Lucy Tunggal

Laminin 5 processing and its integration into the ECM.

Laminins are a family of multi-functional basement membrane proteins. Their C-terminal domain binds to cell surface receptors and is thereby responsible for cell anchorage and the initiation of specific outside-in and inside-out signals. With their N-terminal parts, laminins interact with proteins of the extracellular matrix scaffold to secure the basement membrane to the underlying mesenchymal tissue. Laminins 5A (alpha3Abeta3gamma2), 5B (alpha3Bbeta3gamma2) and 6 (alpha3Abeta1gamma1) are isoforms specific of the basement membrane underneath the epidermis and they undergo a sequential series of extracellular proteolytic changes, which might successively turn on and off one or several of their biological and mechanical functions. Under physiological conditions, such as in adult human skin, epithelial laminins have lost part of the C- and N-terminal domains of the alpha3 and gamma2 chains, respectively. In contrast, in cylindromatosis, a rare inherited disease characterised by major ultrastructural alterations of the basement membrane and altered expression/distribution of integrin receptors, laminin processing has not been completed. Together, these results suggest that laminin processing may regulate signalling pathways and the architecture of the basement membrane by restricting the repertoire of interactions with cell surface receptors and extracellular matrix components.

Keratinocytes from patients lacking collagen XVII display a migratory phenotype.

Acquired or inherited junctional epidermolysis bullosa are skin diseases characterized by a separation between the epidermis and the dermis. In inherited nonlethal junctional epidermolysis bullosa, genetic analysis has identified mutations in the COL17A1 gene coding for the transmembrane collagen XVII whereas patients with acquired diseases have autoantibodies against this protein. This suggests that collagen XVII participates in the adhesion of basal keratinocytes to the extracellular matrix. To test this hypothesis, we studied the behavior of keratinocytes with null mutations in the COL17A1 gene. Initial adhesion of mutant cells to laminin 5 was comparable to controls and similarly dependent on alpha3beta1 integrins. The spreading of mutant cells was, however, enhanced, suggesting a propensity to migrate, which was confirmed by migration assays. In addition, laminin 5 deposited by collagen XVII-deficient keratinocytes was scattered and poorly organized, suggesting that correct integration of laminin 5 within the matrix requires collagen XVII. This assumption was supported by the co-distribution of the two proteins in the matrix of normal human keratinocytes and by protein-protein-binding assays showing that the C-terminus of collagen XVII binds to laminin 5. Together, the results unravel an unexpected role of collagen XVII in the regulation of keratinocyte migration.

Molecular basis of inherited skin-blistering disorders, and therapeutic implications

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.

Defective Laminin 5 Processing in Cylindroma Cells

Cylindromas are benign skin tumors occurring as multiple nodules characteristically well circumscribed by an excess of basement membrane-like material. To determine the molecular defects leading to extracellular matrix accumulation, the ultrastructural, immunological, and biochemical properties of cylindroma tissue and isolated cells were analyzed. In cylindromas, hemidesmosomes are reduced in number, heterogeneous and immature compared to the normal dermal-epidermal junction. Expression of the alpha6beta4 integrin in tumor cells is weaker than in basal keratinocytes of the epidermis. Moreover, although in the epidermis alpha2beta1-integrin expression is restricted to the basal cell layer, it is found in all neoplastic cells within the nodules. Laminin 5 is present throughout the whole thickness of the basement membrane-like zone whereas laminin 10 is restricted to the interface adjacent to the tumor cells. Furthermore, laminin 5 is not properly processed and most of the alpha3A and gamma2 laminin chains remain as 165-kd and 155-kd polypeptides, respectively. Mature laminin 5 is thought to be necessary for correct hemidesmosome and basement membrane formation and its abnormal processing, as well as the low expression of alpha6beta4 integrins, could explain the lack of mature hemidesmosomes. Together, the results show that multiple molecular defects, including alteration of laminin 5 and its integrin receptors, contribute to structural aberrations of the basement membrane and associated structures in cylindromas.

Analysis of the adaptor function of the LIM domain‐containing protein FHL2 using an affinity chromatography approach

Containing four LIM domains and an N‐terminal half LIM domain, FHL2 has been predicted to have an adaptor function in the formation of higher order molecular complexes in the nucleus and the cytoplasm of cells. We expressed recombinant FHL2 in insect cells using the baculovirus system and used it to isolate direct or indirect interaction partners from the cytosolic fraction of fibroblasts by affinity chromatography. These were identified by their peptide mass fingerprints using MALDI‐TOF mass spectrometry. Cytoskeleton‐associated proteins present among the bound proteins were shown to co‐localise with FHL2 in cell lamellipodia by indirect immunofluorescence staining.

Characterization of recombinant and natural forms of the human LIM domain-containing protein FHL2.

FHL2 (Four and a Half LIM domain-containing protein 2) is a member of a small family of proteins with four LIM domains and an N-terminal half LIM domain. It is an intracellular protein thought to function as an adaptor in the formation of multi-protein complexes involved in signaling. To obtain human FHL2 in amounts allowing further characterization, we evaluated different expression systems and chose to express FHL2 with a His6 tag in insect cells using the baculovirus system. The recombinant protein was highly expressed and could be purified to >98% homogeneity as judged by SDS-PAGE analysis. Purified recombinant FHL2 was used to generate antibodies allowing detection and immunoprecipitation of FHL2 from human cells. Both recombinant and natural FHL2 were characterized by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of the recombinant His6-tagged protein obtained by mass spectrometry was 36,995Da, in good agreement with the apparent mass of 36kDa in SDS-PAGE and slightly higher than the 35,981Da calculated from the sequence of the construct. The measured molecular mass of natural human FHL2 was 32,742Da and the calculated mass was 32,192Da. However, the apparent molecular mass in SDS-PAGE is 41kDa, indicating that the natural protein has an abnormal electrophoretic mobility. The results show that both the recombinant and the natural proteins are post-translationally modified and indicate that such modifications may lead to an abnormal electrophoretic behavior of natural human FHL2.

Combined immunomodulation (Propionibacterium avidum KP-40) and lectin blocking (D-galactose) prevents liver tumor colonization in BALB/c-mice

The protective effect of combined treatment (immunomodulation with Propionibacterium avidum KP-40; liver lectin blocking by D-galactose administration) on the liver colonization of RAW 117-H10 lymphosarcoma was investigated in BALB/c-mice. Both, immunomodulation with P. avidum KP-40 as well as liver lectin blocking by D-galactose treatment significantly decreased the number of liver tumor colonies in this experimental model. However, the combination of P. avidum KP-40 and D-galactose obviously proved to be superior to each monotherapy since the liver colonization by RAW 117-H 10 lymphosarcoma could be completely inhibited.

Influence of Propionibacterium avidum KP-40 on the proliferation, maturation, emigration and activity of thymocytes and monocytes

Inactivated cells of Propionibacterium avidum KP-40 could be shown to induce thymocyte proliferation and maturation in BALB/c-mice after intraperitoneal administration of the optimal immunomodulating dosage (1 mg per mouse). The increase in thymus weight and thymocyte numbers per mg organ weight was most pronounced and statistically significant 10 days after P. avidum KP-40 administration. Determinations of lymphatic subsets revealed a considerable up-regulation of mature cells expressing helper/inducer (L3T4+) or cytotoxic/suppressor (Lyt-2+) phenotypes and immature cells presenting both L3T4+/Lyt-2+ antigens. Obviously, P. avidum KP-40 administration accelerated murine thymocyte proliferation and maturation. Counts of BALB/c-mouse peripheral blood lymphocytes (PBL) and monocytes (PBM) revealed statistically significant increases after P. avidum KP-40 administration with peak values after 6-10 days. The determination of activated PBL (expressing interleukin-2 receptors) or PBM (expressing MAC-3 antigens) proved that P. avidum KP-40 induced a potent immunostimulation since counts of these cells were significantly enhanced after P. avidum KP-40 treatment.

Antibacterial in vitro-activity of meropenem against 200 clinical isolates in comparison to 11 selected antibiotics

The antimicrobial activity of meropenem, a new parenteral carbapenem, was tested in vitro by an agar dilution method against 200 clinical isolates (gram-negative/positive aerobes and anaerobes). Meropenem was compared with imipenem, ceftazidime, cefotaxime, piperacillin, ciprofloxacin, gentamicin; and metronidazole, cefoxitin, chloramphenicol, clindamycin, vancomycin when appropriate. Meropenem and imipenem exhibited an extended spectrum of activity with low minimal inhibitory concentrations (MICs). Only one strain each of Enterococcus faecium and Pseudomonas (Xanthomonas) maltophilia were resistant. Of the carbapenems, imipenem was slightly more active against Enterococcus faecalis, Streptococcus agalactiae, and staphylococci, but meropenem was obviously more active against enterobacteriaceae and Clostridium perfringens. Both, meropenem and imipenem had similar activities towards Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Streptococcus pyogenes and Bacteroides sp. All other antibiotics tested were less potent than the carbapenems with the exception of ciprofloxacin which generally exhibited similar antibacterial activities, except for anaerob microorganisms.

Immunprotektion durch ML-1-normiertes Mistelextrakt bei kortisonbehandelten BALB/c-Mäusen

Anhand des etablierten BALB/c-Mausmodells wurden die EinflUsse von Hydrokortisonazetat als Immunsuppressivum und von ML-1, dem galaktosid-spezifischen Hauptmistellektin, als immunprotektiv wirken