Electron microscopy of Coxiella burnetii in tissue culture. Induction of cell types as products of developmental cycle.

An in vitro ultrastructural study was carried out on tissue cultures (J774, murine macrophage-like tumour cell line, and BHK-21, baby hamster kidney cell line) persistently infected with C. burnetii to investigate whether the events of cellular differentiation could be visualized. At a given stage of the developmental cycle, a proportion of the cells within the affected phagolysosomes clearly underwent cellular differentiation. The cells initially showed asymmetrical septation, the primary stage of cellular differentiation, and ended with the formation of the differentiated product, a precursor to the small cell. The results verified our initial observation that the events occurring during growth in a phagolysosome represent stages of a complex developmental cycle consisting not only of i) vegetative growth by typical transverse binary fission, but also ii) cellular differentiation

Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM-P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively. pilM-P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydrophobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod-shape-determining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures

Stimulation of endocytosis in mouse blastocysts by insulin - a quantitative morphological analysis

The effects of insulin on the endocytic activity of mouse blastocysts in vitro were investigated using confocal laser scanning microscopy, quantitative image analysis and electron microscopy. Confocal studies showed that fluorescein isothiocyanate-labelled markers, dextran (fluid phase) and albumin (combined membrane and fluid phase), were endocytosed by blastocysts and localized within vesicles (about 2.5 ?m in diameter) in the outer trophectoderm cells. No labelling was detected in the inner cell mass cells or the blastocoel cavity. Treatment with 170 nmol insulin l?1 stimulated the endocytosis of fluorescently labelled dextran in freshly collected blastocysts, increasing mean vesicle diameter per embryo by 15% (P &lt; 0.05) after incubation with insulin for 2.5 h and mean vesicle number per embryo by 56% (P &lt; 0.01) after 6 h. Both effects were also evident in blastocysts that had been cultured from the late eight-cell stage. Blastocysts incubated for 6 h with insulin displayed increased convolutions in the trophectoderm apical membrane compared with controls, indicating increased membrane activity and suggesting macropinosome formation. Collectively, these results suggest that insulin enhances endocytosis in the trophectoderm by stimulating uptake at the apical membrane into larger and more numerous endocytic vesicles and with some evidence of vesicle fusion. This mechanism may provide a metabolic basis for the stimulation by insulin of biosynthesis, proliferation and morphological development in early embryos. <br/