed in solutions containing a varied amount of Li⁺ reveal a “barrier and well” pattern. The entry/exit barrier for Ca²⁺ is strongly modulated by the Li⁺ concentration of the bath, suggesting a physical explanation for observed kinetic phenomena. Our simulations show that the selectivity of L-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. The reduced system selects for the cation that delivers the largest charge in the smallest ion volume.     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca²⁺] and [Cd²⁺], or [Ca²⁺] and [Ba²⁺], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba²⁺] was added to a B cell line that had been stimulated with anti-immunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca²⁺], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba²⁺. The conductance for Ba²⁺ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca²⁺] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba²⁺.     

Control of insulin receptor affinity by a Ca2+-sensitive binding site

Calcium (Ca²⁺) increased insulin-receptor binding in both membrane and solubilised receptor preparations. Ca²⁺ increased both receptor affinity and initial rate of association of [¹²⁵I]insulin to the receptor preparations. Ca²⁺ had no effect on insulin receptor number in either receptor preparation. The effect of Ca²⁺ on affinity could be mimicked by ions with similar ionic radii and properties (e.g., Ba²⁺, Mg²⁺ and Sr²⁺). EDTA and oleic acid reduced insulin binding and receptor affinity and these effects were reversed by the addition of Ca²⁺. These studies suggest that Ca²⁺ and Ca²⁺-like ions may bind to a site on or near the receptor and may be responsible for a conformational change with a consequent increase in receptor affinity.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Ionophorous properties of the 13 000-Da fragment from sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

The 25 000-Da tryptic fragment from rabbit muscle sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase was subjected to cyanogen bromide digestion, and the four fragments isolated. Only the 13 000-Da fragment induced ionophorous activity in planar thin lipid membranes made with 5:1 (w/w) phosphatidylcholine/cholesterol in decane. The membranes became cation selective, with a selectivity sequence among divalent of Mn²⁺ > Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺. This is different from that of the 25 000-Da fragment (A.E. Shamoo, 1978, J. Memb. Biol. 43, 227–242), it's ‘parent’ 55 000-Da fragment, and the intact enzyme, all of which have the same selectivity sequence. The inhibitory effects of Hg²⁺, Cd²⁺ and Zn²⁺ were also examined. All were inhibitory, with Zn²⁺ being the most effective of these. The heavy-metal-induced inhibition of Ca²⁺ conductance could be reversed by selective chelation of the heavy metals by EDTA. From changes in the selectivity as well as changes in heavy-metal-induced inhibition behavior, we conclude that the ion transport site of the 13 000-Da fragment may not be the same site as that of the parent fragment. It is either a different site altogether or has been physically modified by peptide cleavage.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Biological processes involved in the cycling of elements between soil or sediments and the aqueous environment

The biochemical basis for resistance to toxicity is complicated by the great variety of reactions at the molecular and cellular levels even in closely related organisms and tissues. Several strategies for resistance to intoxication have been identified. Metal ion interactions in biology can be divided into three classes representing fast, intermediate and slow exchange with biological ligands. Examples of those elements in fast exchange include the alkali metals Na⁺ and K⁺, the alkali earth metals Ca²⁺ and Mg²⁺, and, of course, H⁺. Those which can sometimes be in intermediary exchange are Fe²⁺ and Mn²⁺. Examples of those in slow exchange are generally in the active sites of metalloenzymes, e.g., Fe³⁺, Zn²⁺, Ni²⁺, Cu²⁺. In the presented paper, the cycling of one essential element (nickel) and one non-essential element (mercury) are reviewed with special emphasis on their mobilities in the event of in situ sediment contamination.