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Den Herausforderungen des demographischen Wandels stellt sich Ambient Assisted Living (AAL) mittels ICT-basierter Technologien, die das Leben alterer Menschen situationsabhangig und unaufdringlich unterstutzen sollen. Diese Arbeit behandelt Einsatzbeispiele von M2M-Technologien im Bereich AAL. Die anwendungsorientieren Forschungsprojekte Gesundheitsdialog Diabetes, iStoppFalls, Fullstandsmessung mittels Near Field Communication (NFC) und Leichter Wohnen (moduLAAr) werden vorgestellt und erortert.

The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins. Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm. C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments. A fusion toxin containing the N-terminal domain of C2I (residues 1–225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998) Infect. Immun. 66, 1364–1369). We characterized the adaptor function of C2I and its interaction with C2IIa. The fusion toxin GST-C2I1–225-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I1–225 adaptor was positioned in the middle of a fusion protein. Amino acid residues 1–87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells. Residues 1–87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry. An excess of C2I1–87(but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa. Also, the fragment C2I30–431 and the fusion toxin C2I30–225-C3 competed with C2I-Alexa488 for binding to C2IIa. C2I30–225-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1–29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa.

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli . Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa 1–240 ) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa 244–263 ) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia 208–413 but loss of activity of several N-terminally deleted C2I proteins including C2I 103–431 , C2I 190–431 , and C2I 30–431 . The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

Hypersensitivity to nickel (Ni) represents the most common manifestation of contact allergy in humans. The role of metal-specific T cells in this disease is well established, but the molecular interactions involved in their activation are poorly understood. We examined the T cell receptor (TCR) repertoire in T cells activated with either NiSO4 or NiSO4-treated human serum albumin from six allergic patients. For the three most hyperreactive donors, we found a strong over-representation of the TCR BV17 element. TCR sequencing for one of these donors revealed an additional skewing for AV1 as well as a selection for an N region encoded argine at position 95 of the BV17 complementarity determining region (CDR)3. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by contacts with peptide rather than nickel. However, the CDR1 of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni-binding proteins or peptides. We therefore propose that the severe hypersensitivity reactions found in BV17 over-expressors may be the result of Ni2+ ions bridging the germ-line-encoded BV17 CDR1 loop to corresponding sites in the major histocompatibility complex/peptide complex and thereby creating a superantigen-like enhancement of weak TCR-peptide contacts.

The human pathogen Staphylococcus aureus successfully colonizes its primary reservoir, the nasal cavity, most likely by regulatory adaptation to the nose environment. Cotton rats represent an excellent model for the study of bacterial gene expression in the initial phases of colonization. To gain insight into the expression profile necessary for the establishment of colonization, we performed direct transcript analysis by quantitative real-time reverse-transcription polymerase chain reaction on cotton rat noses removed from euthanized animals on days 1, 4, or 10 after instillation of 2 human S. aureus nose isolates. Global virulence regulators (agr, sae) were not active in this early phase, but the essential 2-component regulatory system WalKR seems to play an important role. Accordingly, an elevated expression of walKR target genes (sak, sceD) could be detected. In agreement with previous studies that demonstrated the essential role played by wall teichoic acid (WTA) polymers in nasal colonization, we detected a strongly increased expression of WTA-biosynthetic genes. The expression profile switched to production of the adhesive proteins ClfB and IsdA at later stages of the colonization process. These data underscore the temporal differences in the roles of WTA and surface proteins in nasal colonization, and they provide the first evidence for a regulation of WTA biosynthesis in vivo.