Enhanced Patch-Clamp Technique to Study Antimicrobial Peptides and Viroporins, Inserted in a Cell Plasma Membrane with Fully Inactivated Endogenous Conductances

Many short peptides selectively permeabilize the bacteria plasma membrane, leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. Another class of short (<100 residues) membrane proteins called viroporins, because they are coded by viral genes, permeabilizes the membrane of susceptible cells during infection of by most animal viruses. The permeabilization leads to host cell lyses and the release of the virus mass, replicated at host cell expense, to propagate the infection. Detailed knowledge of the permeabilization properties of these proteins would allow to design, for instance, selective blockers of these pores, that would contrast the spread of the viral infection. In this chapter, the patch-clamp technique is employed to study the mechanism of membrane permeabilization induced by the pore-forming peptides, under strict physiological conditions. This goal is achieved by recording the ion current through the channels formed by these peptides, once inserted in a cell plasma membrane. To avoid contamination by the cell membrane currents, all the endogenous current sources must be blocked. It has been found that the photoreceptor rod outer segment mechanically isolated from the retina of low vertebrates (OS) was the most suitable cell to carry on the above studies, because it was possible to fully block all its endogenous currents without using any drug (such as TTX, TEA, dihydropyridines, etc.), that could obstruct the peptide pores or interfere with the pore formation. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, in which every perfusion parameter (as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles) was controlled by a user-friendly interface. This system allowed rapid application and removal of ions, drugs and peptides on the cells with a controlled timing, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained with representative peptides and with selected analogs, as alamethicin F50/5, the cecoprine-mellitin hybrid peptide, and a 20-aminoacid long fragment of the viroporin poliovirus 2B, it will be shown that the membrane pore formation occurs according to the barrel and stave, toroidal, and carpet model, respectively, that are the most widely-accepted mechanisms of membrane permeabilization. When recording large currents (produced for instance by high concentrations of peptides and/or highly permeable peptides), it is necessary to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. A second problem arises from the asymmetry of the plasma membrane: it is possible that the permeabilization properties of a particular peptide could be different depending upon the side of the membrane to which it is applied. For example, it is conceivable that viroporins are optimized to insert in the intracellular face of the plasma membrane, because they are synthesized in host cell cytosol. These two problems could be circumvented by widening the patch pipette shank, through the calibrated combination of heat and air pressure. These pipettes dramatically reduce series resistance, and allow at the same time to insert pulled quartz or plastic tubes very close to the pipette tip, making it possible the delivery of large molecules to the cytosol with a controlled timing. Finally, it is presented here a simple procedure to consistently attain seals with conventional or pressure polished pipettes, made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures

PLCB2 (phospholipase C, beta 2)

Review on PLCB2 (phospholipase C, beta 2), with data on DNA, on the protein encoded, and where the gene is implicated

Ubiquitination as a key regulatory mechanism for O3-induced cutaneous redox inflammasome activation

NLRP1 is one of the major inflammasomes modulating the cutaneous inflammatory responses and therefore linked to a variety of cutaneous conditions. Although NLRP1 has been the first inflammasome to be discovered, only in the past years a significant progress was achieved in understanding the molecular mechanism and the stimuli behind its activation. In the past decades a crescent number of studies have highlighted the role of air pollutants as Particulate Matter (PM), Cigarette Smoke (CS) and Ozone (O3) as trigger stimuli for inflammasomes activation, especially via Reactive Oxygen Species (ROS) mediators. However, whether NLRP1 can be modulated by air pollutants via oxidative stress and the mechanism behind its activation is still poorly understood. Here we report for the first time that O3, one of the most toxic pollutants, activates the NLRP1 inflammasome in human keratinocytes via oxidative stress mediators as hydrogen peroxide (H2O2) and 4-hydroxy-nonenal (4HNE). Our data suggest that NLRP1 represents a target protein for 4HNE adduction that possibly leads to its proteasomal degradation and activation via the possible involvement of E3 ubiquitin ligase UBR2. Of note, Catalase (Cat) treatment prevented inflammasome assemble and inflammatory cytokines release as well as NLRP1 ubiquitination in human keratinocytes upon O3 exposure. The present work is a mechanistic study that follows our previous work where we have showed the ability of O3 to induce cutaneous inflammasome activation in humans exposed to this pollutant. In conclusion, our results suggest that O3 triggers the cutaneous NLRP1 inflammasome activation by ubiquitination and redox mechanism

Extra virgin olive oil extracts of indigenous Southern Tuscany cultivar act as anti-inflammatory and vasorelaxant nutraceuticals

Extra virgin olive oil (EVOO) is the typical source of fats in the Mediterranean diet. While fatty acids are essential for the EVOO nutraceutical properties, multiple biological activities are also due to the presence of polyphenols. In this work, autochthonous Tuscany EVOOs were chemically characterized and selected EVOO samples were extracted to obtain hydroalcoholic phytocomplexes, which were assayed to establish their anti-inflammatory and vasorelaxant properties. The polar extracts were characterized via 1H-NMR and UHPLC-HRMS to investigate the chemical composition and assayed in CaCo-2 cells exposed to glucose oxidase or rat aorta rings contracted by phenylephrine. Apigenin and luteolin were found as representative flavones; other components were pinoresinol, ligstroside, and oleuropein. The extracts showed anti-inflammatory and antioxidant properties via modulation of NF-κB and Nrf2 pathways, respectively, and good vasorelaxant activity, both in the presence and absence of an intact endothelium. In conclusion, this study evaluated the nutraceutical properties of autochthonous Tuscany EVOO cv., which showed promising anti-inflammatory and vasorelaxant effects

Phospholipase C-beta2 promotes mitosis and migration of human breast cancer-derived cells

Like most human neoplasm, breast cancer has aberrations in signal transduction elements that can lead to increased proliferative potential, apoptosis inhibition, tissue invasion and metastasis. Due to the high heterogeneity of this tumor, currently, no markers are clearly associated with the insurgence of breast cancer, as well as with its progression from in situ lesion to invasive carcinoma. We have recently demonstrated an altered expression of the beta2 isoform of the phosphoinositide-dependent phospholipase C (PLC) in invasive breast tumors with different histopathological features. In primary breast tumor cells, elevated amounts of this protein are closely correlated with a poor prognosis of patients with mammary carcinoma, suggesting that PLC-beta2 may be involved in the development and worsening of the malignant phenotype. Here we demonstrate that PLC-beta2 may improve some malignant characteristics of tumor cells, like motility and invasion capability, but it fails to induce tumorigenesis in non-transformed breast-derived cells. We also report that, compared with the G(0)/G(1) phases of the cell cycle, the cells in S/G(2)/M phases show high PLC-beta2 expressions that reach the greatest levels during the late mitotic stages. In addition, even if unable to modify the proliferation rate and the expression of cell cycle-related enzymes of malignant cells, PLC-beta2 may promote the G(2)/M progression, a critical event in cancer evolution. Since phosphoinositides, substrates of PLC, are involved in regulating cytoskeleton architecture, PLC-beta2 in breast tumor cells may mediate the modification of cell shape that characterizes cell division, motility and invasion. On the basis of these data, PLC-beta2 may constitute a molecular marker of breast tumor cells able to monitor the progression to invasive cancers and a target for novel therapeutic breast cancer strategies

Selective fatty acid amide hydrolase inhibitors as potential novel antiepileptic agents

Temporal lobe epilepsy is the most common form of epilepsy, and current antiepileptic drugs are ineffective in many patients. The endocannabinoid system has been associated with an on-demand protective response to seizures. Blocking endocannabinoid catabolism would elicit antiepileptic effects, devoid of psychotropic effects. We herein report the discovery of selective anandamide catabolic enzyme fatty acid amide hydrolase (FAAH) inhibitors with promising antiepileptic efficacy, starting from a further investigation of our prototypical inhibitor 2a. When tested in two rodent models of epilepsy, 2a reduced the severity of the pilocarpine-induced status epilepticus and the elongation of the hippocampal maximal dentate activation. Notably, 2a did not affect hippocampal dentate gyrus long-term synaptic plasticity. These data prompted our further endeavor aiming at discovering new antiepileptic agents, developing a new set of FAAH inhibitors (3a–m). Biological studies highlighted 3h and 3m as the best performing analogues to be further investigated. In cell-based studies, using a neuroblastoma cell line, 3h and 3m could reduce the oxinflammation state by decreasing DNA-binding activity of NF-kB p65, devoid of cytotoxic effect. Unwanted cardiac effects were excluded for 3h (Langendorff perfused rat heart). Finally, the new analogue 3h reduced the severity of the pilocarpine-induced status epilepticus as observed for 2a

Neuroinhibitory actions of taurine in the main olfactory bulb

The amino acid taurine is abundant in the olfactory bulb (OB), exceeding glutamate and GABA in concentration. In whole-cell patch-clamp recordings in slices of rat OB, we studied the actions of taurine on the principal neurons (PNs), mitral and tufted cells, and the local interneurons, periglomerular (PG) cells. Taurine decreased, in a dose-dependent manner (EC50 = 2.2 mM), the input resistance of PNs and shifted membrane potential towards ECl. The GABAA receptor antagonists, bicuculline and picrotoxin, but not GABAB receptor antagonists, CGP 35348 and CGP 55845A, blocked the taurine effects. This implies that taurine inhibits PNs by increasing GABAA receptor Cl– conductance. PG cells, which also express GABAA receptors, were insensitive to taurine. Olfactory nerve stimulation evoked monosynaptic excitatory responses in PNs and PG cells voltage clamped at ECl or treated with picrotoxin. Taurine (5 mM) and the GABAB receptor agonist baclofen suppressed PNs responses. CGP 55845A, but not bicuculline and the postsynaptic GABAB receptor antagonist CGP 35348, abolished this suppression. The taurine action most likely was due to GABAB receptor-mediated inhibition of presynaptic glutamate release. Neither taurine nor baclofen affected PG cell responses. The results suggest that taurine reduces the excitability of PNs and their sensory input without influencing PG cells. Selective inhibitory actions of taurine in the OB may represent a physiologic mechanism protecting PNs from hyperexcitation. Supported by NIH grant DC04083 (I.K.) and Fondazione Caricento (O.B.)

Mechanism of pore formation of antimicrobial peptides and viroporins inserted in a cell plasma membrane

none4noneRispoli G; Benedusi M; Milani A; Aquila M.Rispoli, Giorgio; Benedusi, Mascia; Milani, Alberto; Aquila, Marc

Adult neurogenesis in the rat olfactory bulb

none3In adult animals, neurons generated within the subventricular zone (SVZ), following the rostral migratory stream (RMS), reach the olfactory bulb (OB), where they apparently give rise to interneurons. In fact, there has never been a functional demonstration that these cells are true neurons. Filling this gap has been impeded largely by the inability to distinguish between newly generated cells (NGC) in living brain tissue using conventional approaches. We have solved this problem combining retrovirus-mediated genetransfer and patch-clamp electrophysiology. The viral vector, including a gene for GFP, was injected into the SVZ. After 2 weeks, GFP+ cells resembling periglomerular cells (PG) were observed in the OB. Using a whole-cell patch-clamp technique, we recorded action potentials from NGC in response to the injection of depolarizing currents, and we have isolated and characterized Na+ and K+ currents. About 95% of the NGC were PG cells; the remaining ended their migration between internal plexiform and mitral cell layers, and were identified as short-axon cells. The progressive maturation of NGC has been characterized in time and space along the RMS. When in the RMS, the cells show only a weak delayed rectifier K+-current, to which an A-current is added when the OB is reached. The mature cells lose the delayed rectifier K+-current, and only display A- and Na+-currents. Finally, by recording action potentials and excitatory synaptic currents in response to stimulation of the olfactory nerve, for the first time we have demonstrated that the NGC fully integrate into the bulbar circuitry, establishing functional synaptic contacts.noneO. Belluzzi; M. Benedusi; J.J. LoTurcoBelluzzi, Ottorino; Benedusi, Mascia; J. J., Loturc

Circadian Deregulation as Possible New Player in Pollution-Induced Tissue Damage

Circadian rhythms are 24-h oscillations driven by a hypothalamic master oscillator that entrains peripheral clocks in almost all cells, tissues and organs. Circadian misalignment, triggered by industrialization and modern lifestyles, has been linked to several pathological conditions, with possible impairment of the quality or even the very existence of life. Living organisms are continuously exposed to air pollutants, and among them, ozone or particulate matters (PMs) are considered to be among the most toxic to human health. In particular, exposure to environmental stressors may result not only in pulmonary and cardiovascular diseases, but, as it has been demonstrated in the last two decades, the skin can also be affected by pollution. In this context, we hypothesize that chronodis-truption can exacerbate cell vulnerability to exogenous damaging agents, and we suggest a possible common mechanism of action in deregulation of the homeostasis of the pulmonary, cardiovascular and cutaneous tissues and in its involvement in the development of pathological conditions