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E Maier

Publications and source records attributed to E Maier.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of a novel mutation in exon 10 of the adrenoleukodystrophy gene.

We have detected a novel mutation in the adrenoleukodystrophy (ALD) gene in skin fibroblasts in primary culture derived from a patient suffering from the adrenocortical insufficiency-only-phenotype of ALD. This nonsense mutation (C2400T/Q672X) is the only mutation reported to date affecting exon 10. It leads to a translation product lacking the 74 C-terminal amino acids. As a consequence of the loss of this region, which immediately follows the putative nucleotide binding domain, the ALD protein (ALDP) was not detectable at all by ALDP-specific monoclonal antibodies. Since ALDP-specific mRNA was readily detected in these fibroblasts, the loss of protein is probably not attributable to RNA instability but may be explained by protein instability. If the Q672X mutation leads in fact to an unstable translation product this would be consistent with the hypothesis that the C-terminus is crucial for ALDP stability.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1998

Neutralizing antibodies protect against lethal flavivirus challenge but allow for the development of active humoral immunity to a nonstructural virus protein.

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.

Animals↗
PubMed1997

In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli.

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3'-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3'-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.

Bacterial Proteins↗
PubMed1997

Characterization of porin from Roseobacter denitrificans.

Porin from Roseobacter denitrificans was isolated and purified to homogeneity. The pore characteristics from this marine bacterium were compared to those of its phylogenetically closely related freshwater bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas blastica. The porin formed weakly cation-selective, general diffusion pores in lipid bilayer membranes. High transmembrane potentials caused channel closing in steps that were of one or two thirds of the initial on-steps indicating that the porin of R. denitrificans comprised three more or less independent channels similar to PhoE and OmpC of Escherichia coli and the porin of Rhodobacter capsulatus. 37b4 Prediction of the secondary structure of the 36 N-terminal amino acid residues indicated two transmembrane beta-strands similar to those of the porins of Rhodobacter capsulatus 37b4 and Rhodopseudomonas blastica. Differences of the single channel conductivities between the porin of R. denitrificans and those of the related freshwater bacteria show that R. denitrificans evolved porin channels that are well adapted to the marine habitat.

Amino Acid Sequence↗
PubMed1997

Purification of OmpU from Vibrio cholerae classical strain 569B: evidence for the formation of large cation-selective ion-permeable channels by OmpU.

The outer membrane of the classical Vibrio cholerae strain 569B was isolated by sucrose density centrifugation. The simple treatment of the isolated outer membrane or the cell envelopes with different detergents allowed the purification of two outer membrane proteins, the 38 kDa OmpU and the 25 kDa OmpV. Furthermore, a 35 kDa outer membrane protein (probably the 35 kDa OmpA-like protein) was purified by two-fold treatment of the cell envelope with 2% SDS solution. A subsequent wash of the SDS-pellet with 2% Genapol buffer yielded in the 38 kDa OmpU protein, which formed SDS-resistant oligomers (66 kDa). The Genapol pellet contained OmpV. Reconstitution experiments with lipid bilayer membranes demonstrated that OmpU was a channel-forming component, whereas OmpV had a small channel-forming ability if any. The OmpU channels appeared to be large and water-filled and had a single-channel conductance of about 2 nS in 1 M KCl for the monomer in a trimer, which means that they have a larger cross-section than enterobacterial porins. The channels showed rapid switching between open and closed configuration. They were slightly cation-selective, which suggests that they contain an excess of negatively charged amino groups.

Adhesins, Bacterial↗
PubMed1996

Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7.

Lipid bilayer experiments were performed with the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933. EHEC-hemolysin caused the formation of transient ion-permeable channels by integration in lipid bilayer membranes composed of asolectin, dioleoylglycerophosphoethanolamine and phosphoserine but not of diphytanoylglycerophosphocholine. Channel formation showed the same characteristics when culture supernatants of E. coli strains EDL 933 or HB101/pEO40, precipitated or purified EHEC-hemolysin were used for these experiments. The EHEC-hemolysin channels had two different states at small transmembrane potential (20 mV): a prestate that represented the first step of channel formation (single-channel conductance 40 pS in 0.15 M KCl) and an open state (550 pS in 0.15 M KCl at pH 6.0). Experiments with different salts suggested that the EHEC-hemolysin-induced channels were cation-selective at neutral pH. The mobility sequence of the cations within the channels resembles their mobility sequence in the aqueous phase. The single-channel data were consistent with the formation of wide, water-filled channels by the EHEC hemolysin. The single channel conductance was strongly pH dependent and increased over 2.5-fold in the pH range 5-8. The analysis of the single-channel data using the Renkin correction factor suggested that the EHEC-hemolysin formed channels with an average diameter of 2.6 nm. This size could be confirmed by the results of osmotic-protection experiments. Neither sucrose nor raffinose inhibited toxin-dependent hemolysis, whereas hemolysis did not occur in the presence of dextran 4 (molecular mass, 4 kDa). Our results demonstrate that EHEC-hemolysin can be considered to be a highly active repeats-in-toxin (RTX)-toxin with a similar but not identical pore-forming capacity as the chromosomal encoded E. coli alpha-hemolysin.

Electric Conductivity↗
PubMed1996

HlyA hemolysin of Vibrio cholerae O1 biotype E1 Tor. Identification of the hemolytic complex and evidence for the formation of anion-selective ion-permeable channels.

Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerae O1 biotype E1 Tor strains by ammonium sulfate precipitation. The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity. The toxin formed a high molecular-mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated. The addition of the E1 Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels, which had long lifetimes at small voltages. The hemolysin channel had a single-channel conductance of 350 pS in 1 M KCl. These results defined hemolysin (HlyA) from V. cholerae as a channel-forming component with properties similar to other cytolytic toxins. The long lifetime of the channel suggested that the channel-forming oligomer did not show a rapid association/dissociation reaction. At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition. The single-channel conductance of the hemolysin (HlyA) from V. cholerae O1 biotype E1 Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm. The hemolysin channel of V. cholerae was found to be moderately anion-selective. The pore-forming properties of hemolysin (HlyA) from V. cholerae O1 biotype E1 Tor were compared with those of aerolysin of Aeromonas sobria and alpha-toxin from Staphylococcus aureus. All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion-selective channels.

Amino Acid Sequence↗
PubMed1996

A novel sequence polymorphism in the promoter region of the human B2-bradykinin receptor gene.

The distribution of a nucleotide polymorphism in the core promoter of the human B2-bradykinin receptor gene was examined in the population of southern Germany. The allelic frequencies were 0.595 for C allele and 0.405 for the T allele. The allele frequencies were in Hardy-Weinberg equilibrium. This new marker provides a valuable tool to assess the risk for putative bradykinin-associated disorders with genetic determinism.

Alleles↗
PubMed1996

Arsenic speciation in marine organisms: from the analytical methodology to the constitution of reference materials.

An analytical procedure for total arsenic and arsenic species quantification in marine organisms has been developed. Fresh materials are freeze-dried and reduced to powders before analysis. Arsenic is determined either by energy dispersive X-ray fluorescence (EDXRF) directly or by inductively coupled plasma optical emission spectrometry (ICP/OES) after microwave digestion. Arsenic speciation is performed on the extracted sample using liquid chromatography coupled to ICP/OES for arsenobetaine and arsenocholine determination and to the hydride generation-quartz furnace atomic absorption spectrometric technique for arsenite, arsenate, monomethylarsonic and dimethylarsinic acids quantification. Special precautions are taken to avoid losses or contaminations as well as to prevent analytical errors during the quantification stage. Other methods are applied and the corresponding results compared for each step of the procedure. The method is finally validated by means of intercomparison studies within the Measurements and Testing Programme of the European Community (formely BCR).

Journal Article↗
PubMed1996

Polymorphisms in the gene for the human B2-bradykinin receptor. New tools in assessing a genetic risk for bradykinin-associated diseases.

The B2-bradykinin receptor gene has been proposed as one of the candidate genes involved in the complex genetic underpinnings of common chronic disorders such as hypertension, ischemic heart disease or allergic asthma. Suitable genetic markers are needed to study these hypotheses. Therefore, it was our aim to identify polymorphic sites in the B2-receptor gene. Up to now, we characterized four polymorphisms: one in the promoter region and three other ones in each of the exons. Possible biological consequences are delineated and preliminary results of allele specific different biological action are shown.

Alleles↗
PubMed1996

Channel-forming activity and channel size of the RTX toxins ApxI, ApxII, and ApxIII of Actinobacillus pleuropneumoniae.

The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were expressed in an Escherichia coli strain. The toxins were concentrated from the supernatants of cell cultures. The addition of the toxins to the aqueous-phase-bathing lipid bilayer membranes resulted in an increase in the membrane conductance when membranes made of asolectin or phosphatidylethanolamine were used. The toxins were relatively inactive in membranes made of other lipids. The membrane activity (i.e., the number of channels formed at a given Apx concentration) was different for each of the three Apx toxins. That of ApxI, which has the strongest cytotoxic activity, was highest, followed by that of ApxIII and ApxII, which is the least cytotoxic. The conductance increases of ApxIII and ApxII were smaller by factors of 10 and 50, respectively, than that of ApxI under otherwise identical conditions. Single-channel experiments demonstrated that all three Apx toxins formed ion-permeable channels of different conductances. The major open state was approximately the same for the two hemolytic toxins ApxI and ApxII (540 and 620 pS in 0.15 M KCI), whereas the single-channel conductance of the nonhemolytic ApxIII was approximately one-fifth of that of the other two toxins (95 pS). Experiments with different salts suggested that the Apx channels of A. pleuropneumoniae were exclusively cation selective because of negative charges localized at the channel mouth. Analysis of the single-channel data using the Renkin correction factor suggested that the Apx toxins formed aqueous channels with different diameters for the three toxins. Pore-forming properties of the Apx toxins were compared with those of other RTX toxins. All of these toxins have common features and form channels that are transient but have different sizes as judged from the different single-channel conductances.

Actinobacillus pleuropneumoniae↗
PubMed1996

Monocytes of individual human subjects display heterogeneous bacterial uptake and antilisterial activity.

Peripheral blood monocytes (Mo) of normal human donors simultaneously exhibit two subsets differing in their functional activity towards the facultative intracellular bacterium Listeria monocytogenes. One subset (on average, 25% of total Mo) was characteristically able to ingest a large number of L. monocytogenes bacteria and permitted intracellular growth of these bacteria. The other Mo subpopulation (on average, 75% of total Mo) was far less active in phagocytosing L. monocytogenes and restricted intracellular L. monocytogenes growth. Electron microscopy revealed that the Listeria-permissive Mo subset allowed the bacteria to escape to the cytosol, a mechanism by which these bacteria evade the lethal attack of phagocytes. The Listeria-restrictive Mo subset, on the other hand, confined the bacteria to the phagolysosomes, where they were exposed to the killing mechanisms of the Mo. Permissiveness for L. monocytogenes growth was further associated with differences in the capacity of the Mo subsets to synthesize tumor necrosis factor alpha TNF-alpha), an important mediator in the defense against intracellular bacteria. Following challenge with L. monocytogenes, the Listeria-restrictive Mo subset secreted two to six times more TNF-alpha than did the Listeria-permissive Mo subset. Enhanced TNF-alpha secretion was paralleled by increased accumulation of TNF-alpha mRNA as assessed by quantitative PCR. Despite these functional differences, the two Mo subsets were indistinguishable with respect to expression of cell surface markers known to be involved in adherence and phagocytosis of microbes. A speculative physiological role of the two Mo subsets may lie in the dual function of Mo as microbicidal effector cells and accessory cells for antigen-specific immune reactions.

Base Sequence↗
PubMed1995

The membrane of leaf peroxisomes contains a porin-like channel.

Spinach leaf peroxisomes were purified by Percoll density gradient centrifugation. After several freeze-thaw cycles, the peroxisomal membranes were separated from the matrix enzymes by sucrose density gradient centrifugation. The purity of the peroxisomal membranes was checked by measuring the activities of marker enzymes and by using antibodies. Lipid bilayer membrane experiments with the purified peroxisomal membranes, solubilized with a detergent, demonstrated that the membranes contain a channel-forming component, which may represent the major permeability pathway of these membranes. Control experiments with membranes of other cell organelles showed that the peroxisomal channel was not caused by the contamination of the peroxisomes with mitochondria or chloroplasts. The peroxisomal channel had a comparatively small single channel conductance of 350 pS in 1 M KCl as compared with channels from other cell organelles. The channel is slightly anion selective, which is in accordance with its physiological function. The single channel conductance was found to be only moderately dependent on the salt concentration in the aqueous phase. This may be explained by the presence of positive point net charges in or near the channel or by the presence of a saturable binding site inside the channel. The possible role of the channel in peroxisomal metabolism is discussed.

Cell Membrane↗
PubMed1995

Interaction with human immunodeficiency virus type 1 modulates innate effector functions of human monocytes.

The effect of human immunodeficiency virus (HIV) type 1 on human mononuclear phagocyte effector functions in response to infection with bacteria of the Mycobacterium avium-intracellular complex (MAC) was investigated. The results showed that interaction of HIV-1 or its constituents with CD4 expressed in the monocyte membrane led to substantial impairment of monocyte capacity to restrict the intracellular growth of MAC. This was accompanied by substantially decreased production of tumor necrosis factor-alpha by HIV-1-exposed and MAC-infected monocytes. However, productive HIV-1 infection of monocytes was not required to induce the observed effects. These studies suggest that HIV-1 may interfere with innate mononuclear phagocyte function. This may be of physiologic importance in the late stages of AIDS, when an impaired T cell immunity can no longer provide proper immune-activating signals, and may help to explain the undue susceptibility to MAC infections in these patients.

HIV-1↗
PubMed1995

Identification of channel-forming activity in the cell wall of Corynebacterium glutamicum.

The cell wall of the gram-positive Corynebacterium glutamicum was prepared. It contained an ion-permeable channel with a single-channel conductance of about 6 nS in 1 M KCl. The mobility sequence of the ions in the channel is similar to that in the aqueous phase, suggesting that it is a water-filled channel wide enough to allow unhindered diffusion of ions. The results indicate that we have identified the hydrophilic pathway through the mycolic acid layer of C. glutamicum.

Cell Fractionation↗
PubMed1995

Sequencing 500 kb of yeast DNA using a GATC 1500 Direct Blotting Electrophoresis System.

We have developed efficient protocols for the construction of shotgun libraries and for high-throughput DNA sequencing at low cost using the optimized GATC 1500 Direct Blotting Electrophoresis system with colorimetric detection on nylon membranes. We can now sequence entire cosmids without any primer walking at low coverage (8-fold to 9-fold). We have demonstrated the efficiency of our protocols while sequencing 500 kb of yeast DNA with the GATC 1500 system. The methods described here have allowed us to achieve the highest data rate within the framework of the European Communities yeast sequencing program. In 1993, only two people were involved in generating and submitting 156,274 bp of final sequence data. Thereof, 39,360 bp have been verified independently in other laboratories, and no sequencing error has been found.

Colorimetry↗
PubMed1994

Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli.

The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

Adenylate Cyclase Toxin↗