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Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier for DNA-dependent processes. Here, we have examined the role of two highly conserved yeast (Sacc...
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Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier for DNA-dependent processes. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, facilitates chromatin transcription (FACT) and Spt6, in regulating transcription. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 histone acetyltransferase Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT depletion reduces RNA polymerase II (Pol II) occupancy genome-wide. Spt6 depletion leads to a reduction in Pol II occupancy toward the 3'-end, in a manner dependent on the gene length. Severe transcription and histone-eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6 Delta 202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with wild-type Pol II occupancies at these genes, indicating critical roles for Spt6 and Spt16 in promoting high-level transcription. Collectively, our results show that both FACT and Spt6 are important for transcription globally and may participate during different stages of transcription.
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Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegan...
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Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and its co-activator FZR-1 (APC/CFZR-1), a ubiquitin ligase, negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in humans) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via the D-box motif. We also show the Slimb/beta TrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by the SKP1-CUL1-F-box (Slimb/beta TrCP)-protein complex (SCFSlimb/beta TrCP)-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that the APC/CFZR-1 and SCFSlimb/beta TrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.
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The IL-2/anti-IL-2 Ab immunocomplex has recently been shown to expand the naturally occurring pool of CD4(+) Foxp3(+) regulatory T cells (Tregs). In this study, we show that administration of the IL-2/anti-IL-2 Ab immunocomplex to...
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The IL-2/anti-IL-2 Ab immunocomplex has recently been shown to expand the naturally occurring pool of CD4(+) Foxp3(+) regulatory T cells (Tregs). In this study, we show that administration of the IL-2/anti-IL-2 Ab immunocomplex to C57BL/6 mice, prior to corneal HSV-1 infection, significantly increased the pool of Foxp3(+) Tregs when measured at early time points postinfection. Increased numbers of Foxp3(+) Tregs on days 2 and 4 postinfection resulted in a marked reduction in the development of severe herpetic stromal keratitis (HSK). When compared with corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 postinfection. Reduced viral load was associated with a 2-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 postinfection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6, and 7 postinfection significantly increased Foxp3(+) Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 postinfection. Our findings demonstrate that increasing Foxp3(+) Tregs early but not late postinfection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.
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The Drosophila trachea is a premier genetic system to investigate the fundamental mechanisms of tubular organ formation. Development of the trachea consists of the well understood early branch specification and migration processes...
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The Drosophila trachea is a premier genetic system to investigate the fundamental mechanisms of tubular organ formation. Development of the trachea consists of the well understood early branch specification and migration processes, and the less clear later branch maturation process including the apical membrane expansion, cytoskeleton rearrangement, luminal matrix clearance, and air-filling. We identified seven members of the Osiris (Osi) gene family with obvious tracheal expression in Drosophila. In addition,.HA-tagged Osi proteins are highly concentrated in vesicle-like structures at and near the apical membrane. Osi proteins are predicted to contain endocytic signals and transmembrane domains. The localization of Osi proteins is consistent with these predictions. Interestingly, the Drosophila tracheal tube maturation process also occurs at the apical membrane. Taken together, the localization of Osi proteins suggest that these proteins are likely involved in tube maturation through vesicular trafficking or interacting with other apical membrane proteins.
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Calcium regulation of flagellar motility is the basis for chemotaxis, phototaxis, and hyperactivation responses in eukaryotic flagellates and spermatozoa. Ca2+ is the internal messenger for these responses, but the coupling betwee...
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Calcium regulation of flagellar motility is the basis for chemotaxis, phototaxis, and hyperactivation responses in eukaryotic flagellates and spermatozoa. Ca2+ is the internal messenger for these responses, but the coupling between Ca2+ and the motor mechanism that generates the flagellar beat is incompletely understood. We examined the effects of Ca2+ on the flagellar curvature at the switch-points of the beat cycle in bull sperm. The sperm were detergent extracted and reactivated with 0.1mM adenosine triphosphate (ATP). With their heads immobilized and their tails beating freely it is possible to calculate the bending torque and the transverse force acting on the flagellum at the switch-points. An increase in the free Ca2+ concentration (pCa 8 to pCa 4) significantly decreased the development of torque and t-force in the principal bending direction, while having negligible effect on the reverse bend. The action of Ca2+ was more pronounced when the sperm were also treated with 4 mM adenosine diphosphate (ADP); it was sufficient to change the direction of bending that reaches the greater curvature. We also observed that the curvature of the distal half of the flagellum became locked in one direction in the presence of Ca2+. This indicates that a subset of the dynein becomes continuously activated by Ca2+ and fails to switch with the beat cycle. Our evidence suggests this subset of dyneins is localized to doublets #1-4 of the axoneme. (c) 2014 Wiley Periodicals, Inc.
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We report the results of an ultrastructural study of Triton X-100-extracted, Mg-adenosine triphosphate (ATP)-reactivated bull sperm. We utilized a rapid fixation method to look for differences in the flagellar apparatus that corre...
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We report the results of an ultrastructural study of Triton X-100-extracted, Mg-adenosine triphosphate (ATP)-reactivated bull sperm. We utilized a rapid fixation method to look for differences in the flagellar apparatus that correlate to the state of motility of reactivated sperm models. In a companion article, we examined the motility characteristics induced in bull sperm models by varying the concentration ratio of ATP and Mg2+ and the stabilizing effect of adenosine diphosphate (ADP) on coordinated beating. Based on the results of that report, we selected four dissimilar states that appeared to represent extremes. One reactivation condition produces vigorous motility similar to live sperm, another produces large amplitude, low frequency beating while the remaining two conditions produce small amplitude vibrations of the flagellum with little coordinated beating. Morphometric analysis of transmission electron micrographs of sperm from these four treatment conditions revealed statistically significant differences between the samples in regard to axoneme diameter, inter-microtubule doublet spacing, and outer dense fiber (ODF) spacing. Our results show that Mg2+ decreases the axoneme diameter and reduces interdoublet spacing, while ATP, uncomplexed with Mg2+, had the opposite effect. We also provide supporting evidence that this may be due to Mg2+ increasing, and ATP decreasing, the interdoublet adhesion of dynein. We also found that 4 mM ADP significantly increases the separation between the ODFs and the space between the ODFs and the central axoneme within the middle piece. We present a hypothetical explanation that is consistent with our results to explain how ATP, ADP, and Mg2+ act to regulate the beat cycle. (c) 2014 Wiley Periodicals, Inc.
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The process of decomposition has received much attention in terrestrial and stream ecosystems, but our understanding of the factors that contribute to this process in wetlands remains relatively poor. Many macroconsumers in wetlan...
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The process of decomposition has received much attention in terrestrial and stream ecosystems, but our understanding of the factors that contribute to this process in wetlands remains relatively poor. Many macroconsumers in wetlands are classically labeled as herbivores, but increasing evidence suggests that they also contribute to the breakdown of dead plant litter depending on the nutritional quality (i.e., nutrient content, density, and toxicity) of the litter. We examined the relative contributions of 2 common North American temperate wetland consumers, the green frog tadpole (Lithobates clamitans) and the Ram's Horn snail (Planorbella trivolvis), to the decomposition of 5 chemically variable plant litter species. Based on anatomical differences (e.g., mouth parts, digestive structures), we hypothesized that snails would have higher consumption rates than tadpoles, but that tadpoles would have higher assimilation efficiency. We also predicted that consumption rates and assimilation efficiency would vary with litter nutritional quality. Overall, consumers exhibited substantial detritivory and caused up to 62% litter mass loss relative to treatments with only microbes. As hypothesized, snails consumed more than tadpoles, but this difference was largely explained by differences in consumer mass. Contrary to our hypothesis, snails and tadpoles exhibited similar assimilation efficiencies. Both litter mass loss and assimilation efficiency by consumers differed among litter species treatments. Litter mass loss tended to be negatively correlated with litter C : N and C: P, whereas assimilation efficiency had no detectable correlation with any measured litter characteristic. Our study demonstrates that studies of energy and nutrient budgets in wetlands should consider both consumer type and litter species to describe ecosystem function fully.
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Motivation: The promise of higher phylogenetic stability through increased dataset sizes within tree of life (TOL) reconstructions has not been fulfilled. Among the many possible causes are changes in species composition (taxon sa...
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Motivation: The promise of higher phylogenetic stability through increased dataset sizes within tree of life (TOL) reconstructions has not been fulfilled. Among the many possible causes are changes in species composition (taxon sampling) that could influence phylogenetic accuracy of the methods by altering the relative weight of the evolutionary histories of each individual species. This effect would be stronger in clades that are represented by few lineages, which is common in many prokaryote phyla. Indeed, phyla with fewer taxa showed the most discordance among recent TOL studies. We implemented an approach to systematically test how the identity of taxa among a larger dataset and the number of taxa included affected the accuracy of phylogenetic reconstruction.
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Past studies have found a heterogeneous distribution of the amphibian chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd). Recent studies have accounted for some of this heterogeneity through a positive association betwee...
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Past studies have found a heterogeneous distribution of the amphibian chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd). Recent studies have accounted for some of this heterogeneity through a positive association between canopy cover and Bd abundance, which is attributed to the cooling effect of canopy cover. We questioned whether leaf litter inputs that are also associated with canopy cover might also alter Bd growth. Leaf litter inputs exhibit tremendous interspecific chemical variation, and we hypothesized that Bd growth varies with leachate chemistry. We also hypothesized that Bd uses leaf litter as a growth substrate. To test these hypotheses, we conducted laboratory trials in which we exposed cultures of Bd to leachate of 12 temperate leaf litter species at varying dilutions. Using a subset of those 12 litter species, we also exposed Bd to pre-leached litter substrate. We found that exposure to litter leachate and substrate reduced Bd spore and sporangia densities, although there was substantial variation among treatments. In particular, Bd densities were inversely correlated with concentrations of phenolic acids. We conducted a field survey of phenolic concentrations in natural wetlands which verified that the leachate concentrations in our lab study are ecologically relevant. Our study reinforces prior indications that positive associations between canopy cover and Bd abundance are likely mediated by water temperature effects, but this phenomenon might be counteracted by changes in aquatic chemistry from leaf litter inputs.
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Sperm flagella derive their motive power from the motor protein dynein. In this study, we show that maintenance of the flagellar beat cycle in detergent-extracted bull sperm models is highly dependent on the ratio of Mg2+ to adeno...
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Sperm flagella derive their motive power from the motor protein dynein. In this study, we show that maintenance of the flagellar beat cycle in detergent-extracted bull sperm models is highly dependent on the ratio of Mg2+ to adenosine triphosphate (ATP). An excess of either ATP un-complexed with Mg2+, or an excess of Mg2+ without an equivalent concentration of ATP, results in the loss of beat amplitude and a reduced curvature development in the beat cycle. In addition, we find that adenosine diphosphate (ADP) can stabilize the beat cycle and permit rhythmic beating across a broader range of ATP and Mg2+ concentrations. We provide evidence that suggests that when ATP is un-complexed with Mg2+, it disrupts the beat cycle by reducing dynein adhesion and thereby, reduces the transmission of dynein-generated force between the doublets. Excess Mg2+ does not act by the same mechanism and induces a condition where the flagellum is more resistant to bending. This is consistent with the idea that high Mg2+ stabilizes rigor bridges, and ATP reduces the microtubule binding affinity of dynein. Our results may explain how intact sperm are able to sustain coordinated flagellar beating under a wide range of metabolic conditions, as intact sperm produce ADP in direct proportion to their consumption of ATP. (c) 2014 Wiley Periodicals, Inc.
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