In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cellspecific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p-and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.
The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G 1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G 1 transition to control cyclin-CDK inactivation and cytokinesis.During the transition from mitosis to G 1 , cytokinesis, disassembly of the mitotic spindle, chromatin decondensation, and DNA licensing must be precisely coordinated to ensure the genomic stability and viability of the cellular progeny (22,29,31,53,67). A major signal that controls these events is the degradation of mitotic cyclins and the inactivation of cyclindependent kinase (CDK) in late mitosis (52, 68). In Saccharomyces cerevisiae, mitotic cyclin degradation and CDK inactivation are regulated by a group of genes that constitute the mitotic exit network (MEN) (45,47). MEN genes encode four protein kinases (Cdc5p, Cdc15p, Dbf2p, and Dbf20p), Cdc14p phosphatase, a GTP binding protein (Tem1p), a GTP exchange factor (Lte1p), and Mob1p, which binds Dbf2p and Dbf20p (35,36,44,57,58,73,75). At the restrictive temperature, conditional alleles of the MEN genes cause cells to arrest in late mitosis with high levels of mitotic cyclin (33,48,58,66,69). The mitotic arrest of several MEN mutants can be suppressed by overexpression of CDK inhibitor SIC1 (18, 33), indicating that CDK inactivation is the major function of the MEN pathway. Indeed, a pivotal step in cyclin and CDK inactivation is mediated by the Cdc14p phosphatase, which is sequestered in the nucleolus during most of the cell cycle until it is released at the end of mitosis (3,60,72). Release of Cdc14p from the nuc...
Peroxisome proliferator-activated receptor-g (PPARg) is a member of the nuclear hormone superfamily and has multiple endogenous and pharmacological ligands, including 15-deoxy-D 12,14 -prostaglandin J 2 and two thiazolidinediones (TZD), rosiglitazone and pioglitazone, which are used clinically to treat type-2 diabetes mellitus. PPARg agonists regulate development, cellular growth and metabolism in various tissues and have been documented to decrease cellular proliferation and to induce apoptosis of various tumour phenotypes, including breast cancer. However, the full spectrum of anti-tumour effects occurs only at suprapharmacological doses. In this study, we investigated the mechanism of rosiglitazone-induced anti-tumour effects of MDA-MB-231 human breast cancer cells, and used that information to predict rosiglitazone-induced sensitization of breast cancer cells to the effects of other compounds. We first confirmed that 100 mM rosiglitazone, but not lower doses, decreases MDA-MB-231 cell viability in vitro. We then used microarray gene expression analysis to determine early rosiglitazone-induced gene expression changes after 4-h exposure, which included 1298 genes that we grouped into functional categories. We selectively confirmed rosiglitazonemediated effects on expression of key regulators of breast cancer proliferation and apoptosis, including p53, p21 and Bax. Finally, we used this information to predict that rosiglitazone would sensitize MDA-MB-231 cells to the anti-tumour effects of CH11, which trimerizes Fas, as well as tumour necrosis factor-a. Moreover, we used the confirmed array data to predict cooperative activity of rosiglitazone and R-roscovitine (CYC202), an inhibitor of multiple cyclin-dependent kinases. We conclude that microarray analysis can determine early TZD-modulated changes in gene expression that help to predict effective in vitro drug combinations.
Objectives: Since the beginning of the COVID-19 pandemic, face masks have been worn by many in public areas and for prolonged periods by healthcare workers (HCWs). This may facilitate bacterial contamination and transmission to and from patients in nursing homes where clinical care areas with strict precautions and residential and activity areas are interconnected. We assessed and compared bacterial mask colonization in HCWs belonging to different demographic categories and professions (clinical and nonclinical) and among HCWs who had worn the mask for different periods of time. Design, setting and participants: We conducted a point-prevalence study of 69 HCW masks at the end of a typical work shift in a 105-bed nursing home serving postacute care and rehabilitation patients. Information collected about the mask user included profession, age, sex, length of time the mask was worn, and known exposure to patients with colonization. Results: In total, 123 distinct bacterial isolates were recovered (1–5 isolates per mask), including Staphylococcus aureus from 11 masks (15.9%) and gram-negative bacteria of clinical importance from 22 masks (31.9%). Antibiotic resistance rates were low. There were no significant differences in the number of clinically important bacteria among masks worn more or less than 6 hours, and there were no significant differences among HCWs with different job functions or exposure to colonized patients. Conclusions: Bacterial mask contamination was not associated with HCW profession or exposure and did not increase after 6 hours of mask wearing in our nursing home setting. Bacteria contaminating HCW masks may differ from those colonizing patients.
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