The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

On the mechanism of inhibition of p27 degradation by 15-deoxy-Δ12,14-prostaglandin J 2 in lymphoblasts of Alzheimer’s disease patients

It has been proposed that neuroinflammation, among other factors, may trigger an aberrant neuronal cell cycle re-entry leading to neuronal death. Cell cycle disturbances are also detectable in peripheral cells from Alzheimer’s disease (AD) patients. We previously reported that the anti-inflammatory 15- deoxy-Δ^12,14-prostaglandin J ₂ (15d-PGJ ₂) increased the cellular content of the cyclin-dependent kinase inhibitor p27, in lymphoblasts from AD patients. This work aimed at elucidating the mechanisms of 15d-PGJ ₂-induced p27 accumulation. Phosphorylation, half-life, and the nucleo-cytoplasmic traffic of p27 protein were altered by 15d-PGJ2 by mechanisms dependent on PI3K/Akt activity. 15d-PGJ ₂ prevents the calmodulin-dependent Akt overactivation in AD lymphoblasts by blocking its binding to the 85-kDa regulatory subunit of PI3K. These effects of 15d-PGJ ₂ were not mimicked by 9,10-dihydro-15-deoxy-Δ^12,14- prostaglandin J ₂, suggesting that 15d-PGJ ₂ acts independently of peroxisome proliferator-activated receptor γ activation and that the α,β-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ ₂ is a requisite for the observed effects. Received 14 July 2008; received after revision 2 September 2008; accepted 12 September 2008     

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca²⁺ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca²⁺], derived from intracellular stores, followed, only in myotubes, by a large Ca²⁺ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca²⁺ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca²⁺ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death. Received 21 January 2009; received after revision 05 March 2009; accepted 11 March 2009     

The discovery of α-Klotho and FGF23 unveiled new insight into calcium and phosphate homeostasis

The traditional view of calcium homeostasis is that it is maintained by two essential reactions. First, changes in extracellular Ca²⁺ are sensed in several distinct cell types, stimulating the secretion of parathyroid hormone (PTH), 1,25(OH)₂ D and calcitonin in response to the body’s requirement. Second, these calcitropic hormones then act on the calcium-translocating cells of the kidney, bone, and intestine to restore calcium balance. Recent progress indicates that α-Klotho and fibroblast growth factor (FGF) 23 are key players that integrate the multi-step regulatory system of calcium homeostasis that rapidly adjusts the extracellular calcium concentration and continuously maintains its concentration within a narrow physiological range. α-Klotho and FGF23 are also found to be major players in the regulatory system of phosphate homeostasis. Here, the demonstration of the molecular functions of α-Klotho and FGF23 has recently given new insight into the field of calcium and phosphate homeostasis. Received 3 April 2008; received after revision 23 May 2008; accepted 5 June 2008     

Conotoxins of the O-superfamily affecting voltage-gated sodium channels

The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While ω-conotoxins and κ-conotoxins block Ca²⁺ and K⁺ channels, respectively, the closely related δ- and μO-conotoxins affect voltage-gated Na⁺ channels (Na_v channels). δ-conotoxins mainly remove the fast inactivation of Na_v channels and, thus, functionally resemble long-chain scorpion α-toxins. μO-conotoxins are functionally similar to μ-conotoxins, since they inhibit the ion flow through Na_v channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na_v channels. Received 27 December 2006; received after revision 30 January 2007; accepted 19 February 2007     

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes: significance in IL-2 production

Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE₂ in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE₂-diminished IL-2 production. The decrease in IL-2 production after PGE₂ and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE₂ in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT₁ antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology. Received 23 September 2008; received after revision 19 November 2008; accepted 21 November 2008     

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca<Superscript>2+</Superscript>/calmodulin and ERK1/2 signaling pathways

We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca²⁺/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca²⁺/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal. F. Bartolomé, N. de las Cuevas: These authors contributed equally to this work. Received 14 February 2007; received after revision 16 April 2007; accepted 23 April 2007     

Flippases: still more questions than answers

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P₄-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The β-subunit of P₄-ATPases shows unexpected similarities between the β- and γ-subunits of the Na⁺/K⁺-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P₄-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P_5A-ATPases are functional homologues of P₄-ATPases. Received 19 June 2008; received after revision 31 July 2008; accepted 15 August 2008     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

CD20-related signaling pathway is differently activated in normal and dystrophic circulating CD133+ stem cells

Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133⁺ cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133⁺ stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca²⁺]_i) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca²⁺]_i is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133⁺ stem cells, supporting the assumption of a “CD20-related calcium impairment-affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133⁺ stem cells. Received 15 October 2008; received after revision 27 November 2008; accepted 05 December 2008     

ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling

Rapid Ca²⁺-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca²⁺ ionophores in megakaryoblastic HEL cells. Ca²⁺ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca²⁺, whereas exogenous Ca²⁺ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca²⁺ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms. A. Arachiche, I. Badirou: These authors contributed equally to this work. Received 18 June 2008; received after revision 24 September 2008; accepted 1 October 2008     

Malignant hyperthermia: Molecular defects in membrane permeability

Summary Malignant hyperthermia (MH), a genetically inherited disorder of skeletal muscle, is due to molecular defect in membrane permeability. The alteration in membrane permeability is suggested to be due to enhanced phospholipase A₂ activity which is responsible for the increased level in sarcoplasmic Ca²⁺. The excess Ca²⁺ is responsible for muscle hyper-rigidity and enhanced rate of glycolysis, resulting in a rapid rate of lactic acid production and a low pH in MH muscle.     

Respiratory burst in peritoneal exudate cells in response to a modified tuftsin

Intraperitoneal administration of tuftsin-M [Thr–Lys–Pro–Arg–NH–(CH₂)₂–NH–CO–C₁₅H₃₁] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O₂ ^–, H₂O₂, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O ₂ ^– and H₂O₂ under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.     

Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens

Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. In this communication we report the production of scorpine in a heterelogous system, using a specific vector containing its cloned gene. The recombinantly expressed scorpine (RScp) in ^{Anopheles gambie} cells showed antibacterial activity against ^{Bacillus subtilis} and ^{Klebsiella pneumoniae}, at 5 and 10 μM, respectively. It also produced 98% mortality in sexual stages of ^{Plasmodium berghei} at 15 μM and 100% reduction in ^{Plasmodium falciparum} parasitemia at 5 μM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic ^Drosophila that overexpresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable. Received 6 May 2008; received after revision 24 July 2008; accepted 29 July 2008     

Cl−/HCO 3 − exchanger is operative in isolated enterocytes from rat jejunum

Enterocytes isolated from rat jejunum were tested for the existence of a Cl^–/HCO ₃ ^– exchange, previously evidenced in basolateral membrane vesicles but not in brush border. Cells were found to retain functional integrity and transport capabilities long enough to allow Cl^– fluxes to be measured. Both efflux and uptake experiments indicate that a Cl^–/HCO ₃ ^– antiport, inhibited by 4,4-diisothiocyanostilbene-2-2-disulfonic acid (DIDS), is functional under resting conditions.     

Control of sperm motility and fertility: Diverse factors and common mechanisms

Spermatozoa generated in the testis are immature and incompetent for fertilization. During their journey toward the egg, the sperm acquire fertility and achieving fertilization. These sperm modifications to ensure fertilization are induced by many female or male extra-sperm factors: for example, sperm motility-activating factors from the egg jelly, sperm attractants from the eggs, and decapacitation factors from the seminal plasma. The factors controlling sperm fertility are myriad and species specific; they may be peptides, sugar chains, or small organic compounds. Nevertheless, the fundamental mechanisms underlying fertilization must be common among all animals; increase in [Ca²⁺]_i triggers all the steps in the process of fertilization, and cAMP plays important roles in many steps. Elucidating the dynamic functional and morphological changes in sperm cells is important for understanding the regulation of fertilization. Here, we introduce the diversity and generality of the control of sperm fertility. Received 28 April 2008; received after revision 13 June 2008; accepted 17 June 2008     

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action. Received 13 June 2008; received after revision 05 November 2008; accepted 11 November 2008     

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.     

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE₂ increased [³H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE₂ increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE₂ obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE₂-induced cell cycle regulatory protein expression and thymidine incorporation. PGE₂ caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE₂-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells. Received 30 January 2009; received after revision 03 March 2009; accepted 10 March 2009