Release of respiratory control in Escherichia coli after bacteriophage adsorption: process independent of DNA injection.

Adsorption of phages T4, T5, and BF23 to previously starved Escherichia coli cells triggered the immediate release of respiratory control. A similar stimulation of respiration was induced after T4 ghost attachment, showing that this process was independent of the mechanism of DNA injection. Rather, this change in the respiratory rate was related to the transient depolarization of the cytoplasmic membrane also induced after phage and ghost adsorption. Both processes were suppressed by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the case of T4 (phage and ghosts) but not of T5 and BF23. The increase in respiratory rate observed after phage adsorption was of a magnitude similar to that induced by protonophores. Since other treatments that depolarize the membrane without a massive proton influx did not increase the rate of respiration of starved bacteria with the same efficiency, these results suggest that phage adsorption induced an entry of protons into the cell cytoplasm.     

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.     

Involvement of calcium in the transient depolarization of E.coli cytoplasmic membrane induced by phage adsorption: a study with the fluorescent calcium indicator QUIN2

Adsorption of phage T4 to its outer membrane receptor (the lipopolysaccharide) triggers a transient depolarization of the cytoplasmic membrane which is prevented upon addition of EGTA, suggesting that calcium is necessary for the transmission of the signal between the two membranes. Using the fluorescent indicator QUIN2, we show that T4 adsorption triggers the release of envelope-bound calcium, the amount of which increases with the number of infecting phages. Since this amount was the same whether the cells were pretreated or not with EDTA-Tris, this suggests that this calcium originated from the high affinity sites of the lipopolysaccharide.     

Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway.

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.     

Ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibits the growth of Escherichia coli at alkaline pH

The growth of Escherichia coli was inhibited by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in a medium of initial pH 8.8. The growth inhibition was reversed by the addition of CaCl(2). E. coli could grow in the presence of EGTA at pH values below 8. The concentration of free calcium ions increases with a decrease in medium pH because of a decrease in the calcium binding capacity of EGTA. So, although the results suggest that calcium ions are essential for the growth of E. coli, the minimum concentration required is very low.     

Phenothiazine binding by a homolog of calpactin, the pp60src tyrosine kinase substrate

Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium-sensitive protein (AMV-p35) that can be isolated with microvillar microfilament cores prepared by Triton X-100 extraction in the presence but not absence of calcium. AMV-p35 can be readily purified from ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV-p35 is related to calpactin I, the pp60src tyrosine kinase substrate. In the presence of calcium, AMV-p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV-p35 also binds phenothiazine in the absence of calcium.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa

Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.     

Effects of antibiotics and other inhibitors on ATP-dependent protein translocation into membrane vesicles.

The effects of several membrane antibiotics and other agents on ATP-dependent protein translocation were examined in membrane vesicles under conditions where no significant proton motive force was present. The membrane perturbants ethanol and procaine abolished ATP-dependent protein translocation. Phenethyl alcohol at low concentrations abolished translocation, whereas at high concentrations it allowed precursors to be translocated but inhibited their processing. Translocation of precursors promoted by phenethyl alcohol was temperature dependent and occurred without an added energy source but was enhanced by ATP. However, such precursors could not be further processed to mature forms upon removal of the alcohol. The membrane-active antibiotics polymyxin B and gramicidin S were strong inhibitors of translocation, whereas gramicidin D, cerulenin, and mycobacillin had no effect even at higher concentrations, indicating some specificity in interference with protein translocation. Duramycin, an antibiotic previously shown to affect protein-lipid interaction, severely impaired protein translocation. These results showed that membrane structures play important roles, either directly or indirectly, in protein translocation. Chelating agents 1,10-phenanthroline and EDTA, but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], also abolished protein translocation.     

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.     

The inhibition of cyclic AMP-mediated exocytosis from rat parotid cells by calcium chelation is due to depression of cellular ATP

The role of second messenger calcium in cAMP-mediated exocytosis from parotid cell aggregates has been assessed following extracellular (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA] and intracellular (quin2) calcium chelation. Only in the former case was inhibition (60-70%) of isoproterenol- and N6,O2-dibutyryl cAMP-stimulated amylase release observed. This inhibition was accompanied by a 70% decrease in cellular ATP levels. Depression of ATP levels with mitochondrial inhibitors was also correlated with inhibition of amylase secretion. Overall, our results suggest that depletion of cellular Ca2+ with EGTA leads to an inhibition of mitochondrial function and that these experimental conditions are therefore inappropriate for the evaluation of the role of cytosolic Ca2+ transients during stimulus-secretion coupling.     

Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction.

Calcium ions are required for fusion of a wide variety of artificial and biological membranes. To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1. Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy. Syncytia did not form in solutions without calcium ions. Addition of calcium ions partially restored the formation of syncytia. EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] blocked syncytium formation in culture media containing calcium ions. Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions. Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM. Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+. Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+. We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.     

Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

There are many uses for solutions with a known free calcium concentration ([Ca2+]free) in the nanomolar range. Most frequently ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) has been used as a buffer for the control of [Ca2+]free; however, under a variety of conditions the use of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for this purpose would be advantageous. The theory and calculations necessary to make solutions with known [Ca2+]free appropriate for given conditions of pH, ionic strength, and temperature for use with EGTA or BAPTA are reviewed. Practical considerations and methods for making such solutions are detailed. The advantages and disadvantages associated with the use of each of the two chelators are discussed. As one example of the application of solutions with free calcium in the nanomolar range, the dissociation constant of the fluorescent indicator fura-2 for calcium has been determined in a physiologic buffer at 22 and 37 degrees C. For practical reasons, the use of BAPTA is advantageous when solutions with different known [Ca2+]free must be used on a daily basis.     

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca(2+)-induced mitochondrial membrane permeabilization and cell death

The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.     

Depolarization reverses age-related decrease of spontaneous transmitter release.

The effect of increasing extracellular Ca concentration on spontaneous transmitter release was studied at soleus nerve terminals of young (10 mo) and old (24 mo) C57BL/6J mice depolarized by high extracellular K concentration ([K]o). By using intracellular recording, miniature end-plate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solutions with four different Ca concentrations: Ca-free/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 0.5, 1.5, and 2.5 mM Ca. In both the normal [K]o Krebs and the Ca-free-high [K]o Krebs solutions, MEPP frequency was lower at old than at young nerve terminals. In the three high [K]o Krebs solutions with Ca, MEPP frequency was progressively higher at old than at young nerve terminals with higher Ca concentrations. Periodic oscillations were observed in MEPP frequency of depolarized nerve terminals. The period of oscillation was inversely proportional to spontaneous transmitter release. These results demonstrate that when the nerve terminal is depolarized, permeability of the terminal membrane to Ca increases because of opening of voltage-dependent Ca channels. In the present study resting MEPP frequency was lower at old than at young terminals. On depolarization, MEPP frequency became higher at old than at young terminals. The study demonstrates that voltage-dependent Ca entry increases during aging at the soleus nerve terminal.     

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.     

A comparative study of mammalian and reptilian alternative pathway of complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi)

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.     

The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA

The apparent calcium association constants (K'Ca) of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 1,2-bis 2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (dibromo-BAPTA) were measured using the method described by Bers (Am. J. Physiol. 242 (1982) C404-408). The purity of the three ligands determined from the chi-intercept of Scatchard plots were 96.3%, 79.0% and 97.3% for EGTA, BAPTA and dibromo-BAPTA, respectively. The impurity of BAPTA was found to be water by drying several samples to constant weight. Increasing temperature from 1 to 36 degrees C led to an increase in K'Ca which was of similar magnitude for the three ligands. Increasing ionic strength from 0.104 to 0.304 M led to a reduction of K'Ca in all cases, though EGTA was affected much less than BAPTA or dibromo-BAPTA. Experimental results were compared with values of K'Ca calculated from the individual association constants of the ligands for calcium and protons which were modified for the experimental conditions using the Debye-Hückel limiting law and the Van't Hoff Isochore to correct for ionic strength and temperature, respectively. The experimental values of K'Ca of EGTA agree well with those in the literature and with the calculated values. Good agreement was also found between the experimental and calculated values of K'Ca for the temperature and ionic strength dependence of BAPTA and dibromo-BAPTA.     

Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers

During implantation, complex embryo-endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo-endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25 x g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids were attached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200-1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst-EEC adhesion. Spheroid-EEC adhesion was inhibited by blocking Ca(2+) influx with extracellular Ca(2+) chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or a Ca(2+) channel blocker (verapamil) but not by interfering with Ca(2+) release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.