Investigation of AlInN barrier ISFET structures with GaN capping for pH detection

In the last decade the interest in nitride-based sensors (gas, ions...) and bio-sensors is increased. In the case of ion sensitive FET (ISFET), gate voltages induced by ions adsorbed onto the gate region modulate the source-drain currents.

Bacteria lacking a multidrug pump: A sensitive tool for drug discovery

Microorganisms express multidrug resistance pumps (MDRs) that can confound antibiotic discovery. We propose the use of mutants deficient in MDRs to overcome this problem. Sensitivity to quinolones and to amphipathic cations (norfloxacin, benzalkonium chloride, cetrimide, pentamidine, etc.) was increased 5- to 30-fold in a Staphylococcus aureus mutant with a disrupted chromosomal copy of the NorA MDR. NorA was required both for increased sensitivity to drugs in the presence of an MDR inhibitor and for increased rate of cation efflux. This requirement suggests that NorA is the major MDR protecting S. aureus from the antimicrobials studied. A 15- to 60-fold increase in sensitivity to antimicrobials also was observed in wild-type cells at an alkaline pH that favors accumulation of cations and weak bases. This effect was synergistic with a norA mutation, resulting in an increase up to 1,000-fold in sensitivity to antimicrobials. The usefulness of applying MDR mutants for natural product screening was demonstrated further by increased sensitivity of the norA− strain to plant alkaloid antimicrobials, which might be natural MDR substrates.

Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118–140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126–140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β-barrel. In contrast, human granulocytes were found to bind α-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor

The RKKEE cluster of charged residues located within the cytoplasmic helix of the bacterial mechanosensitive channel, MscL, is essential for the channel function. The structure of MscL determined by x-ray crystallography and electron paramagnetic resonance spectroscopy has revealed discrepancies toward the C-terminus suggesting that the structure of the C-terminal helical bundle differs depending on the pH of the cytoplasm. In this study we examined the effect of pH as well as charge reversal and residue substitution within the RKKEE cluster on the mechanosensitivity of Escherichia coli MscL reconstituted into liposomes using the patch-clamp technique. Protonation of either positively or negatively charged residues within the cluster, achieved by changing the experimental pH or residue substitution within the RKKEE cluster, significantly increased the free energy of activation for the MscL channel due to an increase in activation pressure. Our data suggest that the orientation of the C-terminal helices relative to the aqueous medium is pH dependent, indicating that the RKKEE cluster functions as a proton sensor by adjusting the channel sensitivity to membrane tension in a pH-dependent fashion. A possible implication of our results for the physiology of bacterial cells is briefly discussed.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′- dibehenate) - A novel adjuvant inducing both strong CMI and antibody responses:a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4°C and 25°C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Biosensor based on excessively tilted fiber grating in thin-cladding optical fiber for sensitive and selective detection of low glucose concentration

We report a highly sensitive, high Q-factor, label free and selective glucose sensor by using excessively tilted fiber grating (Ex-TFG) inscribed in the thin-cladding optical fiber (TCOF). Glucose oxidase (GOD) was covalently immobilized on optical fiber surface and the effectiveness of GOD immobilization was investigated by the fluorescence microscopy and highly accurate spectral interrogation method. In contrast to the long period grating (LPG) and optical fiber (OF) surface Plasmon resonance (SPR) based glucose sensors, the Ex-TFG configuration has merits of nearly independent cross sensitivity of the environmental temperature, simple fabrication method (no noble metal deposition or cladding etching) and high detection accuracy (or Q-factor). Our experimental results have shown that Ex-TFG in TCOF based sensor has a reliable and fast detection for the glucose concentration as low as 0.1~2.5mg/ml and a high sensitivity of ~1.514nm·(mg/ml)−1, which the detection accuracy is ~0.2857nm−1 at pH 5.2, and the limit of detection (LOD) is 0.013~0.02mg/ml at the pH range of 5.2~7.4 by using an optical spectrum analyzer with a resolution of 0.02nm.

Mise au point de complexes liposome/polymère sensibles au pH pour la vectorisation d'agents anticancéreux

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mise au point de complexes liposome/polymère sensibles au pH pour la vectorisation d'agents anticancéreux

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Temporal Control of Ion Channel Activation

Ion channels are a large class of integral membrane proteins that allow for the diffusion of ions across a cellular membrane and are found in all forms of life. Pentameric ligand-gated ion channels (pLGICs) comprise a large family of proteins that include the nicotinic acetylcholine receptor (nAChR) and the γ-aminobutyric acid (GABA) receptor. These ion channels are responsible for the fast synaptic transmission that occurs in humans and as a result are of fundamental biological importance. pLGICs bind ligands (neurotransmitters), and upon ligand-binding undergo activation. The activation event causes an ion channel to enter a new physical state that is able to conduct ions. Ion channels allow for the flux of ions across the membrane through a pore that is formed upon ion channel activation. For pLGICs to function properly both ligand-binding and ion channel activation must occur. The ligand-binding event has been studied extensively over the past few decades, and a detailed mechanism of binding has emerged. During activation the ion channel must undergo structural rearrangements that allow the protein to enter a conformation in which ions can flow through. Despite this great and ubiquitous importance, a fundamental understanding of the ion channel activation mechanism and kinetics, as well as concomitant structural arrangements, remains elusive.

This dissertation describes efforts that have been made to temporally control the activation of ligand-gated ion channels. Temporal control of ion channel activation provides a means by which to activate ion channels when desired. The majority of this work examines the use of light to activate ion channels. Several photocages were examined in this thesis; photocages are molecules that release a ligand under irradiation, and, for the work described here, the released ligand then activates the ion channel. First, a new water-soluble photoacid was developed for the activation of proton-sensitive ion channels. Activation of acid-sensing ion channels, ASIC2a and GLIC, was observed only upon irradiation. Next, a variety of Ru²⁺ photocages were also developed for the release of amine ligands. The Ru²⁺ systems interacted in a deleterious manner with a representative subset of biologically essential ion channels. The rapid mixing of ion channels with agonist was also examined. A detection system was built to monitor ion channels activation in the rapid mixing experiments. I have shown that liposomes, and functionally-reconstituted ELIC, are not destroyed during the mixing process. The work presented here provides the means to deliver agonist to ligand-gated ion channels in a controlled fashion.