Biomedical subjects

E Maier

Publications and source records attributed to E Maier.

At least 19 recordsLinked to original sources

PubMed2006

Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans.

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.

Bacterial Outer Membrane Proteins↗

PubMed2004

[Stationary thromboprophylaxis in casualty surgery. Relevance of postoperative mobility and preexisting risk factors].

The presented thromboprophylactic concept includes weight bearing and ankle motion as well as breathing therapy and drug prophylaxis (antiphlogistics, analgesic drugs, heparin). Routinely performed ultrasound screening of the deep veins (legs and pelvis) before release showed a low DVT incidence of 2.5% in a prospective clinical observation of 841 inpatients. Obesity, venous insufficiency, and a history of previous thromboembolic events were associated with a significantly increased risk of thrombosis (relative risk 4.1, 4,9, and 5.8, respectively) The duration of immobilization also had a relevant influence indicating that early postoperative physiotherapy in traumatology and orthopedic surgery has a widely underestimated thromboprophylactic effect.

Adolescent↗

PubMed2003

[Reformation as proposed solution for the problem of sectioning different levels with 3He-MRT and HR-CT of the chest].

PURPOSE: 3He-MRI of the lung has been shown to be a sensitive method for functional imaging of the lung. A previous study compared 3He-MRI (coronal planes) with CT (transverse planes) by looking for ventilation defects and their pathomorphologic correlation. Anatomic structures, such as lobar fissures and hilar vessels, were used for orientation, but the reliable assignment of ventilation defects to lung segments is problematic. The present work compares multiplanar reformations of 3He-MRI and HR-CT, which were generated from planes determined by the respective method, and investigates their suitability as a solution of this problem. MATERIALS AND METHODS: A total of 16 data sets taken from 15 patients with unilateral lung transplantation and one patient with lung emphysema were retrospectively evaluated. Transverse planes of 3He-MRI and coronal planes of HR-CT were reformatted on an external workstation and images evaluated by two readers in consensus. The evaluation searched for ventilation defects on 3He-MRI and their corresponding defects on HR-CT. The defects were related to anatomic structures, with hilar vessels and tracheobronchial tree selected for 3He-MRI reformations and lobar fissures for HR-CT reformations. RESULTS: All cases were successfully reformatted and all ventilation defects were correctly assigned to anatomic structures. On HR-CT reformations, the lobar fissures were partially visible in 12 of 16 cases and completely visible in the remaining 4 cases. Since reformation compromises the spatial resolution, the reformatted images should be evaluated together with the source images. CONCLUSION: Looking at HR-CT and 3He-MRI images and their reformations enables the detection of ventilation defects and their assignment to lung segments, facilitating the correlation of ventilation defects with a pathomorphologic pattern on HR-CT.

Adult↗

PubMed2002

Lung function of school children with low levels of alpha1-antitrypsin and tobacco smoke exposure.

Exposure to environmental tobacco smoke (ETS) and other air pollutants has been associated with small decrements in lung function. The susceptibility to pollution exposure may, however, vary substantially between individuals. Children with an impaired protease-antiprotease balance may be particularly vulnerable. Therefore this study aimed to investigate the effects of ETS exposure on children with reduced levels of alpha1-antitrypsin (alpha1-AT). Random samples of school children (aged 9-11 yrs) (n=3,526) were studied according to the International Study of Asthma and Allergies in Childhood (ISAAC) phase II protocol, including parental questionnaires, pulmonary function and allergy testing. Blood samples were obtained to measure plasma levels of alpha1-AT and to genotype for pleomorphic protein inhibitor (Pi)Z and PiS alleles. Children with low levels of alpha1-AT (< or = 116 mg x dL(-1)) showed significant, albeit small decrements in baseline lung function. When exposed to ETS, pronounced decrements of pulmonary function, particularly in measures of mid- to end-expiratory flow rates, were seen in these children as compared to exposed children with normal levels of alpha1-AT. The mean levels of % predicted+/-SE in both groups were: maximum expiratory flow at 50% of vital capacity 79.4+/-7.2 versus 99.0+/-1.5, maximum expiratory flow at 25% of vital capacity 67.4+/-10.0 versus 100.3+/-2.1, maximal midexpiratory flow 73.7+/-8.6 versus 99.9+/-1.7. These findings suggest that school children with low levels of alpha1-antitrypsin are at risk of developing pronounced decrements in pulmonary function, particularly if they are exposed to environmental tobacco smoke. Parents of children with heterozygous alpha1-antitrypsin deficiency resulting in significantly reduced blood concentrations should be advised to prevent their children from being exposed to environmental tobacco smoke and dissuade them from taking up smoking.

Child↗

PubMed2001

Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers.

Epsilon toxin is a potent toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in animals. One of the main properties of epsilon toxin is the production of edema. We have previously found that epsilon toxin causes a rapid swelling of Madin-Darby canine kidney cells and that the toxin does not enter the cytosol and remains associated with the cell membrane by forming a large complex (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., and Popoff, M. R. (1997) J. Bacteriol. 179, 6480-6487). Here, we report that epsilon toxin induced in Madin-Darby canine kidney cells a rapid decrease of intracellular K(+), and an increase of Cl(-) and Na(+), whereas the increase of Ca(2+) occurred later. The entry of propidium iodide that was correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test indicates that epsilon toxin formed large pores. In artificial lipid bilayers, epsilon toxin caused current steps with a single-channel conductance of 60 pS in 100 mm KCl, which represented general diffusion pores. The channels were slightly selective for anions, but cations could also penetrate. Epsilon toxin formed wide and water-filled channels permeable to hydrophilic solutes up to a molecular mass of at least 1 kDa, which probably represents the basic mechanism of toxin action on target cells.

Animals↗

PubMed2001

Low pH-induced formation of ion channels by clostridium difficile toxin B in target cells.

Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH </= 5.2) induced uptake of toxin B into the cytosol even in the presence of bafilomycin A1. Toxin B increased (86)Rb(+) release when preloaded Chinese hamster ovary cells were exposed to low pH (pH </= 5.6) for 5 min. Release of (86)Rb(+) depended on the concentration of toxin B and on the pH of the extracellular medium. An antibody directed against the holotoxin prevented channel formation, whereas an antibody against the N-terminal enzyme domain was without effect. The N-terminally truncated toxin B fragment consisting of amino acids 547-2366 increased (86)Rb(+) efflux when cells were exposed to low pH. Toxin B also induced pH-dependent channel formation in artificial lipid bilayer membranes. Clostridium sordellii lethal toxin, another member of the family of large clostridial cytotoxins, also induced increased (86)Rb(+) release at low pH. The results suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation.

Animals↗

PubMed2001

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia.

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127-195 of LcrV and to amino acids 106-173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.

Animals↗

PubMed2001

Identification of the outer membrane porin of Thermus thermophilus HB8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance.

The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.

Biological Transport↗

PubMed2000

Functional characterization of the conserved "GLK" motif in mitochondrial porin from Neurospora crassa.

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a "glycine-leucine-lysine" (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [alpha-(32)P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective beta-barrel that is not directly involved in ATP binding.

Adenosine Triphosphate↗

PubMed2000

The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding.

The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.

Binding Sites↗

PubMed2000

Helical CT angiography with maximum intensity projection in the assessment of aortic coarctation after surgery.

OBJECTIVE: The value of CT angiography and three-dimensional (3D) reconstructions was investigated in the postoperative care after surgical repair of aortic coarctation and compared with conventional angiography. SUBJECTS AND METHODS: Twenty-five patients referred because of suspicion of stenosis in the area of former coarctation were prospectively studied with CT angiography and catheter angiography. We determined the morphometric and morphologic findings such as aortic diameter, stenosis, aneurysm, intimal flaps, circumscribed pouch, or arteriosclerotic plaques with 3D reconstructions, using maximum-intensity-projection (MIP) technique and catheter angiography. The results of both techniques were compared. The ratio of the narrowest diameters of the former coarctation and the descending aorta was correlated with the systolic pullback blood pressure gradient in all patients. RESULTS: The former coarctation was normal in 11 patients, (44%), group A; narrowed in 12 children (48%), group B; and dilated in two children (8%), group C. An intimal flap and a circumscribed pouch were delineated in four subjects. MIP reconstructions and catheter angiography revealed identical results regarding the classification into groups A, B, C; intimal flaps; and circumscribed pouches. Statistical analysis revealed good correlation between the narrowest aortic diameters measured on MIP reconstructions and catheter angiography, whereas no correlation between the systolic pullback blood pressure gradient and the diameter ratio of the former coarctation and the descending aorta was found. CONCLUSION: CT angiography and 3D reconstructions using MIP represent a reliable noninvasive technique to replace diagnostic catheter angiography in the postoperative care of patients with coarctation and provide the clinician with valuable information concerning further invasive procedures.

Adolescent↗

PubMed1999

An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels.

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.

Adenylate Cyclase Toxin↗

PubMed1999

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.

Anions↗

PubMed1999

Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli.

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.

Amino Acid Sequence↗

PubMed1999

[Knowledge and effectiveness of so-called foot sole reflex massage].

Plantar reflex massage is hardly of any importance to insurance medicine. It however helps to explain the affinity of patients for so-called alternative medicine and its methods. A proof of the effectiveness of the pretended radius of curative properties has never been aspired to nor is it scientifically conceivable. Various aspects combine to create the attraction of the method. Two of these result precisely from its unscientific nature. The effect is to be found in the improvement of the general feeling of well-being.

Acupuncture Points↗

PubMed1999

Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin.

The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.

Electric Conductivity↗

PubMed1998

Permeability properties of the porin of spinach leaf peroxisomes.

The membrane of spinach leaf peroxisomes contains an anion-selective channel. Reconstitution experiments were performed with lipid bilayer membranes to study its permeability properties. A variety of different monovalent inorganic and organic anions were found to be permeable through the porin channel. Its single-channel conductance for these different ions suggested that the channel has a minimum diameter of about 0.6 nm. From selectivity measurement in KCl solution a ratio of the anion permeability to cation permeability of less than 0.04 was determined, indicating an almost ideal selectivity of the peroxisomal channel for chloride. The permeation of chloride through the peroxisomal channel could be blocked efficiently by the addition of increasing concentrations of organic anions to the aqueous phase. The results are consistent with a binding site for dicarboxylic anions inside the peroxisomal channel. A particular high stability constant for the binding was obtained for peroxisomal metabolites such as malate, oxaloacetate, succinate, and 2-oxoglutarate, which have to cross the membrane of plant peroxisomes in vivo. Among these solutes maximal binding affinity was determined for C4 dicarboxylic anions. The results indicate that the peroxisomal channel does not form a general diffusion pore similarly to known eukaryotic porins, but has specific properties comparable to specific and inducible porins, which have been characterized in some gram-negative bacteria.

Anions↗

PubMed1998

Vaccination against tick-borne encephalitis virus, a flavivirus, prevents disease but not infection, although viremia is undetectable.

By adoptive transfer of sera or immunoglobulin preparations, vaccine-induced protection against TBEV has been demonstrated to be mediated by antibodies to the surface protein of TBEV, glycoprotein E. Nevertheless, the mechanism of vaccine-induced protection against TBEV remains unclear. Protection by E antibodies without in vitro neutralization was shown by one group, whereas others found a correlation between protection in vivo and neutralization in vitro. Here, the authors confirm in a mouse model of tick-borne encephalitis (TBE) that immunization with the whole-killed virus vaccine protects mice against a subsequent challenge with a highly lethal dose of virus, i.e. 250 LD50 doses. Vaccine-induced immunity, however, is not completely neutralizing as demonstrated by the development of immune responses to a non-structural virus protein absent from the vaccine, yet expressed in the course of virus replication. Antibodies specific for the non-structural protein 1 (NS1) and cytotoxic T-cells could be detected after, but not prior to, virus challenge of vaccinated animals, establishing that protection by this highly effective vaccine is not equivalent with complete neutralization of the challenge virus.

Adoptive Transfer↗