Complex formation betweenγ-immunoglobulin and calmodulin in calcium-free conditions

We show that -immunoglobulin (IgG) binds calmodulin (CaM) in a Ca²⁺-independent manner, with Kd value of (1.7±0.5)×10^–7M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca²⁺ greatly diminished the interaction between IgG and CaM, with IC₅₀=8–9M. These data give a novel insight into protein-protein interactions.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Purification and some properties of hemagglutinin from the Myxomycete,Physarum polycephalum

A new hemagglutinin was isolated from the plasmodium ofPhysarum polycephalum by salting out with ammonium sulphate followed by chromatography on DE-32, DEAE-Toyopearl and hydroxyapatite. This hemagglutinin, named physarumin, was purified 1000-fold over crude extracts. The molecular weight of physarumin was determined to be 22,000 by size exclusion chromatography on Bio-Gel P-60 and 8,700 by SDS-polyacrylamide gel electrophoresis. Physarumin agglutinated rabbit, guinea pig, horse and human erythrocytes. Physarumin-induced hemagglutination was inhibited by fetuin and ₁ glycoprotein, but not by commercially available mono-and disaccharides. Hemagglutinating activity was blocked by EDTA, and was restored by adding Ca²⁺ but not by Mg²⁺.     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004     

Hyposmotic shock-induced discharge in acontia ofCalliactis parasitica is blocked by gadolinium

On acontia ofCalliactis parasitica it was observed that mechanical stimuli applied by a gelatin probe, a method effective in tentacles of Anthozoa, do not induce the discharge of nematocytes. Hyposmotic shock, performed by treatment with NaCl solution 35% hyposmotic with respect to sea water, induces, in the presence of Ca²⁺, the discharge that spreads along the acontial filament, as previously observed following treatment with SCN^–. The hyposmotic shock-induced discharge is blocked by Gd³⁺ at a concentration of 1 M. 10 M Gd³⁺ prevents also the SCN^–-induced discharge. These results suggest the presence of stretch activated cation channels either in nematocytes and/or in supporting cells as well as a possible effect of SCN^– on this class of ion channels.     

The effect of hydrocortisone and thyroxine on development of calcium homeostasis in embryonic intestinal epithelium

Cytoplasmic Ca²⁺ concentration of epithelial cells from 14-day embryonic chick duodena decreased during 72 h of organ culture to a value 54% of that found at 17 days in vivo. The ability of cells to maintain a constant Ca²⁺ concentration when challenged with high extracellular calcium was also significantly reduced. Addition of 1 M hydrocortisone during culture restored both parameters of Ca²⁺ homeostasis to that of 16-day uncultured duodena, and rise in cytoplasmic Ca²⁺ was significant within 4 h of hormone treatment. Thyroxine influenced epithelial Ca²⁺ similarly, but to a lesser degree and only after 48–72 h of culture. These data indicate that glucocorticoids, and possibly thyroid hormones, influence the development of calcium homeostasis in intestinal epithelium.     

17β-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes

Effects of 17-estradiol (E₂) in vitro on Na-dependent Ca²⁺ efflux from, and depolarization-induced Ca²⁺ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E₂ (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca²⁺ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca²⁺ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca²⁺ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E₂, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca²⁺ transport mechanisms in nerve endings.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Metal binding to myosin and to myosin DTNB-light chain

Summary The effects of various divalent cations, Ca²⁺, Mg²⁺ and Mn²⁺ on the intrinsic fluorescence of heavy meromyosin (HMM) and myosin 5,5-dithio-bis-(2-nitrobenzoate) DTNB-light chain of rabbit striated muscle, are compared. At pH 6.4, the fluorescence change induced by the metal ions is present only in the isolated light chain and disappears in HMM, thus indicating an interaction between the heavy and light chains with respect to the binding of the metal ions. Whereas Mg²⁺ binds more strongly than Ca²⁺ to myosin, this order is reversed in the case of the DTNB-light chain.     

Evidence for suppression of Na-dependent Ca2+ efflux from rat brain synaptosomes by ovarian steroids in vivo

Summary The possibility that intracellular Ca²⁺, which mediates neurotransmitter release, regulation of membrane permeability, microtubule polymerization and axonal transport, is influenced by gonadal steroids via a Na–Ca exchange mechanism was examined. The resting Ca²⁺ uptake into synaptosomes was measured using crude synaptosomal pellets (P₂ fraction), isolated from the brain stem, mesencephalic reticular formation (MRF), nucleus caudatus (NC) and the hippocampus of intact, long-term ovariectomized (OVX) and OVX plus progesterone (P) or estradiol-17 benzoate (EB) treated adult female rats. Irrespective of the brain structure investigated, the uptake was 1) markedly increased in synaptosomes from OVX animals in comparison to intact controls, and 2) reduced to near control values in synaptosomes from OVX rats treated s.c. with a single dose of 2 mg P or 5 g EB. Since Ca²⁺ influx into synaptosomes was shown earlier to depend on external sodium concentration, which was the same in all experiments described in this work, the results obtained indicate that ovarian steroids modulate basal synaptic activity in the rat brain by suppressing Na-dependent Ca²⁺ efflux from the nerve cell.     

The effects of various agents in vitro on homocarnosine-carnosine synthetase from rat brain

Summary The present paper reports the first evidence that homocarnosine-carnosine synthetase from rat brain requires free sulfhydryl groups for activity. The activity of the synthetase can be stabilized by dithioerythritol and inhibited strongly by Cu²⁺, Cd²⁺, Hg²⁺, Zn²⁺, and to a lesser extent by Ca²⁺, Ni²⁺, Li, chlorpromazine, -methyl DOPA and norepinephrine at the concentrations tested.Acknowledgment. This work was supported by United States Public Health Service Grant No. NS-06137.     

Sr2+ can become incorporated into an agonist-sensitive,cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium

We have explored the properties of a Ca²⁺-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca²⁺-influx pathway allowed Sr²⁺ to enter the cytoplasm but permitted only a minimal influx of Ba²⁺. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr²⁺, but not Ba²⁺, could become incorporated into the agonist-sensitive, cytoplasmic Ca²⁺ store. Further evidence for this was provided by experiments using ionomycin, a Ca²⁺ ionophore which has no affinity for Sr²⁺.     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

Chromate reduction inStreptomyces

Summary Streptomyces species 3M grew in peptone yeast extract medium with 1000 g/ml K₂Cr₂O₇. Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr⁶⁺ in the reaction mixture. The level of Cr⁶⁺ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000×g for 1 h, in the presence of NADH. The reducing enzyme was associated with this cell fraction. The enzyme was constitutive and reduced Cr⁶⁺ to Cr³⁺.     

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione

Alcian blue and plumbagin induced transient Ca²⁺ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca²⁺ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca²⁺. Mg²⁺ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca²⁺ release. These results suggest that oxidation of sulfhydryls on Ca²⁺ release channels induces Ca²⁺ release even in the presence of GSH in situ.     

Effect of X-537A on the phosphorylated protein in sarcoplasmic reticulum vesicles

Summary The effect of the antibiotic X-537A on the phosphorylated ATPase (EP) was investigated. The results show that X-537A depresses the level of EP which is dependent on the Ca²⁺ gradient, while the Ca²⁺-independent EP is not affected. Acknowledgments. This work was supported by grants from the Instituto de Alta Cultura of the Portuguese Ministry of Education (No. CB/2) and the Calouste Gulbenkian Foundation.     

Zinc ions block the second but not the first phasic response to repetitive application of carbachol and histamine in guinea-pig taenia caeci

In the presence of Zn²⁺ (0.3 mM), carbachol (10^–6 M) or histamine (10^–5 M) induced the phasic response in guinea-pig taenia caeci while the tonic response was markedly inhibited. However, when the muscles were kept in Zn²⁺-containing medium following the first stimulation with either carbachol or histamine, neither application of carbachol nor of histamine elicited another phasic contraction. Caffeine (25 mM) did not induce contraction in the presence of Zn²⁺. After the washing out of caffeine in the presence of Zn²⁺, however, the muscle did then develop the phasic response on the application of carbachol or histamine. In conclusion, Zn²⁺ did not affect the carbachol or histamine-induced Ca²⁺ release from the storage sites. However, when Zn²⁺ was continuously present, Ca²⁺ was not supplied to the storage sites. Furthermore, carbachol and histamine mobilized a common cellular Ca²⁺ store, but they activated Ca²⁺ release channels different from the ones activated by caffeine in the Ca²⁺ storage sites.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

The role of the <Emphasis Type="Italic">ATP2C1</Emphasis> gene in Hailey–Hailey disease

Hailey–Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis, characterized by a chronic course of repeated and exacerbated skin lesions in friction regions. The pathogenic gene of HHD was reported to be the ATPase calcium-transporting type 2C member 1 gene (ATP2C1) located on chromosome 3q21–q24. Its function is to maintain normal intracellular concentrations of Ca²⁺/Mn²⁺ by transporting Ca²⁺/Mn²⁺ into the Golgi apparatus. ATP2C1 gene mutations are reportedly responsible for abnormal cytosolic Ca²⁺/Mn²⁺ levels and the clinical manifestations of HHD. Environmental factors and genetic modifiers may also affect the clinical variability of HHD. This article aims to critically discuss the clinical and pathological features of HHD, differential diagnoses, and genetic and functional studies of the ATP2C1 gene in HHD. Further understanding the role of the ATP2C1 gene in the pathogenesis of HHD by genetic, molecular, and animal studies may contribute to a better clinical diagnosis and provide new strategies for the treatment and prevention of HHD.