Biomedical subjects

Francesco Tombola

Publications and source records attributed to Francesco Tombola.

13 recordsLinked to original sources

PubMed2007

Red wine and green tea reduce H pylori- or VacA-induced gastritis in a mouse model.

AIM: To investigate whether red wine and green tea could exert anti-H pylori or anti-VacA activity in vivo in a mouse model of experimental infection. METHODS: Ethanol-free red wine and green tea concentrates were administered orally as a mixture of the two beverages to H pylori infected mice, or separately to VacA-treated mice. Gastric colonization and gastric inflammation were quantified by microbiological, histopathological, and immunohistochemical analyses. RESULTS: In H pylori-infected mice, the red wine and green tea mixture significantly prevented gastritis and limited the localization of bacteria and VacA to the surface of the gastric epithelium. Similarly, both beverages significantly prevented gastric epithelium damage in VacA-treated mice; green tea, but not red wine, also altered the VacA localization in the gastric epithelium. CONCLUSION: Red wine and green tea are able to prevent H pylori-induced gastric epithelium damage, possibly involving VacA inhibition. This observation supports the possible relevance of diet on the pathological outcome of H pylori infection.

Animals↗

PubMed2006

The twisted ion-permeation pathway of a resting voltage-sensing domain.

Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.

Animals↗

PubMed2006

How does voltage open an ion channel?

Neurons transmit information through electrical signals generated by voltage-gated ion channels. These channels consist of a large superfamily of proteins that form channels selective for potassium, sodium, or calcium ions. In this review we focus on the molecular mechanisms by which these channels convert changes in membrane voltage into the opening and closing of "gates" that turn ion conductance on and off. An explosion of new studies in the last year, including the first X-ray crystal structure of a mammalian voltage-gated potassium channel, has led to radically different interpretations of the structure and molecular motion of the voltage sensor. The interpretations are as distinct as the techniques employed for the studies: crystallography, fluorescence, accessibility analysis, and electrophysiology. We discuss the likely causes of the discrepant results in an attempt to identify the missing information that will help resolve the controversy and reveal the mechanism by which a voltage sensor controls the channel's gates.

Animals↗

PubMed2005

How far will you go to sense voltage?

Despite tremendous progress in the study of voltage-gated channels, the molecular mechanism underlying voltage sensing has remained a matter of debate. We review five new studies that make major progress in the field. The studies employ a battery of distinct approaches that have the common aim of measuring the motion of the voltage sensor. We interpret the results in light of the recent crystal structure of the mammalian potassium channel Kv1.2. We focus on the transmembrane movement of the voltage sensor as a key element to the detection of membrane potential and to the control of channel gating.

Animals↗

PubMed2005

The properties of the mitochondrial megachannel in mitoplasts from human colon carcinoma cells are not influenced by Bax.

This paper explores the relationship between Bax and the mitochondrial permeability transition pore (PTP). Isolated human colon tumor (HCT116) Bax- mitochondria exposed to recombinant Bax exhibited a slow, cyclosporin A-sensitive swelling, but only at [Bax]>200 nM. The amount of Bax incorporated was much higher than that found in organelles isolated from HCT116 Bax+ staurosporine- or etoposide-treated apoptotic cells, casting doubts on the significance of the putative PT induction for apoptosis. Bax did not influence the electrophysiological properties of an approximately 1 nS channel ascribed to the Ca2+-dependent mitochondrial permeability transition pore. These observations indicate that the PTP is independent of Bax.

Calcium↗

PubMed2005

Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores.

Voltage-gated ion channels sense voltage by shuttling arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged arginines but excludes solution ions. We find that substituting the first S4 arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K(+) channel at rest. The cationic current does not flow through the central K(+) pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane arginine permeation. We propose that when S4 moves it ratchets between conformations in which one arginine after another occupies and occludes to ions the narrowest part of this pathway.

Amino Acid Substitution↗

PubMed2005

The cooperative voltage sensor motion that gates a potassium channel.

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.

Animals↗

PubMed2005

Molecular handles for the mechanical manipulation of single-membrane proteins in living cells.

We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell. The technology was successfully tested by mechanical manipulation of biotinylated extracellular residues of channels in oocytes using an atomic force microscope under conditions which preserve function of the channels. Binding forces of approximately 80 pN at 100 nN/s were measured.

Animals↗

PubMed2004

Bax does not directly participate in the Ca(2+)-induced permeability transition of isolated mitochondria.

The mitochondrial permeability transition pore and Bax have both been proposed to be involved in the release of pro-apoptotic factors from mitochondria in the "intrinsic" pathway of apoptosis. The permeability transition pore is widely thought to be a supramolecular complex including or interacting with Bax. Given the relevance of the permeability transition in vivo, we have verified whether Bax influences the formation and/or the properties of the Ca(2+)/P(i)-induced permeability transition by using mitochondriaisolated from isogenic human colon cancer bax(+/-) and bax(-/-) HCT116 cell lines. We used mitochondria isolated from both types of cells and from Bax(+) cells exposed to apoptotic stimuli, as well as Bax-less mitochondria into which exogenous Bax had been incorporated. All exhibited the same behavior and pharmacological profile in swelling and Ca(2+)-retention experiments. Mitochondria from a bax(-)/bak(-) cell line also underwent an analogous Ca(2+)/P(i)-inducible swelling. This similarity indicates that Bax hasno major role in regulating the Ca(2+)-induced mitochondrial permeability transition.

Animals↗

PubMed2003

Plant polyphenols inhibit VacA, a toxin secreted by the gastric pathogen Helicobacter pylori.

VacA is a major virulence factor of the widespread stomach-dwelling bacterium Helicobacter pylori. It causes cell vacuolation and tissue damage by forming anion-selective, urea-permeable channels in plasma and endosomal membranes. We report that several flavone derivatives and other polyphenols present in vegetables and plants inhibit ion and urea conduction and cell vacuolation by VacA. Red wine and green tea, which contain many of the compounds in question, also potently inhibit the toxin. These observations suggest that polyphenols or polyphenol derivatives may be useful in the prevention or cure of H. pylori-associated gastric diseases.

Anti-Bacterial Agents↗

PubMed2002

The vacuolating toxin of Helicobacter pylori mimicks the CFTR-mediated chloride conductance.

Cystic fibrosis (CF) is caused by defects of the CF transmembrane conductance regulator (CFTR), which acts both as an anion-selective channel and as a regulator of other proteins. The relative contribution of these two functions in CF disease is debated. The toxin VacA forms channels with properties similar to those of the CFTR, and we report here that it can insert into the membrane of various cells originating from respiratory epithelia, generating a chloride conductance comparable to that produced by activation of the CFTR. VacA may therefore become a valuable tool in the study of CF pathogenesis.

Bacterial Proteins↗

PubMed2002

The Vibrio cholerae haemolysin anion channel is required for cell vacuolation and death.

Several strains of Vibrio cholerae secrete a haemolytic toxin of 63 kDa, termed V. cholerae cytolysin (VCC). This toxin causes extensive vacuolation and death of cells in culture and forms an anion-selective channel in planar lipid bilayers and in cells. Here, we identify inhibitors of the VCC anion channel and show that the formation of the anion channel is necessary for the development of the vacuoles and for the cell death induced by this toxin. Using markers of cell organelles, we show that vacuoles derive from different intracellular compartments and we identify the contribution of late endosomes and of the trans-Golgi network in vacuole biogenesis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

DNA interacts with Bacillus subtilis mechano-sensitive channels in native membrane patches.

Much recent evidence indicates that systems devoted to the transmembrane transport of proteins and/or genetic material in bacteria comprise proteins capable of forming large pores as a key element. In several cases these pores have been observed in electrophysiological experiments after purification and reconstitution of the proteins in artificial bilayers. A comparison of their properties with those of large mechanosensitive channels observed by patch-clamping bacterial proto- or spheroplasts suggests that the latter may be formed by such transport machines. In support of this hypothesis, this paper reports that the properties of high-conductance channels in the membrane of Bacillus subtilis are altered by DNA through specific interactions. Thus, the previously demonstrated interaction between DNA and the same channels reconstituted in planar bilayers, which in that system results in the transmembrane translocation of the genetic material, takes place also in the native membrane.

Bacillus subtilis↗