Biomedical subjects

M Colombini

Publications and source records attributed to M Colombini.

At least 19 recordsLinked to original sources

PubMed2006

Searching for the molecular arrangement of transmembrane ceramide channels.

Ceramides have been implicated in the initiation of apoptosis by permeabilizing the mitochondrial outer membrane to small proteins, including cytochrome c. In addition, ceramides were shown to form large metastable channels in planar membranes and liposomes, indicating that these lipids permeabilize membranes directly. Here we analyze molecular models of ceramide channels and test their stability in molecular dynamics simulations. The structural units are columns of four to six ceramides H-bonded via amide groups and arranged as staves in either a parallel or antiparallel manner. Two cylindrical assemblies of 14 columns (four or six molecules per column) were embedded in a fully hydrated palmitoyloleoyl-phosphatidylcholine phospholipid bilayer, and simulated for 24 ns in total. After equilibration, the water-filled pore adopted an hourglass-like shape as headgroups of ceramides and phospholipids formed a smooth continuous interface. The structure-stabilizing interactions were both hydrogen bonds between the headgroups (including water-mediated interactions) and packing of the hydrocarbon tails. Ceramide's essential double bond reduced the mobility of the hydrocarbon tails and stabilized their packing. The six-column assembly remained stable throughout a 10-ns simulation. During simulations of four-column assemblies, pairs of columns displayed the tendency of splitting out from the channels, consistent with the previously proposed mechanism of channel disassembly.

Apoptosis↗

PubMed2006

Dihydroceramide hinders ceramide channel formation: Implications on apoptosis.

Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to small proteins. The self-assembly of ceramide to form channels could be the means by which intermembrane space proteins are released to induce apoptosis. Dihydroceramide desaturase converts dihydroceramide to ceramide. This conversion may be removing an inhibitor as well as generating a pro-apoptotic agent. We report that both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. One tenth as much dihydroceramide was sufficient to inhibit the permeabilization of the outer membrane by about 95% (C2) and 51% (C16). Similar quantities inhibited the release of carboxyfluorescein from liposomes indicating that other mitochondrial components are not necessary for the inhibition. The apoptogenic activity of ceramide may thus depend on the ceramide to dihydroceramide ratio resulting in a more abrupt transition from the normal to the apoptotic state when the de novo pathway is used in mitochondria.

Animals↗

PubMed2003

[Jeiuno-ileal diverticula complicated by perforation. Clinical case].

The diverticular disease is rarely located in the small intestine (0.1-1.4%). The most important feature is due to the lack of a typical symptomatology which may appear only on the occasion of the complications it may incur (perforation, haemorrhage and so on). It isn't also infrequent that the surgeon may observe intestinal diverticula accidentally, on the occasion of laparotomies carried out in emergency or for other pathologies. The literature on intestinal diverticula is reviewed and personal experience in a clinical case presenting as acute abdomen is described.

Abdomen, Acute↗

PubMed2003

[Day Surgery. Five years of experience and activity].

BACKGROUND: The aim of this paper is to evaluate the management of an integrated unit of Day Surgery in a General Surgery Department. METHODS: An outline of the first 5 years of activity is presented. Setting of investigation: little provincial hospital. Patients have been selected on a basis of Day Surgery specific features: a total of 1,294 patients. Pathologies treated: inguinal hernias, phlebectasias, phimosis, borsitis, arthritic cysts, tendon cysts, carpal tunnel, Dupuytren's disease ("crispatura tendinum"), lymphadenitis, mammary nodules, hemorrhoids, hydroceles, varicoceles, adipomas. Patients' selection parameters, surgical operation type and modalities, postoperative course, protected discharges from hospital, follow-up and complications have all been carefully recorded. RESULTS: The most numerous operations were related to hernial pathology (54.32%) and to adipomas (10.81%). The form of anesthesia was mainly loco-regional. Only 4 cases (0.3%) had to be hospitalized for the night after operation. COMPLICATIONS: 1 serious wound infection, needing removal of the hernial prosthesis; 1 painful "tumefaction" on the inguinal wound for hernioplastic operation; 3 "seromas" in inguinal hernioplastics. CONCLUSIONS: Advantages of Day-Surgery: cut in health costs due to the reduction of admissions to hospital; reduction in hospital infections and in surgical wounds; reduction in drug use; thinning of waiting lists; increase of available beds for other pathologies; reduction of disability days; high appreciation index.

Adolescent↗

PubMed2002

VDAC channels differentiate between natural metabolites and synthetic molecules.

VDAC provides the major permeability pathway through the mitochondrial outer membrane by forming voltage-gated channels with pore radius of 1.2-1.5 nm. We find that VDAC can select among comparably-charged molecules with a much smaller effective radius, 0.4-0.5 nm. The molecules studied were the nucleotides, ATP, UTP, NADH and synthetic anions, tetraglutamate (T-Glu) and 1-hydroxypyrene-3,6,8-trisulfonate (HPTS). VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl (buffered to pH 8.0). The nucleotides decreased the conductance of VDAC for NaCl demonstrating that they could permeate into the channel. In contrast, T-Glu and HPTS did not change the single-channel conductance, indicating exclusion from the channel. Reversal potential measurements report near ideal selectivity of Na + over T-Glu. The nucleotides increased single-channel noise as they penetrated into the channel, while T-Glu had no effect. HPTS increased noise, but unlike NADH, this was not voltage-dependent when HPTS was added asymmetrically, indicating no penetration into the channel. The differences in effective size and charge cannot explain the difference in permeation characteristics. Thus VDAC must select among these based on shape and charge distribution. We propose that the electrostatic environment within the channel has been evolutionarily selected to favor the passage of adenine nucleotides.

Adenosine Triphosphate↗

PubMed2001

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane.

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.

Adenosine Diphosphate↗

PubMed2001

Actin modulates the gating of Neurospora crassa VDAC.

VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nM caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. alpha-Actin, from mammalian muscle, has a stronger effect than beta- and gamma-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase- did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism.

Actins↗

PubMed2001

Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy.

Lachesis muta muta and Bothrops atrox snakes are responsible for most accidents occurring in the Amazon. The clinical features of the accidents are similar; however, there are still controversies about the efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the antigenic cross-reactivity between these venoms using polyclonal and monoclonal antibodies and the efficacy of B. atrox and L. m. muta experimental antivenoms in cross-neutralizing the main toxic activities of each venom. Electrophoretic patterns differed consistently between the species. However, antigenic cross-reactivity was extensive except for a few bands. Several species-specific monoclonal antibodies were obtained by immunization of Balb/c mice with L. m. muta whole venom or B. atrox and L. m. muta specific antigens. The monoclonal antibodies specific to L. m. muta recognized different bands of this venom and the antibodies specific to B. atrox recognized a complex pattern on whole venom by Western blotting. These antibodies are important tools for developing an immunoassay able to discriminate patients bitten by these snakes. The experiments involving cross-neutralization of the main activities of the venoms showed that hemorrhage and blood incoagulability induced by B. atrox venom were similarly neutralized by both B. atrox and L. m. muta antivenoms. However, B. atrox antivenom partially neutralized the hemorrhage and completely failed in neutralizing coagulopathy induced by L. m. muta venom. Therefore, antigenic variation between B. atrox and L. m. muta venoms does occur and the use of specific antivenom is suggested for patients bitten by Lachesis snakes.

Animals↗

PubMed2000

The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis.

We report that physiological concentrations of both short- and long-chain ceramides, despite being lipids, form large stable pores in membranes. Some of these pores should be large enough to allow cytochrome c to permeate. Dihydroceramide differs from ceramide by the reduction of one double bond, and yet both its apoptogenic and channel-forming activities are greatly reduced. A structural model provides insight into how ceramides might form pores. According to a mathematical model, both the individual conductance of the channels and the overall membrane conductance are directly related to the overall concentration of ceramide in the membrane. Slight changes in concentration have dramatic effects on the size of the channels formed, providing an easy way for rapidly altering membrane permeability by changing the activity of local synthetic and catabolic enzymes. A possible role for these channels in apoptosis is discussed.

Apoptosis↗

PubMed2000

Positive cooperativity without domains or subunits in a monomeric membrane channel.

The monomeric VDAC channel shows an accelerated pH titration of its transport properties with a Hill coefficient of about 2. This manifests itself as a sharp peak in conductance noise as well as a fast change in channel selectivity with pH. On the basis of the known structure of this channel, we propose that this cooperativity arises from a mechanically linked mobile pair of ionizable groups. Concerted movement of these groups between two states changes the distance from nearby electrostatic charge to influence the pK of the groups. This model of pH-dependent motion produces positive cooperative behavior that fits the observations without need for subunits or identifiable domains within the protein. The mathematical formalism has never required such domains, but these are generally considered an essential part of cooperative behavior in proteins. The present proposal reduces the size of a cooperative unit to a minimum, extending the limits of what is perceived to be possible. Together with large-scale conformational transitions, these subtle cooperative structural changes may allow proteins to adapt, with high sensitivity, to changes in their environment. They might also be relatively easy to engineer into a protein.

Allosteric Regulation↗

PubMed2000

Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival.

Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the mitochondrial matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across the mitochondrial membranes. Unexpectedly, we find that this defect results from loss of outer mitochondrial membrane permeability to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany closure of the voltage-dependent anion channel (also known as mitochondrial porin). Loss of outer membrane permeability (i) results in the accumulation of stored metabolic energy within the intermembrane space in the form of creatine phosphate, (ii) is prevented by the outer mitochondrial membrane proteins Bcl-x(L) and Bcl-2, and (iii) can be reversed by growth factor readdition. If outer membrane impermeability persists, the disruption of mitochondrial homeostasis culminates in loss of outer mitochondrial membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that outer membrane permeability is regulated under physiological conditions has important implications for the understanding of bioenergetics and cell survival.

Adenosine Diphosphate↗

PubMed1999

Mouse VDAC isoforms expressed in yeast: channel properties and their roles in mitochondrial outer membrane permeability.

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed.

Animals↗

PubMed1999

Meningococcal PorA/C1, a channel that combines high conductance and high selectivity.

Class 1 porins (PorA/C1) from Neisseria meningitidis achieve both high selectivity and high conductance. The channel is highly selective (24:1 Na+ over Cl-), suggesting a highly negatively charged selectivity filter. The trimeric nature of PorA/C1 accounts for part of the enormous conductance in 200 mM NaCl (0.97nS). However, the currents that can be achieved exceed the simple infinite-sink calculation for a pore 0.7 nm in radius (estimated from nonelectrolyte permeability). The conductance is linear with salt activity from 20 mM to 2.0 M NaCl with no sign of saturation at low salt. Impermeant polymers reduce the conductance in a manner consistent with their ability to reduce bulk conductivity. Extrapolating from the known structure of homologous porins, the selectivity filter is likely to be small and localized. If small and highly negatively charged ( approximately 9 charges), the predicted conductance would be an order of magnitude higher than that observed. The rate at which ions reach the selectivity filter seems to limit overall ionic flux. PorA/C1 rectifies strongly, and this rectification can be accounted for by calculated differences in the voltage and concentration profiles in the access regions. Thus, it appears that the conductance of this channel is determined by the access resistance and the selectivity by a highly-conductive filter.

Amino Acid Sequence↗

PubMed1998

The topology of VDAC as probed by biotin modification.

The outer membrane of mitochondria contains channels called VDAC (mitochondrial porin), which are formed by a single 30-kDa protein. Cysteine residues introduced by site-directed mutagenesis at sites throughout Neurospora crassa VDAC (naturally devoid of cysteine) were specifically biotinylated prior to reconstitution into planar phospholipid membranes. From previous studies, binding of streptavidin to single biotinylated sites results in one of two effects: reduced single-channel conductance without blockage of voltage gating (type 1) or locking of the channels in a closed conformation (type 2). All sites react with streptavidin only from one side of the membrane. Here, we extend this approach to VDAC molecules containing two cysteines and determine the location of each biotinylated residue with respect to the other within the membrane. When a combination of a type 1 and a type 2 site was used, each site could be observed to react with streptavidin. Two sets of sites located on opposite surfaces of the membrane were identified, thereby establishing the transmembrane topology of VDAC. A revised folding pattern for VDAC, consisting of 1 alpha helix and 13 beta strands, is proposed by combining these results with previously obtained information on which sites are lining the aqueous pore.

Amino Acid Sequence↗

PubMed1998

Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1-1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages (Vo=15 mV) than the other (Vo=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less (n=1.3) than that observed for the other process or for the wild-type channel (n=1.5-1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiological function.

Bacterial Outer Membrane Proteins↗

PubMed1998

The role of yeast VDAC genes on the permeability of the mitochondrial outer membrane.

In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the POR1 gene (delta por1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1 in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability. The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria.

Genes, Fungal↗

PubMed1998

The voltage-gating process of the voltage-dependent anion channel is sensitive to ion flow.

The voltage-dependent anion channel (VDAC) is a voltage-gated channel from the mitochondrial outer membrane. It has two gating processes: one at positive potentials and the other at negative potentials. The energetics of VDAC gating are quite different when measured in the presence or absence of an ion gradient. A positive potential on the high-salt side results in channel closure at lower transmembrane potentials. The midpoint potential (V0) shifted from 25 to 5.7 mV, with an activity gradient for KCl of 0.6 versus 0.06. The opposite occurred for negative potentials on the high-salt side (V0 shifted from -25 to -29 mV). Thus the salt gradient favored closure for one gating process and opening for the other. These results could be explained if part of the electrochemical potential of the gradients present were transferred to the gating mechanism. If the kinetic energy of the ion flow were coupled to the gating process, the effects of the gradient would depend on the mass and velocities of these ions. This was tested by using a series of different salts (KCl, NaCl, LiCl, KBr, K acetate, Na butyrate, and RbBr) under an identical activity gradient. The kinetic energy correlated very well with the measured shifts in free energy of the channel gating. This was true for both polarities. Thus the gating of VDAC is influenced by ion flow. These results are consistent in sign and direction with the voltage gating process in VDAC, which is believed to involve the movement of a positively charged portion of the wall of the channel out of the membrane.

Anions↗

PubMed1998

The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes.

The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora crassa VDAC (naturally lacks cysteine). These were chemically biotinylated and reconstituted into planar phospholipid membranes. In the 19 sites examined, only two types of results were observed upon streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state. The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening. The binding sterically interferes with ion flow. The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions. The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface. No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited. From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.

Amino Acid Sequence↗