Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Loosening and unfolding of E. coli 50 S ribosomal subunits: Dependence on magnesium content and temperature

Reversible change of 50 S ribosomal subunits to 40 S particles takes place in cold buffered 0.5 M NH₄Cl solutions either containing Mg⁺⁺ (up to 0.1 M), or free from Mg⁺⁺ and even supplemented with EDTA (1 mM). The 40 S particles were stable only within a definite temperature range. Heating of the samples caused completely irreversible unflding of the 40 S particles. This melting appeared to be co-operative and took place within a very narrow range of temperature, which for samples containing Mg⁺⁺ was a linear function of the log of Mg⁺⁺ concentration.The results suggest that two types of bonds maintained the compact structure of the ribosomal subunits: ionic bonds involving Mg⁺⁺ and heat-labile weak interactions between ribosomal components.     

3H-mepyramine binding to histamine H1-receptors in guinea pig lung: Characteristics and regulation by ions

The antogonist [³H]-mepyramine is used to label histamine H₁-receptors in guinea pig lung. Scatchard analysis reveals two classes of binding sites. Monovalent cations decrease steady-state binding (Na⁺ > Li⁺ > K⁺), while divalent cations (Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, Ba⁺⁺) exhibit a biphasic curve, increasing binding at low concentrations and decreasing it at higher levels. Na⁺ decreases both affinity and number of binding sites. Dissociation curve shows two components, and Na⁺ accelerates the rate of dissociation of the slower component. GTP does not affect the binding of the antagonist ³H-Mepyramine.     

Ultrastructural and histological study of degenerative changes leading to black gills in grass shrimp exposed to a dithiocarbamate biocide

The presence of a carbohydrate-specific opsonin, distinguishable from hemolymph agglutinin, was demonstrated in the American lobster, Homarus americanus. Hemolymph opsonin, measured by the enhancement of hemocyte phagocytosis of sheep erythrocytes, was found to be more heat and acid labile than the agglutinin. Both opsonization and agglutination of sheep erythrocytes were inhibited by monosaccharides; however, maximal effects on opsonization were observed with d-(+)mannose, which did not affect agglutination. On the other hand, N-acetyl-d-glucosamine significantly inhibited agglutination but had little effect on opsonization. Fractionation of the molecules was accomplished by differential adsorption to Sephadex G-200 using a 0.15 m NaCl buffer. Hemolymph recovered in the column effluent was enriched for opsonic activity and devoid of agglutinin. However, both opsonin and agglutinin could be detected in the effluent when the column was equilibrated with a 0.48 m NaCl buffer. Neither agglutinin nor opsonin were able to pass an ultrafiltration membrane capable of retaining molecules greater than 3 × 10⁵ daltons.     

Partial myeloperoxidase deficiency in preleukemia

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.     

Dielectric properties of polyelectrolytes. III. Effect of divalent cations on dielectric increment of polyacids

Dielectric dispersion of polyacrylic acid (PAA) and polystyrene sulfonic acid (PSS) was measured in the presence of divalent cations. Effects of divalent ions were studied by neutralization with varying ratios of sodium hydroxide and divalent base concentration, addition of salts of divalent cations, and neutralization with divalent bases only. Two dispersion regions were observed in all cases, i.e., low-frequency dispersion (10²–10⁴ Hz) and high-frequency dispersion (10⁵–10⁶ Hz). The dielectric increment increases in the presence of sodium and alkaline earth metal ions together, but not with sodium and transition metal ions. This is due to the increment of low-frequency dispersion and is attributable to the fluctuation of bound counterions which is explained by our theory previously reported.¹ In the case of PAA neutralized with large fractions of divalent ions, or with divalent ions only, the increment is very small because of reduction of the fluctuation by interaction between bound ions at the neighboring sites and reduction of the effective length of polyion probably due to chelation by divalent ions. There are some differences among the effects of Mg⁺⁺, Ca⁺⁺, and Ba⁺⁺ on dielectric increment which may result from affinity or chelating ability of these ions.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

Calcium as a possible modulator of Kupffer cell phagocytic function by regulating liver-specific opsonic activity

Recently we described some properties of organ-specific serum opsonins which differentiate between liver- and spleen-specific opsonic activities, and reported that, on dialysis of serum, its liver opsonic activity is enhanced by 2- to 3-fold, whereas spleen-specific activity is reduced by 20-30% of that of control serum (Moghimi, S.M. and Patel, H.M. (1989) Biochim. Biophys. Acta 984, 379-383). This observation suggests that serum contains dialysable factors which regulate liver- as well as spleen-specific opsonic activities. Our results from EGTA-treated serum suggest that dialysable factor(s) could be divalent cations such as Ca2+, Mn2+, Mg2+ or Co2+, and among them, calcium may be the key regulatory factor for liver-specific opsonic activity. The regulatory mechanism of spleen-specific opsonic activity seems to be complex, since addition of dialysate or calcium or magnesium to the dialysed serum does not restore its activity; probably the removal of divalent cations has induced an irreversible conformational change in spleen-specific opsonin. In conclusion, we propose that the blood calcium concentration may play an important role in modulating hepatic phagocytic function by modifying liver-specific opsonic activity in serum. An increase in the physiological concentration of calcium will suppress and a decrease will enhance this opsonic activity.     

The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe

Summary The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca⁺⁺ and Mg⁺⁺. The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca⁺⁺ in coupled mitochondria and Mg⁺⁺ in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn⁺⁺ indicate that the probe is primarily visualizing actively accumulated Ca⁺⁺ on the inner surface of the inner membrane. By employing the Ca⁺⁺ transport inhibitor, Tb⁺⁺⁺, the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.     

The response to oxidative stress induced by magnesium deficiency in kidney bean plants

To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg⁺⁺) deficiency on the formation of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg⁺⁺ deficiency also was examined via two-dimensional electrophoresis. The formation of H₂O₂ and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg⁺⁺. Protease activity in Mg⁺⁺-deficient plants was also higher than in those grown with sufficient Mg⁺⁺. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg⁺⁺-deficient conditions. Therefore, a deficiency in Mg⁺⁺ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg⁺⁺ deficiency.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

The effect of multivalent cations on the membrane potential of the Ehrlich ascites tumor cell

The effect of di- and trivalent cations on the membrane potential of the Ehrlich ascites tumor cell has been investigated using micro-electrode techniques. In solutions free of multivalent cations the average membrane potential for 46 cells was 8.3 ± 0.5 mv (SE). However, the potentials were not stable and decayed with a half-time of about six seconds. Addition of Ca⁺⁺ decreased the rate of decay and concomitantly increased the membrane potential. The magnitude of these effects was a function of the Ca⁺⁺ concentration. At the optimum concentration (2 mM ), the half-time of decay was increased to 12 seconds and the membrane potential was raised to 17.8 ± 1.7 mv (SE). The related alkaline-earth cations, Sr⁺⁺, Ba⁺⁺ and Mg⁺⁺ had similar effects on both the stability and magnitude of the membrane potential. The effect of La⁺⁺⁺, which was qualitatively similar to that of the divalent cations, was also concentration dependent. However, 100-fold lower concentrations were adequate to achieve comparable effects. Moreover, membrane potentials were stable for up to ten minutes in La⁺⁺⁺-containing solutions. Variations in intracellular Cl^? content induced by temperature changes were paralleled by changes in membrane potentials. However, the potentials were not those expected for a simple Cl^? electrode.     

Effect of Mg++ and polyamines on proflavine binding to T2 DNA

Proflavine binding may be used as a probe of the environment and interactions of DNA. In this paper we report the effects of the divalent cations Mg⁺⁺ and putrescine and the trivalent cation spermidine on the proflavine–Na DNA binding equilibrium. Difference spectroscopy at 430 nm was used to determine apparent proflavine–DNA binding constants K at several concentrations of each cation for temperatures between 15 and 43°C, and at a constant total ionic strength of 0.1M. Mg⁺⁺, putrescine, and spermidine all have greater effects on K than expected on the basis of ionic strength alone in the order spermidine > Mg⁺⁺ ? putrescine. van't Hoff analysis of K(T) enabled calculation of ΔH° and ΔS°, which are affected differently by each cation. These differences are discussed qualitatively in terms of such concepts as release of condensed counterions, localized or unlocalized condensation, hydration, and restriction of molecular and internal rotation.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

Role of divalent cations in plasma opsonin-dependent and-independent erythrophagocytosis by hemocytes of the Asian clam,Corbicula fluminea

The in vitro phagocytosis-promoting properties of hemolymph from the freshwater clam, Corbicula fluminea, are described. Hemocytes were capable of phagocytosing aldehyde-fixed erythrocytes (RBCs) of seven vertebrate species with equal facility, but only in the presence of homologous clam plasma. The plasma factors mediating erythrophagocytosis were heat sensitive. Pretreatment (opsonizing) of target RBCs with plasma also resulted in enhancement of hemocyte particle uptake in the absence of plasma. Opsonin-dependent phagocytosis required the presence of divalent cations, especially calcium, although not in free ionic form. Evidence suggests that the plasma opsonin may normally exist as a divalent cation-macromolecular complex since opsonizing activity was retained after dialysis against Tris-buffered saline (TBS), but was lost following TBS/EDTA or TBS/EGTA dialysis. We also have identified an opsonin-independent phagocytosis mechanism in which Corbicula hemocytes are able to ingest nonopsonized RBCs in the absence of homologous plasma. Extracellular calcium or magnesium in the incubation medium is needed for particle uptake, although the direct binding of free ions to the target RBC surface does not appear to be mediating enhanced phagocytosis. From the present data, it is concluded that hemocyte recognition of aldehyde-fixed RBCs can be accomplished by either of two mechanisms: (1) by the coating of cells with plasma factors capable of triggering the phagocytic process (opsonization) or (2) by a plasma opsonin-independent mechanism in which extracellular divalent cations (e.g., Ca²⁺ or Mg²⁺) in the incubation buffer stimulate uptake of nonopsonized RBCs. The factors regulating in vitro erythrophagocytosis by clam hemocytes are considered to be analogous to those involved in nonimmune opsonin-dependent and -independent phagocytosis in mammalian macrophages.     

Binding of cations by microsomes from rabbit skeletal muscle

Fragmented sarcoplasmic reticulum and transverse tubular system, as isolated in the microsomal fraction from rabbit skeletal muscle, bind H⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺. The binding depends on a cation exchange type of interaction between these cations and the chemical components of the membranous systems of the muscle cell. The monovalent and divalent cations exchange quantitatively for each other at the binding sites on an equivalent basis. Scatchard plots of the H⁺ binding data indicate that the binding groups can be resolved into two major components in terms of their pK values. Component 1 has a pK value of 6.6 and a capacity for H⁺ binding of 2.2 meq/g N . The second component has a much higher H⁺ binding capacity (7–8 meq/g N ), but its pK value, 3.4, is non-physiological. The binding of cations other than H⁺ at a neutral pH occurs at the binding sites making up component 1. The order of affinity of the cations for the microsome binding sites is H⁺ » Zn⁺⁺ > Ca⁺⁺ > Mg⁺⁺ » Na⁺ = K⁺ as reflected by the apparent respective pK_M values: 6.6, 5.2, 4.7, 4.2, 1.3, 1.3. Caffeine, which causes contracture and potentiates the twitch of skeletal muscle, does not interfere with the binding of Ca⁺⁺ by the microsomes at neutral pH.     

The penetration of some cations into muscle

The influx of Na⁺, K⁺, Rb⁺, and Cs⁺ into frog sartorius muscle has been followed. The results show that a maximum rate is found for K⁺, while Na⁺ and Cs⁺ penetrate much more slowly. Similar measurements with Ca⁺⁺, Ba⁺⁺, and Ra⁺⁺ show that Ba⁺⁺ penetrates at a rate somewhat greater than that of either Ca⁺⁺ or Ra⁺⁺. All these divalent cations, however, penetrate at rates much slower than do the alkali cations. The results obtained are discussed with reference to a model that has been developed to explain the different penetration rates for the alkali cations.     

Perturbational effects of inorganic cations on human erythrocyte membranes

Summary The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy of incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni⁺⁺, Co⁺⁺ and Mn⁺⁺ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.