The effects of magnesium, calcium, EDTA, and pH on the in vitro adhesion of Staphylococcus epidermidis to plastic.

The effects of increasing concentrations of magnesium (Mg2+), calcium (Ca2+) or EDTA, and pH on the adhesion of five slime-positive strains of Staphylococcus epidermidis (Se+) to plastic were examined using an in vitro microwell assay. The addition of Mg2+ (as either MgSO4 or MgCl2) to the bacterial suspension in concentrations as low as 16 microM significantly enhanced the adhesion of all test strains to plastic (P < 0.001). Similarly, the addition of Ca2+ (as CaCl2) in concentrations exceeding 128 microM produced a significant increase in the adhesion of all test strains, but not to the extent observed with Mg2+. In contrast, the adhesion of all test strains to plastic was significantly reduced in the presence of EDTA at concentrations greater than 8 mM. However, EDTA in concentrations as low as 0.25 mM caused a significant decrease in the adhesion of two strains of Se+. The effect of pH was variable, but at a pH of 5.0 and 6.0, the adhesion of all test strains was significantly reduced compared to control values at a pH of 7.0. Two strains showed a significant increase in adhesion at a pH of 8.0. We also compared the effects of these variables on the adherence of a slime-negative phase variant derived from a slime-positive parent strain. With the exception of pH, the adhesion of both strains in response to increasing divalent cations or EDTA was similar. These data indicate that, in addition to hydrophobic interactions, ligand-specific binding, and slime production, pH and divalent cations, especially Mg2+, are important determinants of the adhesion of S. epidermidis to plastic surfaces in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of Ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

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Comparison of three methods detection of slime production by Staphylococcus aureus and Staphylococcus epidermidis

In this article, slime production of Staphylococcus aureus and Staphylococcus epidermidis strains from infective skin lesions was evaluated by three different methods: Congo red agar method (CRA), Christensen tube method (CT) and spectrophotometric method (SC). All strains by CT method interpreted as negative (dark-claret or red colonies of the surface). 12 (37.5%) strains of S. aureus, 16 (50.0%) strains of S. epidermidis produced slime as shown by CT method, 6 (18.7%) strains of S. aureus, 8 (25,0%) strains of S. epidermidis by SC method. They also found a correlation of slime production by CT and SC method (p > 0.05).     

Slime-producing Staphylococcus epidermidis and S. aureus in acute bacterial conjunctivitis in soft contact lens wearers

In recent years, an increase in ocular pathologies related to soft contact lens has been observed. The most common infectious agents were Staphylococcus spp. Some strains produce an extracellular polysaccharidic slime that can cause severe infections. Polysaccharide synthesis is under genetic control and involves a specific intercellular adhesion (ica) locus, in particular, icaA and icaD genes. Conjunctival swabs from 97 patients with presumably bacterial bilateral conjunctivitis, wearers of soft contact lenses were examined. We determined the ability of staphylococci to produce slime, relating it to the presence of icaA and icaD genes. We also investigated the antibiotic susceptibility and Pulsed Field Gel Electrophoresis (PFGE) patterns of the clinical isolates. We found that 74.1% of the S. epidermidis strains and 61.1% of the S. aureus strains isolated were slime producers and showed icaA and icaD genes. Both S. epidermidis and S. aureus slime-producing strains exhibited more surface hydrophobicity than non-producing slime strains. The PFGE patterns overlapped in S. epidermidis strains with high hydrophobicity. The similar PFGE patterns were not related to biofilm production. We found scarce matching among the Staphylococcus spp. studied, slime production, surface hydrophobicity and antibiotic susceptibility.     

Slime production by Staphylococci isolated from prosthesis-associated infections.

Clinical isolates of Staphylococcus epidermidis are frequently referred to produce a biofilm, known as slime, involved in adherence to medical devices and in resistance to host defences. A high frequency of slime producing Staphylococcus aureus strains was never reported, at least in the case of human isolates. In the present study the production of slime by clinical isolates of S. aureus and S. epidermidis from catheter associated infections and from post-surgical infections was studied by a sensitive method based on culturing the isolates on Congo red agar. The study demonstrates that in nosocomial surgical infections, considered separately from catheter-associated infections, S. aureus emerges as a more prevalent etiologic agent than S. epidermidis, with a proportion of slime producing strains markedly high.     

Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase.

Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase), the first prokaryotic UDP-GT and a key enzyme in the biosynthesis of the antibiotic blasticidin S, was purified 870-fold. It has optimum activity at a pH of 8.4 to 8.6, Kms of 6.0 (UDP-glucuronic acid) and 243 (cytosine) microM, and a maximum rate of metabolism of 14.6 mumol/min/mg. The apparent M(r) is 43,000. Activity was slightly enhanced by Mg2+ or Ca2+ but was not inhibited by EDTA. Activity was strongly inhibited by UDP. Cytosylglucuronic acid differs from eukaryotic UDP-glucuronosyltransferases in being a soluble protein with no apparent phospholipid requirement.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes

To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.     

CD11b is a calcium-dependent epitope in human neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.     

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.     

Extracellular Mg2+ induces an intracellular Ca2+ wave during oocyte activation in the marine shrimp Sicyonia ingentis.

In contrast to most systems in which oocyte activation is triggered by the fertilizing sperm, Sicyonia ingentis oocytes are activated by seawater Mg2+ during spawning. S. ingentis oocytes were spawned into Mg(2+)-free seawater and microinjected with the fluorescent Ca2+ indicator Fluo-3 to study the effects of added Mg2+ on intracellular Ca2+ levels. The Mg2+ induced a wave of fluorescence across the oocyte that traveled at a speed of 13 +/- 3 microns/sec. Extracellular Ca2+ was not required for induction of the wave. Treatment with Ca2+ ionophore in Mg(2+)-free medium or a localized injection (0.3% oocyte volume) of 3-5 microM Ca2+ also initiated the wave; injection of 250 mM Mg2+ (up to 1.5% oocyte volume) had no effect. Microinjection of 750 microM EGTA (final) suppressed the Mg(2+)-induced wave, while an identical concentration of EDTA had no inhibitory effect. Subsequent to the initial Mg(2+)-induced intracellular Ca2+ increase, a second Ca2+ increase was observed at approximately 15 min postspawning; the timing of this second increase appeared to be independent of when the Mg(2+)-induced wave was initiated, thus an event associated with spawning may be involved. While oocytes in normal seawater were monospermic, those in Mg(2+)-free seawater were polyspermic, suggesting a role for the Mg(2+)-induced Ca2+ wave in regulating sperm entry into the oocyte.     

Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.     

A specific role for Ca2+ in the oxidation of exogenous NADH by Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.     

Ca2+-sensitivity of actomyosin ATPase purified from Physarum polycephalum.

A contractile protein closely resembling natural actomyosin (myosin B) of rabbit skeletal muscle was extracted from plasmodia of the slime mold, Physarum polycephalum, by protecting the SH-groups with beta-mercaptoethanol or dithiothreitol. Superprecipitation of the protein induced by Mg2+-ATP at low ionic strength was observed only in the presence of very low concentrations of free Ca2+ ions, and the Mg2+-ATPase [EC 3.6.1.3] reaction was activated 2- to 6-fold by 1 muM of free Ca2+ ions. Crude myosin and actin fractions were separated by centrifuging plasmodium myosin B in the presence of Mg2+-PPi at high ionic strength. The crude myosin showed both EDTA- and Ca2+-activated ATPase activities. The Mg2+-ATPase activity of crude myosin from plasmodia was markedly activated by the addition of pure F-actin from rabbit skeletal muscle. Addition of the F-action-regulatory protein complex prepared from rabbit skeletal muscle as well as the actin fraction of plasmodium caused the same degree of activation as the addition of pure F-actin only in the presence of very low concentrations of Ca2+ ion     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus

Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.     

The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity

NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.     

Characterization of Mg2+-ATPase from slow-twitch muscle membranes

SR vesicles from rabbit slow-twitch muscle reveal high activity (0.7-0.9 mumol/mg X min) of "basic" or Mg2+-ATPase. This enzyme differs in its biochemical properties from the well characterized Ca2+ pump ATPase. It is active in millimolar concentration of magnesium or calcium. The activity is inhibited by various detergents except for digitonin. This enzyme seems to be an integral membrane protein since it remains in the membrane after removal of peripheral proteins with EDTA. It can be partially solubilized from the membrane using digitonin without a decrease in specific activity. Ion exchange chromatography on DEAE-Sephacel of the post digitonin supernatant allows us to obtain a 5-fold increase in Mg2+-ATPase specific activity concomitantly with the enrichment in two proteins of Mr = 30,000 and 150,000.     

Calcium uptake in skeletal muscle mitochondria. I. The effects of chelating agents on the mitochondria from fatigued rats.

Female Wistar rats were used to determine the effects of the chelating agents, EDTA and EGTA, on the in vitro 45Ca2+ accumulation by mitochondria isolated from the skeletal muscle of fatigued animals. The rats were divided into three groups: sedentary-rested (SR), trained-rested (TR), trained-exhausted (TE). The trained groups were exercised on a treadmill for 1 h daily, five times a week, for 22 weeks. At the conclusion of the training program, the TE group was rapidly exercised to exhaustion immediately following their daily 1-h run. In the TR group EDTA reduced 45Ca2+ binding while both EDTA and EGTA appeared to increase mitochondrial Ca2+ and Mg2+ content. In the TE group, EDTA reduced endogenous mitochondrial Ca2+ and Mg2+ content, while both EDTA and EGTA increased 45Ca2+ binding. Since chelating Ca2+ and Mg2+ from the membrane may affect the structure and function of the mitochondria, it is suggested that the use of chelating agents during the isolation of mitochondria from the skeletal muscle of trained rats be viewed with caution.