Cellular dynamic simulator: an event driven molecular simulation environment for cellular physiology.

In this paper, we present the Cellular Dynamic Simulator (CDS) for simulating diffusion and chemical reactions within crowded molecular environments. CDS is based on a novel event driven algorithm specifically designed for precise calculation of the timing of collisions, reactions and other events for each individual molecule in the environment. Generic mesh based compartments allow the creation / importation of very simple or detailed cellular structures that exist in a 3D environment. Multiple levels of compartments and static obstacles can be used to create a dense environment to mimic cellular boundaries and the intracellular space. The CDS algorithm takes into account volume exclusion and molecular crowding that may impact signaling cascades in small sub-cellular compartments such as dendritic spines. With the CDS, we can simulate simple enzyme reactions; aggregation, channel transport, as well as highly complicated chemical reaction networks of both freely diffusing and membrane bound multi-protein complexes. Components of the CDS are generally defined such that the simulator can be applied to a wide range of environments in terms of scale and level of detail. Through an initialization GUI, a simple simulation environment can be created and populated within minutes yet is powerful enough to design complex 3D cellular architecture. The initialization tool allows visual confirmation of the environment construction prior to execution by the simulator. This paper describes the CDS algorithm, design implementation, and provides an overview of the types of features available and the utility of those features are highlighted in demonstrations.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

Mitochondrial defects in primary leukemia cells: Biological consequences and therapeutic implications

Mitochondria are actively engaged in the production of cellular energy sources, generation of reactive oxygen species (ROS), and regulation of apoptosis. Mitochondrial DNA (mtDNA) mutations/deletions and other mitochondrial abnormalities have been implicated in many diseases, especially cancer. Despite this, the roles that these defects play in cancer development, drug sensitivity, and disease progression still remain to be elucidated. The major objective of this investigation was to evaluate the mechanistic relationship between mitochondrial defects and alterations in free radical generation and chemosensitivity in primary chronic lymphocytic leukemia (CLL) cells. This study revealed that the mtDNA mutation frequency and basal superoxide generation are both significantly higher in primary cells from CLL patients with a history of chemotherapy as compared to cells from their untreated counterparts. CLL cells from refractory patients tended to have high mutation frequencies. The data suggest that chemotherapy with DNA-damaging agents may cause mtDNA mutations, which are associated with increased ROS generation and reduced drug sensitivity. Subsequent analyses demonstrated that CLL cells contain significantly more mitochondria than normal lymphocytes. This abnormal accumulation of mitochondria was linked to increased expression of nuclear respiratory factor-1 and mitochondrial transcription factor A, two key free radical-regulated mitochondrial biogenesis factors. Further analysis showed that mitochondrial content may have therapeutic implications since patient cells with high mitochondrial mass display significantly reduced in vitro sensitivity to fludarabine, a frontline agent in CLL therapy. The reduced in vitro and in vivo sensitivity to fludarabine observed in CLL cells with mitochondrial defects highlights the need for novel therapeutic strategies for the treatment of refractory disease. Brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport that is being developed as an anticancer agent, effectively induces apoptosis in fludarabine-refractory CLL cells through a secretory stress-mediated mechanism involving intracellular sequestration of pro-survival secretory factors. Taken together, these data indicate that mitochondrial defects in CLL cells are associated with alterations in free radical generation, mitochondrial biogenesis activity, and chemosensitivity. Abrogation of survival signaling by blocking ER to Golgi protein transport may be a promising therapeutic strategy for the treatment of CLL patients that respond poorly to conventional chemotherapy. ^