Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA.

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.     

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa.

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.     

Requirement for calcium ions in mycobacteriophage I3 DNA injection and propagation

Ca²⁺ ions are absolutely necessary for the propagation of mycobacteriophage I3 in synthetic medium. These ions are required for successful infection of the host and during the entire span of the intracellular development of the phage. A direct assay of the phage DNA injection using ³²[P] labelled phage, showns that Ca²⁺ ions are necessary for the injection process. The injection itself is a slow process and takes 15 min to complete at 37°C. The bacteria infected in presence of Ca²⁺ tend to abort if the ions are subsequently withdrawn from the growth medium. The effect of calcium withdrawal is maximally felt during the early part of the latent period; however, later supplementation of Ca²⁺ ions salvage phage production and the mature phage progeny appear after a delayed interval, proportional to the time of addition of Ca²⁺.Abbreviations moi multiplicity of infection - PFU plaque forming units - EGTA ethylene-glycol-bis (-aminoethyl ether) N,N-tetraacetic acid     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 x 10(-10) to 1 x 10(-6) M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 x 10(-6) M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10(-6) M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10(-10) - 10(-6) M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activity in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Identification of beta-N-acetylhexosaminidase A in mouse tissues with the fluorigenic substrate 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate.

The ionic mechanism of inositol trisphosphate (InsP3)-induced Ca2+ release was investigated in microsomes (microsomal fractions) isolated from rat brain. InsP3 stimulated Ca2+ release from microsomes incubated in media containing 100 mM-KCl. The InsP3-induced Ca2+ release was insensitive to a variety of Ca2+-channel blockers; however, the K+-channel blockers tetraethylammonium chloride (TEA; 1 mM) and 9-tetraethylammonium chloride (9-TEA; 1 mM) blocked InsP3-induced Ca2+ release. Moreover, addition of InsP3 increased 86Rb+ influx into the microsomes. The influx of 86Rb+ also was sensitive to TEA and 9-TEA. The above results suggest that InsP3-induced Ca2+ release requires an opposite flow of K+ ions, and modulation of K+ channels by TEA and 9-TEA may underlie the inhibition of InsP3-induced Ca2+ release from brain microsomes by these agents.     

The role of Ca2+ ions in a phage infection of Corynebacterium glutamicum

It was shown that neither uncouplers of oxidative phosphorylation, nor lack of Ca2+ ions affected the normal MC-2 phage absorption on Corynebacterium glutamicum cells, while the phage development was repressed under these conditions. Simultaneous measurement of Ca2+, K+ and H+ ion flows, as well as measurement of membrane potential showed that the addition of the phage into the experimental medium led to significant depolarization of the membrane from -160 mV to -100 mV due to the penetration of Ca2+ ions into the cells followed by K+ and H+ efflux. The (Ca2+) to (K+ + H+) ratio was shown to be 1 : 1. Phage DNA is supposed to be injected into the host cells as a positively charged (Ca2+-DNA) complex.     

The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.     

Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Kinetic properties of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli 080

Results on the kinetics of 7 alpha-hydroxysteroid dehydrogenase 7 alpha-HSDH showed that this enzyme could oxidize all bile acids having an -OH group at the C-7 position. Lineweaver-Burk plots showed Michaelis constant (Km) values of 0.83 and 0.12 mM for cholic acid and chenodeoxycholic acid, respectively. The effect of enzyme concentration on the reaction velocity showed a constant increase in the enzyme activity with increase in enzyme-protein concentration. 7 alpha-HSDH was activated by Na+, K+, Ca2+, and Mn2+ ions and by reducing agents having a thiol group (dithiothreitol, 2-mercaptoethanol). Co2+, Hg2+, Fe3+, Mg2+, Zn2+, Ba2+, and Cu2+ ions, chelating agents (potassium oxalate, heparin, EDTA) oxidizing agents (sodium perchlorate, sodium periodate, sodium persulphate), and detergents (Tween 20, Tween 40, Tween 80, Triton X-100, sodium lauryl sulphate) were inhibitory to 7 alpha-HSDH activity.     

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.     

Effect of guanosine triphosphate on the release of Ca2+ from intracellular store sites of saponin-treated human peripheral lymphocytes

The effects of guanosine triphosphate (GTP) on the release and uptake of Ca2+ in nonmitochondrial intracellular store sites of human peripheral lymphocytes were examined. GTP in the presence of 3% polyethylene glycol released Ca2+ from the intracellular store sites of lymphocytes in a dose-dependent manner, and the maximal release was obtained at 10 microM GTP. GDP and 5'-GMP also enhanced the release of Ca2+. On the other hand, Ca2+ uptake in the presence of oxalate by saponin-treated lymphocytes was stimulated by GTP and this stimulation was abolished when polyethylene glycol was concomitantly present. The dose dependence of the stimulated Ca2+ uptake by GTP was much the same as that of the Ca2+ released by GTP. These results indicate that GTP has an inherent activity to release Ca2+ as well as to stimulate the uptake of Ca2+ in nonmitochondrial intracellular store sites of saponin-treated lymphocytes. The stimulatory effect of polyethylene glycol on GTP-mediated Ca2+ release may occur by inhibiting functions of the Ca2+ pump.     

The heavy metal ions Ag+ and Hg2+ trigger calcium release from cardiac sarcoplasmic reticulum

Heavy metal ions have been shown to induce Ca2+ release from skeletal sarcoplasmic reticulum (SR) by binding to free sulfhydryl groups on a Ca2+ channel protein and are now examined in cardiac SR. Ag+ and Hg2+ (at 10-25 microM) induced Ca2+ release from isolated canine cardiac SR vesicles whereas Ni2+, Cd2+, and Cu2+ had no effect at up to 200 microM. Ag(+)-induced Ca2+ release was measured in the presence of modulators of SR Ca2+ release was compared to Ca2(+)-induced Ca2+ release and was found to have the following characteristics. (i) Ag(+)-induced Ca2+ release was dependent on free [Mg2+], such that rates of efflux from actively loaded SR vesicles increased by 40% in 0.2 to 1.0 mM Mg2+ and decreased by 50% from 1.0 to 10.0 mM Mg2+. (ii) Ruthenium red (2-20 microM) and tetracaine (0.2-1.0 mM), known inhibitors of SR Ca2+ release, inhibited Ag(+)-induced Ca2+ release. (iii) Adenine nucleotides such as cAMP (0.25-2.0 mM) enhanced Ca2(+)-induced Ca2+ release, and stimulated Ag(+)-induced Ca2+ release. (iv) Low Ag+ to SR protein ratios (5-50 nmol Ag+/mg protein) stimulated Ca2(+)-dependent ATPase activity in Triton X-100-uncoupled SR vesicles. (v) At higher ratios of Ag+ to SR proteins (50-250 nmol Ag+/mg protein), the rate of Ca2+ efflux declined and Ca2(+)-dependent ATPase activity decreased gradually, up to a maximum of 50% inhibition. (vi) Ag+ stimulated Ca2+ efflux from passively loaded SR vesicles (i.e., in the absence of ATP and functional Ca2+ pumps), indicating a site of action distinct from the SR Ca2+ pump. Thus, at low Ag+ to SR protein ratios, Ag+ is very selective for the Ca2+ release channel. At higher ratios, this selectivity declines as Ag+ also inhibits the activity of Ca2+,Mg2(+)-ATPase pumps. Ag+ most likely binds to one or more sulfhydryl sites "on" or "adjacent" to the physiological Ca2+ release channel in cardiac SR to induce Ca2+ release.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.     

Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).