Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

Social sustainability and collaborative learning.

The social dimension is central to sustainable development of agri-food systems. If farmers are not satisfied with their situation or motivated to continue farming, many of today's environmental goals will be impossible to achieve. Between 1997 and 2003, several case studies were carried out on social sustainability, the importance of recognition in the farming system, and the potential role of increased collaboration between actors. The main hypothesis was that improved recognition is a basis for sustainable social conditions. Our findings show that many farmers today perceive an impoverished social situation. They believe they lack control over decisions, which hinders their ability to continue farming. Public images and political decisions show a lack of respect for farmers' skills and knowledge. However, increased collaboration among actors is believed to be one important way forward, creating stronger relationships and networks, as well as a stronger identity for farmers. Our findings emphasize the need for authorities and other organizations to support farmers and to facilitate collaborative learning and decision-making processes for socioecological sustainability.

Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import.

The Cdc25 phosphatases play crucial roles in cell cycle progression by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Cdc25A is an important regulator of the G1/S transition but functions also in the mitotic phase of the human cell cycle. In this paper, we investigate the sub-cellular localisation of exogenously expressed Cdc25A. We show that YFP-Cdc25A is localised both in the nucleus and the cytoplasm of HeLa cells and untransformed fibroblasts. Cell fusion assays and fluorescence loss in photobleaching (FLIP) assays reveal that the localisation is dynamic and the protein shuttles between the nucleus and the cytoplasm. We demonstrate that nuclear export of Cdc25A is partly mediated by an N-terminal nuclear export sequence (NES), in a manner not sensitive to the Exportin 1-inhibitor leptomycin B. A nuclear localisation signal (NLS) is also characterised, mutation of which leads to cytoplasmic localisation of Cdc25A. Our results imply that the Cdc25A phosphatase may interact with substrates and regulators both in the nucleus and the cytoplasm.

Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress.

Cdc25 phosphatases are essential regulators of the cell cycle. In mammalian cells, the Cdc25B isoform activates cyclin A- and cyclin B1-containing complexes and is necessary for entry into mitosis. In this report, we characterise the subcellular localisation of Cdc25B by immunofluorescence in combination with RNA interference to identify specific antibody staining. We find that endogenous Cdc25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle. Cdc25B starts to appear in S-phase cells and accumulates until prophase, after which the protein disappears. We characterise a nuclear export sequence in the N-terminus of Cdc25B (amino acids 54-67) that, when mutated, greatly reduces the ability of Cdc25B to shuttle in a fluorescence loss in photobleaching assay. Mutation of the nuclear export sequence makes Cdc25B less efficient in inducing mitosis, suggesting that an important mitotic function of Cdc25B occurs in the cytoplasm. Furthermore, we find that when cells are exposed to cycloheximide or ultraviolet irradiation, Cdc25B partially translocates to the cytoplasm. The dependence of this translocation event on a functional nuclear export sequence, an intact serine 323 residue (a 14-3-3 binding site) and p38 mitogen-activated protein kinase activity indicates that the p38 pathway regulates Cdc25B localisation in different situations of cellular stress.

Neisseria meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

Neisseria meningitidis colonizes the upper respiratory tract (URT), enters the blood stream, and reaches the cerebrospinal fluid (CSF). In the present study, we show that bacteria isolated from the URT adhere better to human epithelial cells, compared with bacteria from blood or CSF, which suggests that important changes of virulence-associated proteins take place during bacterial dissemination. Phase variation in the pilus adhesin PilC and sequence variation in the pilus subunit PilE occurred among strains from 1 patient. Changes were not found in the invasion-associated opacity proteins or in lipooligosaccharides. PilC was frequently expressed in serogroup B strains and in URT strains but was often switched off in other serogroups and in CSF strains. Strains lacking PilC showed impaired adhesion to epithelial cells. These data argue that N. meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

CD46 in meningococcal disease.

The human-specific bacterial pathogen Neisseria meningitidis is a major cause of sepsis and/or meningitis. The pili of N. meningitidis interact with CD46, a human cell-surface protein involved in regulation of complement activation. Transgenic mice expressing human CD46 were susceptible to meningococcal disease, because bacteria crossed the blood-brain barrier in these mice. Development of disease was more efficient with piliated bacteria after intranasal, but not intraperitoneal, challenge of CD46 transgenic mice, suggesting that human CD46 facilitates pilus-dependent interactions at the epithelial mucosa. Hence, the human CD46 transgenic mice model is a potentially useful tool for studying pathogenesis and for vaccine development against meningococcal disease.

The phase-variable pilus-associated protein PilC is commonly expressed in clinical isolates of Neisseria gonorrhoeae, and shows sequence variability among strains.

PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.