Effect of peptidoglycan preparations on phage inactivation: decrease in phage 187 binding using monosaccharides]

Monosaccharides were found to be capable to decreasing the binding of phage 187 with peptidoglycan. Galactosamine hydrochloride and glucosamine hydrochloride proved to be the most effective, followed in order of decreasing activity by N-acetylgalactosamine, N-acetylglucosamine, glucose and galactose; mannose produced a weak effect. Ca2+ ions increased the reduction of phage peptiglycan binding with N-acetylgalactosamine and N-acetylglucosamine. Both of these monosaccharides also reduced the adsorption of phage 187 by the Staphylococcus aureus cells. In the medium enriched with Ca2+ ions; in the absence of Ca2+, they were, however, incapable of releasing the phage irreversibly bound with the peptidoglycan.     

Characterization of calcium-activated bifunctional peptidase of the psychrotrophic Bacillus cereus

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, 60 degrees C. The endopeptidase activity was stimulated by Ca++, Co++, Mn++, Mg++, and Ni++, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM Ca++, and was partially restored by Co++ and Mn++, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of Ca++ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the Km value of endopeptidase is 0.315 mM and Vmax is 0.222 mmol of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.     

Role of divalent cations in the resumption of meiosis of rat oocytes.

Resumption of meiotic maturation was induced in follicle-enclosed rat-oocytes by treatment with the divalent cationophore A23187 (10(-5)M). However, the same effect was attained by incubation in Ca++-deficient medium, even in the presence of EDTA or EGTA (1mM). The stability of the first polar body was increased under Ca++-deficient conditions. Neither the ionophore nor Ca++-deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

Agonist-induced myopathy at the neuromuscular junction is mediated by calcium

Inactivation of cholinesterases at mammalian neuromuscular junctions (nmj) produces extensive muscle "necrosis." Fine-structurally, this myopathy begins near the nmj with an increase in large-diameter vesicles in the soleplasm, the dissolution of Z-disks, dilation of mitochondria, destruction of sarcoplasmic reticulum, and often a highly specific contracture of the muscle under the endplate. Since a Ca++-activated protease which specifically removes Z-disks is known to exist in mammalian skeletal muscle, we tested the possibility that the myopathy after esterase inactivation is due to the prolongation of acetylcholine lifetime and thus of Ca++ influx. We first produced the myopathy near endplates by inactivating esterases with diisopropylfluorophosphate (DFP) followed by nerve stimulation for 1--2 h in vitro. The myopathy was later mimicked by bath application of carbamylcholine without esterase inhibitors. This myopathy could be prevented by inactivating the acetylcholine receptors (AChR) with alpha-bungarotoxin (alpha-BGT) or by removing Ca++ from the bath with EGTA. These results favor the hypothesis that esterase inhibition leads to an agonist-induced myopathy, which is mediated by Ca++ and requires an intact AChR.     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

Characterization of modular bacteriophage endolysins from Myoviridae phages OBP, 201φ2-1 and PVP-SE1

Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201φ2-1gp229 (Pseudomonas chlororaphis phage 201φ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (K(aff) = 1.26 × 10(6) M(-1)). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90 °C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2-3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201φ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Isolation and functional characterization of rat pancreatic acini.

The Authors describe an optimized procedure for the isolation of rat pancreatic acini and the preliminary results concerning the functional characterization of the cells. Isolation is carried out by two sequential digestive steps in a KREBS modified medium containing collagenase, separated by an intermediate step in which acini separation is fostered by incubation in a Ca++ free medium containing the Ca++ chelator EDTA. Final separation is obtained through the application of mechanical forces by aspirating the suspension in plastic pipettes. The choice of the collagenase, the duration and the entity of the mechanical dissociation strength are the main modifications to the classic procedure and allow to obtain a very high yield of cells maintaining their ability to secrete enzymes for a long period (6-7 hours). Functional characterization is completed mainly by the determination of the amylase release stimulated by increasing doses of cholecystokinin.     

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.     

Evidence for a holin-like protein gene fully embedded out of frame in the endolysin gene of Staphylococcus aureus bacteriophage 187.

We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to an N-acetylmuramoyl-L-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5' region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage lambda Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by lambdagt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

The phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase is a Ca++-calmodulin dependent process

Regulation of the in vitro phosphorylation process of the photosynthetic form (G form) of Sorghum leaf Phosphoenolpyruvate carboxylase (PEPC: EC 4.1.1.31) was studied. Results established that: 1) PEPC was efficiently phosphorylated on seryl residues in crude leaf extract 2) Pyruvate, orthophosphate dikinase (EC 2.7.9.1.) which has been supposed to interfere with the process, was found not to be significantly phosphorylated in our experimental conditions 3) KF, as well as both Ca++ and Mg++ ions increased the radioactive signal detected 4) addition of EDTA or EGTA nullified it and Ca++ alone was found to reverse the inhibitory effect exerted by both chelators 5) addition of anti-Calmodulin antibodies to the medium also abolished the PEPC phosphorylation. Present data demonstrated that the post-translational modification of the C4-plant photosynthetic PEPC is a Ca++/Calmodulin dependent process.     

Ca++ dependence of the contraction of coronary artery segments in response to norepinephrine after beta-adrenergic blockade

The effect of external calcium concentration on the NE-induced contraction after beta-adrenergic blocking was studied in vitro. It resulted that the effect of NE was enhanced by increase, or reduced by decrease of calcium concentration. NE-induced contraction was not abolished when the bathing fluid was Ca++-free. The disappearance of the NE effect was only obtained in preparations treated with EDTA and perfused with Ca++-free Ringer-Locke solution. It is concluded that NE induced contraction after beta-adrenergic blocking is Ca++-dependent and on the tissue bound Ca++.     

The role of Ca++ in regulation of oxoglutarate dehydrogenase complex activity from bovine adrenal cortex

Binding of Ca2+ ions by EGTA is established to increase the oxoglutarate dehydrogenase complex K'm for 2-oxoglutarate up to 25 mM in spite of the Mg2+ ions presence in the medium. The maximum reaction rate remains unchanged. Addition, besides EGTA of an equimolar Ca2+ amount to the medium induces a more than 100-fold decrease in K'm. A positive effect of Ca2+ ions is intensified by ADP. Ca2+ counteracts the inhibition of the oxoglutarate dehydrogenase complex activity by NADH. When chelating Ca2+ by EGTA with NADH available, a nonhyperbolic dependence of the reaction rate on the 2-oxoglutarate concentration is observed. When Ca2+ is absent, signs of a positive cooperative interaction of the enzyme with ADP and NADH are observed under conditions when 2-oxoglutarate concentration is much lower than the saturating one.     

Permeability properties of isolated enterocytes from rat small intestine

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.     

Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts.

The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.     

Inhibition of Ca++-stimulated respiration by uncouplers.

In a phosphate medium at pH 6.6 low concentrations of uncouplers such as p-trifluoromethoxyphenylhydrazone carbonylcyanide and 2,4-dinitrophenol inhibit the oxidation of beta-hydroxybutyrate and succinate, when added during Ca++-accumulation. The inhibition depends on the amount of accumulated Ca++, and is released by N,N,N',N'-tetramethyl-p-phenylendiamine plus ascorbate as substrate. Under identical conditions the uncouplers have no inhibitory effect when added to mitochondria during state 3 respiration or during accumulation of Sr++. Inhibition of respiration by the decrease of transmembranal succinate transport or by accumulation of oxaloacetate can be excluded. It is suggested that accumulation of Ca++ in the presence of phosphate induces structural alteration of the mitochondrial membrane, which on the one hand changes the accessibility or sensitivity of dehydrogenases to uncouplers and causes leakage of accumulated Ca++ on the other.     

The Ca++ permeability of the apical membrane in neuromast hair cells

The mechanosensitivity of eel (Anguilla anguilla) neuromasts was measured by the impulse responses of single afferent nerve fibers to mechanical stimuli. It is dependent on the potential across the skin and on the ions in the water outside the apical membrane of the sensory cells. The mechanosensitivity decreases to zero when the skin is polarized by 10-100 mV cathodal DC (skin surface negative); it increases with increasing (10-60 mV) anodal DC and remains remarkably constant with higher polarization (Fig. 1). The mechanosensitivity increases with increasing concentrations of Ca++ outside the apical membrane of the sensory cells. Na+ and K+ have no influence. Addition of La , Co++, Mg++, D 600 and A-QA 39 inhibits the mechanosensitivity; the degree of inhibition varies with the inhibitor and the ratio [Ca++]/[inhibitor], indicating that the inhibition is competitive (Figs. 2, 3). We conclude that the apical membrane is specifically permeable to Ca++ ('late Ca channel') and that the inward receptor current through the apical membrane is carried by Ca++. Streptomycin also inhibits mechanosensitivity by competing with Ca++. With streptomycin, however, anodal polarization reduces, rather than increases, the mechanosensitivity (Fig. 4).(ABSTRACT TRUNCATED AT 250 WORDS)