Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities.

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.     

Life and death in aluminium-exposed cultures of rat lactotrophs studied by flow cytometry

Prolonged exposure to aluminium may impact health. Aluminium’s deleterious effects are mostly attributed to its selective accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size. Concentration-dependent effects of AlCl₃ were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl₃, viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%. The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%) remained unaltered after exposure to AlCl₃ concentrations up to 300 μM, while in single cells, viability was halved at 30 μM. In conclusion, our finding indicates that prolonged exposure to aluminium may lead to significant loss of pituitary cells.     

Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b

In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.     

LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain

Human leucine-rich repeat kinase 1 (LRRK1) is a multi-domain protein of unknown function belonging to the ROCO family of complex proteins. Here, we report the molecular characterization of human LRRK1 and show, for the first time, that LRRK1 is both a functional protein kinase and a GDP/GTP-binding protein. Binding of GTP to LRRK1 is specific, requires the GTPase-like Roc domain, and leads to a stimulation of LRRK1 kinase activity. LRRK1 is the first example of a GTP-regulated protein kinase harboring both the kinase effector domain and the GTP-binding regulatory domain. Hence, we propose a model in which LRRK1 cycles between a GTP-bound active and a GDP-bound inactive state. Moreover, we mutated LRRK1 to mimic mutations previously identified in LRRK2/dardarin, the only human paralogue of LRRK1, that have been linked to autosomal-dominant parkinsonism. We demonstrate that three of four mutations analyzed significantly downregulate LRRK1 kinase activity. Ultimately, the results presented for LRRK1 may contribute to the elucidation of LRRK2's role in the pathogenesis of Parkinson's disease.