Regulation of tumor phenotypes by caveolin-1 and sphingolipid-controlled membrane signaling complexes

Aberrant (glyco)sphingolipid expression deeply affects several properties of tumor cells that are involved in tumor progression and metastasis formation: cell adhesion (to the extracellular matrix or to the endothelium of blood vessels), motility, recognition and invasion of host tissues. In particular, (glyco)sphingolipids might contribute to the modulation of integrin-dependent interactions of tumor cells (determining their adhesion, motility and invasiveness) with the extracellular matrix as well as with host cells present in the stromal compartment of the tumor. A model based on solid experimental evidence has been proposed: (glyco)sphingolipids at the cell surface interact with plasma membrane receptors (e.g., integrin receptors and growth factor receptors) and adapter molecules (including tetraspanins) forming signaling complexes that are able to influence the activity of signal transduction molecules oriented at the cytosolic surface of the plasma membrane (mainly the Src kinases pathway members). The function of these signaling complexes appears to be strictly dependent on their (glyco)sphingolipid composition, and likely on specific sphingolipid-protein interactions. From this point of view, particularly intriguing is the connection between (glyco)sphingolipids and caveolin-1, a membrane protein that plays multiple roles as a suppressor of tumor growth and metastasis in ovarian, breast and colon human carcinomas.     

Molecular interaction in the mouse PAG between NMDA and opioid receptors in morphine-induced acute thermal nociception

Previous evidence demonstrates that low dose morphine systemic administration induces acute thermal hyperalgesia in normal mice through μOR stimulation of the inositol signaling pathway. We investigated the site of action of morphine and the mechanism of action of μOR activation by morphine to NMDA receptor as it relates to acute thermal hyperalgesia. Our experiments show that acute thermal hyperalgesia is blocked in periaqueductal gray with the μOR antagonist CTOP, the NMDA antagonist MK801 and the protein kinase C inhibitor chelerythrine. Therefore, a site of action of systemically administered morphine low dose on acute thermal hyperalgesic response appears to be located at the periaqueductal gray. At this supraspinal site, μOR stimulation by systemically morphine low dose administration leads to an increased phosphorylation of specific subunit of NMDA receptor. Our experiments show that the phosphorylation of subunit 1 of NMDA receptor parallels the acute thermal hyperalgesia suggesting a role for this subunit in morphine-induced hyperalgesia. Protein kinase C appears to be the key element that links μOR activation by morphine administration to mice with the recruitment of the NMDA/glutamatergic system involved in the thermal hyperalgesic response.     

A gene-specific cerebral types 1, 2, and 3 RyR protein knockdown induces an antidepressant-like effect in mice

Elevation of baseline intracellular calcium levels was observed in platelets or lymphoblasts of patients with bipolar affective disorders suggesting an altered intracellular Ca(2+) homeostasis in the pathophysiology of mood disorders. The role of supraspinal endoplasmic ryanodine receptors (RyRs), which allow mobilization of intracellular Ca(2+) stores, in the modulation of depressive states was, then, investigated. Ryanodine and FK506 reduced the immobility time in the mouse forced swimming test showing an antidepressant-like profile comparable with that produced by amitriptyline and clomipramine. We generated types 1, 2, and 3 RyR knockdown mice by using selective antisense oligonucleotides (aODN) to investigate the role of each RyR isoform. A gene-specific cerebral RyR protein level reduction in knockdown animals was demonstrated by immunoblotting, immunoprecipitation, and immunohistochemical experiments. Repeated intracerebroventricular administration of aODNs complementary to the sequence of the types 1, 2, or 3 RyR produced an antidepressant-like response in the forced swimming test. The aODN-induced reduction of immobility time was temporary and reversible and did not impair motor coordination, spontaneous mobility, and exploratory activity. These findings identify cerebral RyRs as critical targets underlying depressive states and should facilitate the comprehension of the pathophysiology of mood disorders and help developing of new therapeutical strategies.     

Abscisic acid is an endogenous stimulator of insulin release from human pancreatic islets with cyclic ADP ribose as second messenger

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic beta cells and inflammatory cells is increasingly recognized as a pathogenetic mechanism in the metabolic syndrome and type II diabetes, we investigated the effect of ABA on insulin secretion. Nanomolar ABA increases glucose-stimulated insulin secretion from RIN-m and INS-1 cells and from murine and human pancreatic islets. The signaling cascade triggered by ABA in insulin-releasing cells sequentially involves a pertussis toxin-sensitive G protein, cAMP overproduction, protein kinase A-mediated activation of the ADP-ribosyl cyclase CD38, and cyclic ADP-ribose overproduction. ABA is rapidly produced and released from human islets, RIN-m, and INS-1 cells stimulated with high glucose concentrations. In conclusion, ABA is an endogenous stimulator of insulin secretion in human and murine pancreatic beta cells. Autocrine release of ABA by glucose-stimulated pancreatic beta cells, and the paracrine production of the hormone by activated granulocytes and monocytes suggest that ABA may be involved in the physiology of insulin release as well as in its dysregulation under conditions of inflammation.     

Excitation-Contraction Coupling: The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of Ca_V1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both Ca_V1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human Ca_V1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in Ca_V1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca²⁺ release. The prominent role of VSD-I in governing Ca_V1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished Ca_V1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of Ca_V activation, which accounted for both time- and voltage-dependent properties of Ca_V1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how Ca_V1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human Ca_V1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.     

Antimalarial activity of novel pyrrolizidinyl derivatives of 4-aminoquinoline

Two pyrrolizidinylalkyl derivatives of 4-amino-7-chloroquinoline (MG2 and MG3) were prepared and tested in vitro against CQ-sensitive and CQ-resistant strains of Plasmodium falciparum and in vivo in a Plasmodium berghei mouse model of infection. Both compounds exhibited excellent activity in all tests and low toxicity against mammalian cells. Preliminary studies of the acute toxicity and of the metabolism of the most active compound MG3 indicate a promising profile as a new antimalarial drug candidate.     

Novel amodiaquine congeners as potent antimalarial agents

To develop new classes of antimalarial agents, the possibility of replacing the phenolic ring of amodiaquine, tebuquine, and isoquine with other aromatic nuclei was investigated. Within a first set of pyrrole analogues, several compounds displayed high activity against both D10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. The isoquine structure was also modified by replacing the diethylamino group with more metabolically stable bicyclic moieties and by replacing the aromatic hydroxyl function with a chlorine atom. Among these compounds, two quinolizidinylmethylamino derivatives (6f and 7f) displayed high activity against both CQ-S and CQ-R strains.     

Effect of different primate species on germination of Ficus (Urostigma) seeds

We examined the germination of Ficus seeds (subgenus Urostigma) after defecation by six primate species (New World monkeys, Old World monkeys, and apes). Seeds from figs (control) and primate feces were placed in a thermostatically controlled chamber for 30 days. Seeds defecated by Alouatta palliata, A. pigra, and Cercopithecus aethiops showed significantly higher germination rates than control seeds. In addition, seeds from A. palliata feces germinated significantly faster than control seeds and seeds from C. aethiops and Pan troglodytes. These differences may be due to the different digestive characteristics of the six primate species. Zoo Biol 23:273–278, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of prelamin A processing inhibitors on the differentiation and activity of human osteoclasts

Osteoclast differentiation is a complex process involving cytoskeleton and nuclear reorganization. Osteoclasts regulate bone homeostasis and have a key role in bone degenerative processes. Osteolysis and osteoporosis characterize a subset of laminopathies, inherited disorders due to defects in lamin A/C. Laminopathies featuring bone resorption are characterized, at the molecular level, by anomalous accumulation of the unprocessed lamin A precursor, called prelamin A. To obtain a suitable cell model to study prelamin A effects on osteoclasts, prelamin A processing inhibitors FTI-277 or AFCMe were applied to peripheral blood monocytes induced to differentiate towards the osteoclastic lineage. Previous studies have shown that treatment with FTI-277 causes accumulation of non-farnesylated prelamin A, while AFCMe inhibition of prelamin A maturation causes accumulation of a farnesylated form. We demonstrate that monocytes subjected to FTI-277 treatment and mostly those subjected to AFCMe administration, differentiate towards the osteoclastic lineage more efficiently than untreated monocytes, in terms of number of multinucleated giant cells, mRNA expression of osteoclast-related genes and TRACP 5b activity. On the other hand, the bone resorption activity of osteoclasts obtained in the presence of high prelamin A levels is lower with respect to control osteoclasts. This finding may help the understanding of the osteolytic and osteoporotic processes that characterize progeroid laminopathies.