H(2)O(2) is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport ...chains. Depending on the concentration H(2)O(2) can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H(2)O(2) may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H(2)O(2) generation coincided with the onset of secondary wall deposition, (b) inhibition of H(2)O(2) production or scavenging the available H(2)O(2) from the system prevented the wall differentiation process, and (c) exogenous addition of H(2)O(2) prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H(2)O(2) generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H(2)O(2), whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H(2)O(2).
Grasslands occupy large areas in the northern Chihuahaun Desert. These grasslands, dominated by
Bouteloua eriopoda
, are subjected to periodic drought, infrequent fire and grazing by herbivores. ...Previous work shows that
B. eriopoda
is sensitive to disturbance but much work has been based on aboveground responses. We evaluated seasonal and annual recovery of belowground production and biomass following fire at two sites in ungrazed
B. eriopoda
-dominated grassland in Central New Mexico, USA. At one site, we quantified belowground standing crop and net primary production in burned and unburned areas during the first full growing season following wildfire the previous summer. At a second site, we measured annual below- and aboveground net primary production in burned and unburned grassland from 2005 through 2010 following a fire in 2003. At the first site, belowground standing crop did not change seasonally nor differ between burned/unburned areas. Patch types were different in that belowground standing crop was higher in soils under clumps of
B. eriopoda
than patches of unvegetated soil. Patterns of belowground biomass and daily production differed between patch types and over time in burned/unburned areas. Biomass was higher in soils below clumps of
B. eriopoda
than beneath unvegetated soil patches throughout the monsoon season. Patterns of belowground biomass and daily production differed in burned and unburned areas. Earlier in the growing season, biomass in the burned area was greater than in the unburned area. By early August, biomass increased rapidly in the unburned area and was higher than in the burned area. Daily rates of belowground production generally declined throughout the growing season with a large increase in rate of production in the unburned site in early August. At the second site's measured inter-annual responses, annual belowground production did not differ consistently between burned/unburned grasslands nor over time, nor was belowground production correlated with aboveground production. Our results demonstrate that despite the years required for aboveground production to recover following fire in
B. eriopoda
-dominated grassland, belowground standing crop and production was unchanged the year following fire. These results emphasize that aboveground production is not a reliable proxy for belowground production in this grassland.
Targeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP‐tagged Cdc24 proteins or fragments was assayed in deletion mutants of ...Cdc24p‐interacting proteins. The boi2Δ, ent2Δ, and hua1Δ mutants showed localization defects. The tos2Δ skg6Δ double mutant displayed aberrant pre‐anaphase localization to the mother‐bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.
Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a ...member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature‐sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity‐purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post‐docking phase of the reaction, we show that Cdc42p action follows Ypt7p‐dependent tethering, but precedes the formation of trans‐SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.
Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin ...rearrangements and signal transduction pathways in all eukaryotic cells 1, and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane 2,3 at sites of polarized growth 4. We show that Cdc24p labeled with green fluorescent protein (GFP–Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP–Cdc24p also localized to the nucleus and GFP–Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother–bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother–bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the Gβ subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.
The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae . Cells expressing the cdc42
V44A effector domain mutant allele ...displayed morphological defects of highly elongated and multielongated budded cells indicative
of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei
indicative of a G 2 /M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and
septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42
V44A cells; however, Cdc42 V44A p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42pâs interactions
with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the
Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42
V44A morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression
of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest
that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G 2 /M transition and that Cdc42 V44A p differentially interacts with a number of effectors and regulators.
Targeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP-tagged Cdc24 proteins or fragments was assayed in deletion mutants of ...Cdc24p-interacting proteins. The boi2Δ, ent2Δ, and hua1Δ mutants showed localization defects. The tos2Δ skg6Δ double mutant displayed aberrant pre-anaphase localization to the mother-bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.
To investigate the inherent clinical risks associated with the presence of cerebral microhemorrhages (CMHs) or cerebral microbleeds and characterize individuals at high risk for developing ...hemorrhagic amyloid-related imaging abnormality (ARIA-H), we longitudinally evaluated families with dominantly inherited Alzheimer disease (DIAD).
Mutation carriers (n = 310) and noncarriers (n = 201) underwent neuroimaging, including gradient echo MRI sequences to detect CMHs, and neuropsychological and clinical assessments. Cross-sectional and longitudinal analyses evaluated relationships between CMHs and neuroimaging and clinical markers of disease.
Three percent of noncarriers and 8% of carriers developed CMHs primarily located in lobar areas. Carriers with CMHs were older, had higher diastolic blood pressure and Hachinski ischemic scores, and more clinical, cognitive, and motor impairments than those without CMHs.
ε4 status was not associated with the prevalence or incidence of CMHs. Prevalent or incident CMHs predicted faster change in Clinical Dementia Rating although not composite cognitive measure, cortical thickness, hippocampal volume, or white matter lesions. Critically, the presence of 2 or more CMHs was associated with a significant risk for development of additional CMHs over time (8.95 ± 10.04 per year).
Our study highlights factors associated with the development of CMHs in individuals with DIAD. CMHs are a part of the underlying disease process in DIAD and are significantly associated with dementia. This highlights that in participants in treatment trials exposed to drugs, which carry the risk of ARIA-H as a complication, it may be challenging to separate natural incidence of CMHs from drug-related CMHs.
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