Biomedical subjects

Daniel Keller

Publications and source records attributed to Daniel Keller.

6 recordsLinked to original sources

PubMed2006

Engineered covalent leucotoxin heterodimers form functional pores: insights into S-F interactions.

The staphylococcal alpha-toxin and bipartite leucotoxins belong to a single family of pore-forming toxins that are rich in beta-strands, although the stoichiometry and electrophysiological characteristics of their pores are different. The different known structures show a common beta-sandwich domain that plays a key role in subunit-subunit interactions, which could be targeted to inhibit oligomerization of these toxins. We used several cysteine mutants of both HlgA (gamma-haemolysin A) and HlgB (gamma-haemolysin B) to challenge 20 heterodimers linked by disulphide bridges. A new strategy was developed in order to obtain a good yield for S-S bond formation and dimer stabilization. Functions of the pores formed by 14 purified dimers were investigated on model membranes, i.e. planar lipid bilayers and large unilamellar vesicles, and on target cells, i.e. rabbit and human red blood cells and polymorphonuclear neutrophils. We observed that dimers HlgA T28C-HlgB N156C and HlgA T21C-HlgB T157C form pores with similar characteristics as the wild-type toxin, thus suggesting that the mutated residues are facing one another, allowing pore formation. Our results also confirm the octameric stoichiometry of the leucotoxin pores, as well as the parity of the two monomers in the pore. Correctly assembled heterodimers thus constitute the minimal functional unit of leucotoxins. We propose amino acids involved in interactions at one of the two interfaces for an assembled leucotoxin.

Amino Acid Sequence↗

PubMed2006

Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.

Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor-acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA-HlgB or HlgB-HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA-HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB-HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

Amino Acid Substitution↗

PubMed2005

Sensitive and specific detection of staphylococcal epidermolysins A and B in broth cultures by flow cytometry-assisted multiplex immunoassay.

Two of the most common bacterial skin infections of young infants and children are bullous impetigo due to Staphylococcus aureus and its more acute form, staphylococcal scalded skin syndrome. Epidermolysin A (ETA), ETB and, possibly, ETD are responsible for these diseases, which may appear as epidemics in pediatric patients. We tested the reliability of a flow cytometry-assisted multiplex immunoassay (Bio-Plex system) for the detection of ETA and ETB. The Bio-Plex system was found to be highly specific and highly sensitive for toxin concentrations of between 2 and 80,000 pg/ml. The results of this assay were 100% identical to the results of a PCR-based method. We demonstrated that this test did not generate any cross-reactions with ETD-producing isolates. The level of detection of ETB by this test differed according to culture conditions and from isolate to isolate; these results must be taken into account for diagnostic purposes.

Exfoliatins↗

PubMed2004

Crystallization and preliminary crystallographic data of a leucotoxin S component from Staphylococcus aureus.

Class S proteins of staphylococcal bicomponent pore-forming leucotoxins play an important role in membrane targetting and cell specificity. Wild-type and recombinant S components of the Panton-Valentine leucocidin (LukS-PV) were expressed in Staphylococcus aureus and Escherichia coli, respectively, and purified. Both proteins were crystallized in two crystal forms with Jeffamine M-600 as the precipitant at 285 K using the hanging-drop vapour-diffusion method and seeding techniques. Crystals belong to space group P2 (or P2(1)) and P4(1) (or P4(3)), with unit-cell parameters a = 72.3, b = 95.1, c = 108.1 A, beta = 106.4 degrees and a = b = 94.8, c = 306.2 A, respectively. A full set of X-ray diffraction data was collected to 2.1 A from a single tetragonal crystal of the wild-type protein at 100 K.

Crystallography, X-Ray↗

PubMed2002

Network therapy for cocaine abuse: use of family and peer supports.

Cocaine-dependent subjects were treated by psychiatric residents in a 24-week sequence of Network Therapy. This approach, developed for practitioners in solo practice, employs a cognitive-behavioral orientation in sessions with family and peers as well as in individual sessions. Of 47 subjects, 73% of all observed weekly urines were negative for cocaine, and 20 (45%) of the subjects had negative toxicologies in the last 3 scheduled samples. A positive outcome was associated with the number of network (but not individual) sessions attended and completion of the full treatment sequence. Results suggest the utility of Network Therapy, even in the hands of relatively naive therapists.

Adult↗

PubMed1995

[Pharmacokinetic Simulations for Teaching]

A new pharmacokinetic simulation program called PharmaSim has been developed. It is focused on the simulation of drug plasma levels based on compartment models. The major advantage lies in the easy visualization of curves and the high speed of the simulations. When the value of a parameter changes the display of the corresponding curve reacts almost instantly (delay time < 0.1 sec.). This allows to visualize the effects of one parameter in a formula, a concept which was hard to illustrate so far. The simulations are embedded in a HyperCard-line electronic book. The contents of the tutorial pages - texts and graphics - as well as the preset values for the simulations can be saved in script files. This makes PharmaSim a tool with which many different tutorials can be created.

Journal Article↗