Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

Long-term effects of Macroternies termites, herbivores and annual early fire on woody undergrowth community in Sudanian woodland, Burkina Faso

Termites, herbivores and fire are recognized as major guilds that structure woody plant communities in African savanna and woodland ecosystems. An understanding of their interaction is crucial to design appropriate management regimes. The aim of this study was to evaluate the long-term impacts of herbivore, fire and termite activities on regeneration of trees. Permanent experimental quadrats were established in 1992 in the Sudanian woodland of Burkina Faso subjected to grazing by livestock and annual early fire and the control. Within the treatment quadrats, an inventory of the woody undergrowth community was conducted on termitaria occupied by Macrotermes subhyalinus, extended termitosphere (within 5 m radius from the mound base) and adjacent area (beyond 5 m from the mound base). Hierarchical analysis was performed to determine significant differences in species richness, abundance and diversity indices among vegetation patches within fire and herbivory treatments. Grazed quadrats had significantly (P < 0.001) more species and stem density of woody undergrowth than non-grazed quadrats but maintained similar level of species richness and stem density of woody undergrowth on termitaria. There were not significant differences (P>0.05) in species richness and stem density between burnt and unburnt quadrats. Termitaria supported a highly diverse woody undergrowth with higher stem density than either the extended termitosphere or rest of quadrats. The density of woody undergrowth was significantly related with mature trees of selected species on termitaria (R-2 = 0.593; P<0.001) than that on the extended termitosphere (R-2 = 0.333; P<0.001) and adjacent area (R-2 = 0.197; P<0.001). It can be concluded that termites facilitate the regeneration of woody species while grazing and annual early fire play a minor role in the regeneration of woody species. The current policy that prohibits grazing should be revised to accommodate the interests of livestock herders. (C) 2014 Elsevier GmbH. All rights reserved.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+-permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+-permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells

Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two-and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model.

A Computational Study of the Factors Influencing the PVC-Triggering Ability of a Cluster of Early Afterdepolarization-Capable Myocytes

Premature ventricular complexes (PVCs), which are abnormal impulse propagations in cardiac tissue, can develop because of various reasons including early afterdepolarizations (EADs). We show how a cluster of EAD-generating cells (EAD clump) can lead to PVCs in a model of cardiac tissue, and also investigate the factors that assist such clumps in triggering PVCs. In particular, we study, through computer simulations, the effects of the following factors on the PVC-triggering ability of an EAD clump: (1) the repolarization reserve (RR) of the EAD cells; (2) the size of the EAD clump; (3) the coupling strength between the EAD cells in the clump; and (4) the presence of fibroblasts in the EAD clump. We find that, although a low value of RR is necessary to generate EADs and hence PVCs, a very low value of RR leads to low-amplitude EAD oscillations that decay with time and do not lead to PVCs. We demonstrate that a certain threshold size of the EAD clump, or a reduction in the coupling strength between the EAD cells, in the clump, is required to trigger PVCs. We illustrate how randomly distributed inexcitable obstacles, which we use to model collagen deposits, affect PVC-triggering by an EAD clump. We show that the gap-junctional coupling of fibroblasts with myocytes can either assist or impede the PVC-triggering ability of an EAD clump, depending on the resting membrane potential of the fibroblasts and the coupling strength between the myocyte and fibroblasts. We also find that the triggering of PVCs by an EAD clump depends sensitively on factors like the pacing cycle length and the distribution pattern of the fibroblasts.