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.     

PHOSPHORUS DYNAMICS IN THE MONIMOLIMNION OF SWARTVLEI

SUMMARY

The exchange of phosphorus between the bottom sediment and monimolimnion of Swartvlei, a meromictic, humic lake, was investigated during the last three months of 1980. The concentrations of oxygen, dissolved salts, phosphorus and Fe⁺⁺ in the water column were monitored, and electrode potentials in the bottom mud were measured, at approximately weekly intervals. At the same time laboratory experiments were performed, using Jenkin core samples, to observe the effect of changing oxygen concentration and salinity on phosphate exchange between sediment and water, and on electrode potentials at the sediment-water interface. Phosphorus was released under unaerobic conditions at a rate of 2,5 mg P m^?2 d^?1 and was taken up again under aerobic conditions at 1,6 mg P m^?2 d^?1 These values were in agreement with existing observed data on changes in phosphate concentration.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : II. THE INFLUENCE OF SALTS.

It is shown by the older experiments by Loeb and by the experiments reported in this paper that the effect of salts on the membrane potentials, osmotic pressure, swelling of gelatin chloride, and that type of viscosity which is due to the swelling of protein particles, depends only on the valency but not on the chemical nature of the anion of the salt, and that the cation of the salt has no effect on these properties, if the pH of the protein solution or protein gel is not altered by the salt. The so called Hofmeister series of salt effects on these four properties are purely fictitious and due to the failure of the former authors to measure the hydrogen ion concentration of their protein solutions or gels and to compare the effects of salts at the same pH of the protein solution or the protein gel. These results confirm the older experiments of Loeb and together they furnish a further proof for the correctness of the idea that the influence of electrolytes on the four properties of proteins is determined by membrane equilibria. Such properties of proteins which do not depend on membrane equilibria, such as solubility or cohesion, may be affected not only by the valency but also by the chemical nature of the ions of a salt.     

Extraction of an actomyosin-like pootein from amoebae of Dictyostelium discoideum

An actomyosin-like protein has been extracted from amoebae of Dictyostelium discoideum, V-12. The purified protein exhibited a reversible change in viscosity upon addition of ATP, indicating an ATP sensitivity of 75–85% and a specific viscosity of 0.1. At low ionic strength in the presence of Mg⁺⁺ and ATP the amoeba protein displayed the phenomenon of superprecipitation. The protein extract was found to be an adenosinetriphosphatase (ATP'ase) hydrolyzing ATP to ADP and inorganic phosphate. Both Mg⁺⁺ and Ca⁺⁺ at low ionic strength accelerated the ATP ase activity whereas at high ionic strength only Ca⁺⁺ stimulated ATP hydrolysis. The ATP'ase activity was inhibited by ethylene-diamine-tetraacetic-acid, Mersayl and p-chloromercuribenzoate. The physico-chemical and enzymatic properties of the extracted amoeba protein are qualitatively comparable to those of muscle actomyosin, and very similar in quantitative properties to smooth muscle actomyosin and the actomyosin-like proteins of blood platelets, leucocytes and slime mold plasmodia. The significance of the presence of this actomyosin-like protein in Dictyostelium amoebae is discussed in relation to amoeboid form and movement.     

Lead and some other metals in the histochemical demonstration of rat liver glycogen phosphorylase activity

Summary Lead in a concentration of 0.25 mM was tried as a histochemical trapping agent for inorganic phosphate, liberated in the reversible reaction catalyzed by the liver glycogen phosphorylase.The reaction product, formed during the incubation and made visible with ammonium sulphide, was totally extracted with -amylase. Iodine staining after incubation was completely negative.The inhibitory effect on liver phosphorylase activity of several other metals was also studied histochemically. It was found that the inhibition generally increased with the molecular weight and concentration of the metals. It is concluded that Fe⁺⁺ could be useful as a trapping agent instead of lead.     

The acute and subacute effects of cadmium on calcium homeostasis and bone trace metals in the rat

The effect of acute and subacute administration of cadmium chloride on calcium homeostasis and the trace metal content of the bone was investigated in the male rat. A single subcutaneous injection of cadmium chloride (1.5 mg Cd⁺⁺/kg) produced a decreased plasma concentration of calcium and a decrease in the femur concentration of both calcium and zinc. Repeated administration of cadmium chloride (1.5 mg Cd⁺⁺/kg daily, for 28 days) caused a marked hypocalciuria that persisted throughout the period of cadmium treatment. There was an accompanying increased excretion of alkaline phosphatase into the urine, and plasma inorganic phosphate was also elevated in these animals. Both of these effects are considered to be evidence of kidney damage.A possible mechanism for this cadmium-induced effect may involve a disturbance of the renal biotransformation of vitamin D, and decreased bioavailability of the essential trace metals due to metallothionein synthesis and excessive loss into the urine.     

The kinetics of the renaturation of deoxyribonucleic acid denatured in the presence of copper(II) ions

DNA which has been heat denatured in the presence of Cu⁺⁺ ions can be completely and rapidly renatured by increasing the ionic strength of the solution above a critical value. A kinetic study of this renaturation recation was carried out by following the associated UV absorbance change and also by following the change in free Cu⁺⁺ ion concentration by means of a specific Cu⁺⁺ ion activity electrode. The data obtained could be fitted to first-order kinetics for a considerable extent of the reaction and the rate constant was found to increase with temperature and ionic strength, but to decrease markedly as the bulk viscosity of the solution was increased. At temperatures greater than 5°C the reaction rate depended on the time elapsing between denaturation and the commencement of the renaturation reaction. As there was good agreement between the rate constants obtained by following the decrease in hyperchromism and by following the increase in free Cu⁺⁺ ion concentration, it is concluded that under the conditions employed, the rate of renaturation is determined by the rate of release of Cu⁺⁺ ions from the denatured DNA-Cu⁺⁺ complex.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Salt effects on the cross-linking mechanism of cupric-induced sol—gel transition in alginate solutions

The viscosity behaviour of alginate-Cu²⁺-NaCl systems has been experimentally examined at various concentrations of cupric and sodium salts. Dependence of the intrinsic viscosity of alginate as a function of NaCl concentration is discussed to supplement the previous study which shows a similar behaviour to that found for other polyelectrolytes in aqueous solution in the presence of an added salt. The effects of sodium ions on the cupric association in cupric-induced alginate solutions were investigated by means of viscosity measurements. The mechanisms of complex formation in the presence of the simple added salt were studied. It was found that, at a given NaCl concentration, the viscosity of the mixture will pass through a maximum with increasing cupric concentration. The amounts of cupric cations corresponding to the maximum depends on the concentration of NaCl in the solution. Comparison of salt effects on the viscosity behaviour of alginate solutions during sol—gel transition reveals that an optimum NaCl concentration of 10⁻² mol 1⁻¹ exists where the viscosity of the mixture gives a maximum value at a certain cupric amount. This result indicates that salt effects play an important role in the sol—gel transition of the polyelectrolyte solutions. The observed phenomenon was interpreted in terms of conformational change of polyelectrolyte chain due to the addition of salt resulting in a different cross-linking mode in the system.     

A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster , and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading.
Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.     

Stabilization of ribose-5-phosphate isomerase from tobacco

The highly purified ribose phosphate isomerase from tobaccoleaves is heat labile. 0.2% of various kinds of proteins stabilizedisomerase activity when Mg⁺⁺ was present. 1x10^–3% polyethyleneglycol2,000 showed the same effect as the proteins did. Smaller polyediyleneglycolswere less effective. Polyhydroxyl compounds showed litde effect.Mn⁺⁺ or Sr⁺⁺ was also effective as a stabilizer instead of Mg⁺⁺. (Received March 8, 1976; )     

ELECTRON STAINS : I. Chemical Studies on the Interaction of DNA with Uranyl Salts

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO₂⁺⁺/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.     

Decondensation of sperm nuclei of australian marsupials: Effects of air drying and of calcium and magnesium

Spermatozoa of six species of Australian marsupials have been studied. The nucleus is highly unstable when compared with those of eutherian mammals. When thin films of spermatozoa in buffered saline are air-dried on glass slides, the nucleus disintegrates and flattens, leaving the acrosome, midpiece, and tail intact. This spreading of the nucleus can be inhibited by seminal plasma proteins and by bovine serum albumin, but is potentiated by detergents. The nucleus also decondenses spontaneously in the presence of high concentrations (>0.25M) of calcium and magnesium salts, leaving the head membranes, acrosome, midpiece, and tail intact. This is inhibited by EDTA. In some species, certain areas of the nucleus appear more resistant t o Ca⁺⁺/Mg⁺⁺ treatment, and the initial stages of decondensation are uneven. Ultrastructurally the Ca⁺⁺/Mg⁺⁺ dispersed chromatin shows a moderately fine, branching, fibrillar structure, interspersed with dense granules. Treatment with disulphide bond cleaving agents together with detergents results in rapid and complete dispersal of the chromatin and acrosome, and slow digestion of midpiece and tail structures. Treatment with HCl, NaCl, KCl, EDTA, detergents, and sucrose has no effect on nuclear integrity, but treatment with NaOH (0.9–1.0M) results in complete digestion of the whole sperm. These findings are discussed in the light of evolutionary differences between marsupial and eutherian mammals in terms of sperm structure and composition.     

Lead toxicity and phosphate deficiency in chlamydomonas

The addition of lead salts to phosphate-containing Chlamydomonas reinhardtii media caused precipitation of Pb₃(PO₄)₂, effectively removing phosphate from solution. The effect of Pb²⁺ on growth of Chlamydomonas in liquid cultures depended strictly on the ratio of the equivalents of Pb²⁺ to phosphate present. When the amount of Pb²⁺ approached equivalency with phosphate, cell growth was initially slow as cells adhered to the surface of the precipitated Pb₃(PO₄)₂. Later, cells grew at a normal rate, spread throughout the solution, and reached the same densities obtained in the absence of Pb²⁺. Cells did not survive when the amount of Pb²⁺ in the culture exceeded the equivalents of phosphate.

Elemental analysis showed that in the presence of equivalent Pb²⁺ and phosphate, considerable Pb²⁺ remained in solution. The concentration of dissolved Pb²⁺ did not vary significantly when the amount of Pb²⁺ added to the culture was increased slightly, from an amount which permitted growth to an amount which completely prevented growth. The concentration of phosphate was decreased to an undetectable level when the amount of Pb²⁺ approached equivalency with phosphate.

In the presence of the chelating agent nitrilotriacetic acid, higher concentrations of Pb²⁺ remained in phosphate-containing media. The chelated Pb²⁺ did not retard the growth of Chlamydomonas.

It appears that Pb²⁺ is not toxic to Chlamydomonas, but kills cells by depriving them of phosphate.

     

Prospects of Inducing Resistance in Fodder Species against Toxic Ions and Metals

ABSTRACT

Experiments were carried out with various salts and their combinations to ascertain the impact of these salts on seedling traits of fodder species and to identify tolerant species. Length-based traits showed a repressed effect, whereas weight-based traits were increased under salt stress. Furthermore, accumulation of Na⁺, Ca²⁺, and Cl^? ions and metals (Cu²⁺, Fe²⁺, and Al³⁺) increased in various organs of seedlings due to various salt treatments. Contrastingly, K⁺, K⁺/Na⁺, and Ca²⁺/Cl^? decreased, showing priority for specific salts. Seedling traits, such as shoot length sensitivity and shoot biomass, provide an effective mean of selection for tolerant or susceptible genotypes. Diverse types of tolerance mechanisms were present in cultivars to detoxify the effect of ions and metals. Cultivars that showed low susceptibility index, high shoot biomass, and high metal concentration were salt includers and could be utilized for bioremediation of the affected areas, whereas tolerant cultivars that showed low susceptibility index, metals concentration, and comparable shoot biomass to that of the control were salt excluders and could be utilized for fodder purposes.     

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10^?3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum.The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li⁺, Ca²⁺ and Mg²⁺.The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10^?2 m to 2 m.The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be ?14·6 kcal/mol of O₂.     

Preparation and characterization of milk calcium salts by using casein phosphopeptide

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.     

Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells

A number of metals have been shown to be involved in the etiology of animal and human neoplasms. The molecular mechanisms have not yet been determined, but the observed plethora of genetic effects observed following treatment of mammalian cells with metals clearly indicates the possibility that metals can exert their effects at least partially at the level of DNA metabolism. Several studies have suggested that metal treatment may inhibit normal DNA repair processes in procaryotic and eucaryotic cells but a systematic study of this question has not previously been conducted. The present study surveyed the ability of 15 metal salts to interfere with repair of X-ray or UV-induced DNA damage in HeLa cells. Hg⁺⁺, As⁺⁺⁺, Cu⁺⁺, Ni⁺⁺, Co⁺⁺, and Cd⁺⁺ were shown to inhibit the excision of pyrimidine dimers from DNA and to do so in a dose-dependent fashion. Inhibition of repair by only Ni⁺⁺ and Co⁺⁺ resulted in the accumulation of long-lived DNA strand breaks suggestive of a block in the gap-filling stage of repair. Ability to inhibit repair was not correlated with cytotoxicity. X-ray repair was sensitive to Hg⁺⁺, Ni⁺⁺, As⁺⁺⁺, Ga⁺⁺, Zn⁺⁺, and Mo(VI). All inhibitory metals inhibited closure of single strand DNA breaks. Ga⁺⁺ appeared, in addition, to inhibit a later step involving chromatin reconstitution. These findings support the notion that interference of DNA repair processes may be a consequence of exposure of mammalian cells to certain metals. This may be a factor in the etiology of metal-associated carcinogenesis.     

Anti-juvenile Hormone Effects of an Imidazole Compound (KK-42) in Different Larval Instars of Bombyx mori

The non-Newtonian behavior and dynamic viscoelasticity of solutions of the Ca salt of xanthan were measured with a rheogoniometer. The Ca salt of xanthan showed pseudoplastic behavior < 0.1 % but was plastic >0.3%. Compared with native, Na, and K salts of xanthan, the Ca salt had higher dynamic viscoelasticity at high concentrations. The apparent viscosity of Ca salt of xanthan was very large at low temperatures and decreased with increasing temperature. These suggest very strong intermolecular association of the xanthan (Ca salt) molecules, probably due to the formation of ionic force between adjacent charged trisaccharide side-chains via Ca²⁺ on different molecules. Possible structures for quaternary association including intramolecular association of xanthan molecules (Ca and K salts) in aqueous solution were proposed.     

Studies on an Alkaline Proteinase of Aspergillus sydowi

An alkaline proteinase of Aspergillus sydowi (Bainier et Sartory) Thom et Church has been purified approximately 4.5-fold from a culture filtrate by fractionation with ammonium sulfate, treatment with acrynol and Alumina gel C_γ, and DEAE-Sephadex column chromatography. The purified proteinase obtained as needle crystals was monodisperse in both the ultracentrifuge and the electrophoresis on polyacrylamide gel.

The optimum pH and temperature for the activity were 8.0 and 40°C, respectively. Fifty per cent of the activity was lost at 45°C within ten minutes and 95% at 50°C. At 5°C, the enzyme was highly stable at the range of pH 6 to 9. None of metallic salts tested promoted the activity, but Zn⁺⁺, Ni⁺⁺ and Hg⁺⁺ were found to be inhibitory. Sulfhydryl reagent, reducing and oxidizing reagents tested except iodine had no effect on the activity, but potato inhibitor, DFP and NBS caused a marked inhibition.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca⁺⁺ in the enzyme solution. The protective effect of Ca⁺⁺ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca⁺⁺ for the protective effect at pH 7.1, 7.5 and 7.8 were 10^?2, 10^?3 and 10^?4 M, respectively. Conversely, at higher pH values such as 9.0, Ca⁺⁺ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution.

The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2 m trichloroacetic acid solution, with 14~17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chrysogenum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slightly on casein hydrolyzed by the above-mentioned proteinases.