Biomedical subjects

G Molle

Publications and source records attributed to G Molle.

At least 37 records · Page 2Linked to original sources

PubMed1999

Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish.

Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16-0.18 microM) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.

Animals↗

PubMed1999

Isolation, amino acid sequence and functional assays of SGTx1. The first toxin purified from the venom of the spider scodra griseipes.

A new toxin (SGTx1) was purified from the venom of the spider Scodra griseipes by a combination of gel filtration and reverse-phase chromatography. The complete amino acid sequence of SGTx1, TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF, was established by direct automated Edman degradation, and is in perfect agreement with the molecular mass of 3775 Da found by mass spectrometry. The primary structure of SGTx1 exhibited sequence identity with other spider toxins such as hanatoxin (76%), TxP5 toxin (32%) and huwentoxin (26%). The six cysteines in the sequence suggested three disulfide bridges, the presence of which was demonstrated by mass spectrometry after dithiothreitol reduction. Analysis of secondary structure using circular dichroism spectrometry yielded more than 50% beta-sheet and about 15-20% beta-turn. The extent of the beta-content and the presence of disulfide bridges suggest a structure of interconnected beta-strands. In addition, a study of membrane/toxin interactions was carried out by reconstitution in planar lipid bilayers and by antibacterial assays. SGTx1 displays moderate pore-forming ability (conductance of about 100 pS in 1 M NaCl), but antibacterial activity was not observed against Gram-positive or Gram-negative strains. As a preliminary assay, the activity of SGTx1 was investigating using electrophysiological measurements. At 0.15 microM, SGTx1 reversibly inhibits more than 40% of outward potassium currents in rat cerebellum granular cells. This result is reminiscent with the effect described for hanatoxin extracted from the venom of Grammostola spatulata.

Amino Acid Sequence↗

PubMed1998

Ionic channels formed by a primary amphipathic peptide containing a signal peptide and a nuclear localization sequence.

The peptide SP-NLS (Ac-Met-Gly-Leu-Gly-Leu-His-Leu-Leu-Leu-Ala10-Ala-Ala-Leu-Gln-Gly- Ala -Lys-Lys-Lys-Arg20-Lys-Val-NH-CH2-CH2-SH) is composed of a hydrophobic signal sequence (SP, Met-1 to Ala-16) followed by a polycationic nuclear localization sequence (NLS, Lys-17 to Val-22) terminated by a cysteamide group. Designed to act as drug carrier this primary amphipathic peptide proved cytotoxic and bactericidal when used at high concentrations, probably by inducing the formation of ion channels. In this work, we show that indeed SP-NLS exhibits a pore-forming activity when incorporated into planar lipid bilayers and Xenopus laevis oocyte plasma membranes, with conductance values of 25 pS in 0.1 M NaCl. In both membranes, the insertion of the peptide was voltage-triggered whereas the induced conductances proved almost voltage-independent. Moreover, SP-NLS ion channels were selective for monovalent cations (K+>Na+>Li+>tetraethylammonium+>choline+). The ion channel activity of this type of peptides thus provides some insight on their toxicity but also on the mechanism involved for their membrane crossing process.

Amino Acid Sequence↗

PubMed1998

Alamethicin-like behaviour of new 18-residue peptaibols, trichorzins PA. Role of the C-terminal amino-alcohol in the ion channel forming activity.

The influences of peptide length, absence of a Glx (Gln/Glu) residue and the C-terminal amino alcohol on liposome permeabilization and ion-channel characteristics in planar lipid bilayers were examined with two 18-residue peptaibols, PA V and PA IX. As compared to the 20-residue alamethicin, both peptides belonging to the newly isolated trichorzin family, lack a proline in the N-terminal part and one of the two Gln/Glu residues in the C-terminal part of the sequence. The two analogues studied here differ among themselves in their C-terminal amino alcohol (tryptophanol for PA V and phenylalaninol for PA IX). These alpha-helical peptaibols modify to a similar extent the permeability of liposomes, as measured by leakage of a previously entrapped fluorescent probe. Monitoring tryptophanol fluorescence, a greater embedment of the peptide PA V is observed in cholesterol-free bilayers. Macroscopic conductance studies for PA V and PA IX display alamethicin-like current-voltage curves, with a similar voltage dependence, but a smaller mean number of monomers per conducting aggregate is estimated for the tryptophanol analogue, PA V. Single-channel recordings indicate faster current fluctuations for PA IX, while amplitude histograms show lower conductance levels for PA V. Apart from underlining the role of the mismatch between helix length and bilayer hydrophobic thickness, these results stress that the C-terminal tryptophanol favours a stabilization of the conducting aggregates.

Alamethicin↗

PubMed1998

Influence of the secondary structure on the pore forming properties of synthetic alamethicin analogs: NMR and molecular modelling studies.

Synthetic alamethicin analogs, in which all Aib residues had been replaced by Leu (L2) then proline 14 replaced by an alanine (L5), were studied in SDS micelles using circular dichroism and NMR spectroscopy. Nuclear Overhauser effects were used as constraints for molecular modelling. The structures determined for both peptides in SDS micelles were compared with those previously obtained in methanol in order to establish a secondary structure/ionophore activity relationship. Our results indicated that a shortening of peptide helices could be responsible for the observed decrease in ion channel lifetimes. However, the length of helices may not by itself explain the drastic destabilization of channels when Pro14 of alamethicin is replaced by Ala in L5. Indeed analysis of the helical wheel of L5 reveals heterogeneity in the amphipathicity depending on the medium. Thus, loss of amphipathicity seems to underly the observed destabilization of channels.

Alamethicin↗

PubMed1998

Synthesis and characterization of a new biotinylated gramicidin.

A new linear gramicidin analog bearing a biotinyl group grafted on C-terminal part was designed to study ligand-receptor interactions. The C-terminal alcohol in the native peptide was first replaced by an amino group. Then the peptide was synthesized on a polystyrene resin functionalized by the 2-chlorotrityl chloride following a biotinylation performed in solution. This new N'-biotinyl-(EDA)15-Gramicidin A was reconstituted in planar lipid bilayers and exhibited channel activities similar to those of natural gramicidin, with unitary conductance value about 30 ps in 1 M KCl. Furthermore this ionophore activity was quenched by addition of streptavidin in the surrounding medium. Our system is an outstanding tool for monitoring ligand-receptor interactions and could be used for designing a new biosensor.

Amino Acid Sequence↗

PubMed1998

A major outer membrane protein of Rahnella aquatilis functions as a porin and root adhesin.

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60 degrees C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand beta-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.

Amino Acid Sequence↗

PubMed1997

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers.

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.

Alamethicin↗

PubMed1997

Ion channel stability and hydrogen bonding. Molecular modelling of channels formed by synthetic alamethicin analogues.

Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

Alamethicin↗

PubMed1997

Interaction of the 14-residue peptaibols, harzianins HC, with lipid bilayers: permeability modifications and conductance properties.

Harzianins HC are a series of 14-residue peptaibols containing three Aib-Pro motives separated by sequences of two usual amino acids (Aib-Pro-Xaa-Xaa)n. They are organized in a subtype of the 3(10)-helix, which results in an approximate length of about 27-30 A for the helical rods, allowing them to span a bilayer. Permeabilization of small unilamellar vesicles composed of zwitterionic lipids (egg phosphatidylcholine/cholesterol 7/3 and 8/2) by harzianins HC was observed as well as voltage-gated macroscopic conductance and single-channel formation in planar lipid bilayers (DOPE/POPC 7/3) The permeabilization process was shown to increase with increasing the helix global hydrophobicity. The ion channel-for ming properties appeared rather favoured by an increase in the peptide amphipathicity. The set of conductance levels increasing in geometrical progression, reflecting the sequential uptake and release of monomers which is characteristic of the barrel-stave model for ion-channels described for alamethicin was not observed. The passage of ions through the bilayer would rather be the result of a set of aggregates with fixed numbers of monomers formed in the bilayer. The permeability process and the voltage-gated properties could thus result from different mechanisms showing that harzianins HC can permeabilize membranes via bilayer destabilization or channels, depending on the membrane system, composition and application of voltage.

Amino Acid Sequence↗

PubMed1997

Cavocaval liver transplantation without venovenous bypass and without temporary portocaval shunting: the ideal technique for adult liver grafting?

The influence of the implantation technique on the outcome was studied prospectively in a series of 116 consecutive adult patients undergoing primary liver transplantation during the period January 1991-June 1994. Thirty-eight patients (32.8%; group 1) underwent classical orthotopic liver transplantation (OLT) with replacement of the recipient's inferior vena cava (R-IVC) and with venovenous bypass (VVB). Thirty-nine patients (33.56%) had a piggy-back OLT with preservation of the R-IVC (group 2); bypass was used in 17 of them (43.6%) because of poor hemodynamic tolerance of R-IVC occlusion. Thirty-nine patients (33.6%) had OLT without VVB and with side-to-side cavocaval anastomosis (group 3). The three techniques were performed irrespective of the anatomical situation and of the status of the recipient at the time of transplantation. The following parameters were assessed in all patients: implantation time, blood product use, morbidity (e.g., hemorrhagic, thoracic, gastrointestinal, neurological, and renal complications), and outcome. Thirty-one patients underwent detailed intraoperative hemodynamic assessment. The early (< 3 months) post-transplant mortality of 10.3% (12/116 patients) was unrelated to the implantation technique. Group 3 had a significantly shorter mean implantation time, a reduced need for intraoperative blood products, and a lower rate of reoperation due to intra-abdominal bleeding. After excluding two immediate perioperative deaths and eight patients requiring early retransplantation because of primary nonfunction, the frequency of immediate extubation was significantly higher in group 3. Detailed hemodynamic assessment did not show a difference between 6 group 1 patients and 17 group 3 patients, indicating that partial lateral clamping of the IVC fulfills the function of venous bypass. Similar results were obtained in 6 group 2 patients who did not have IVC occlusion. Cavocaval OLT has become our preferred method of liver implantation. It allows the transplantation to be performed without VVB, regardless of the anatomical situation and of the condition of the patient at the time of transplantation. Moreover, it avoids all of the potential complications and costs of VVB.

Adult↗

PubMed1997

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

Alamethicin↗

PubMed1996

The inositol 1,4,5-trisphosphate-gated Ca2+ channel: effect of the protein thiol reagent thimerosal on channel activity.

The solubilized partially purified Ins(1,4,5)P3-sensitive Ca2+ channel from rat cerebellum has been reconstituted into planar lipid bilayer membranes by the 'tip-dip' method [Ehrlich (1992) Methods Enzymol. 207, 463-471] allowing low noise current records. Single-channel events have been recorded. In the presence of 10 microM Ins(1,4,5)P3, 50 microM ATP, and 0.2 microM Ca2+ the Ins(1,4,5)P3 receptor channel opens to a conductance level of 53 pS. In the presence of 100 microM thimerosal (TMS), a sulphydryl-oxidizing agent, three subconductance levels (60 pS, 80 pS and 120 pS) were observed. More than one population of mean open times was found, both in the absence and presence of TMS, although TMS affected the length of the open time by decreasing the short opening significantly from 4.05 ms to 2.78 ms and increasing the longer open time from 27.8 ms to 94.8 ms. The results indicate that TMS enhances Ins(1,4,5)P3-induced Ca2+ release by both altering the open times of the channel significantly and causing a shift to higher subconductance levels.

Adenosine Triphosphate↗

PubMed1996

Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio).

A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31-kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave Pcl/Pk ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018-0.18 microM) against different strains of gram-negative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp.; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

Amino Acid Sequence↗

PubMed1996

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.

Alamethicin↗

PubMed1995

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3.

The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).

Amino Acid Sequence↗

PubMed1995

Influence of proline-14 substitution on the secondary structure in a synthetic analogue of alamethicin.

Due to the bend introduced by proline 14 in the conformation of alamethicin (AcUPUAUAQUVUGLUPV UUEQFol), the role of this residue was assumed essential in the barrel-stave model for voltage-gated ion channels. Taking advantage of a previous synthetic alamethicin analogue (L2), in which all eight alpha-aminoisobutyric (U) were replaced by leucines (AcLPLALAQLV LGLLPV LLEQFol), another analogue (L5) was synthesized in order to test the effects of proline-14 substitution by an alanine (AcLPLALAQLVLGLLPVLLEQFol). Previous conductance experiments showed that both high voltage dependence and multistate behavior were conserved. In order to complement these functional results, a conformational study of L5 has been undertaken and compared to L2 using CD, high field nmr, and molecular dynamics. Results show that L5 presents a better ordered structure than L2 particularly in the region of the substitution and in the C-terminal part. These results are discussed as regards the previous hypothesis of the nonessential character of helix bending for the gating of voltage-dependent ion channels.

Alamethicin↗

PubMed1995

Sequencing of hydrophobic peptides as lithiated adducts using liquid secondary-ion mass spectrometry without tandem mass spectrometry.

Lithium cationized hydrophobic peptides in the molecular weight range 1900-2650 Da have been studied by liquid secondary-ion mass spectrometry. The mass spectra exhibit characteristic lithiated sequence ions. N-terminal [a(n)+Li-H]+ and [dn+Li-H]+ ions together with limited C-terminal [ypro+Li+H]+ ions lead to an easy sequencing of the peptides studied, without the need for tandem mass spectrometry.

Amino Acid Sequence↗