Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.
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The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation. (+info)
Phosphatase 2A and polo kinase, two antagonistic regulators of cdc25 activation and MPF auto-amplification.
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The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. We have studied the Cdc2/Cdc25 feed-back loop using cell-free systems derived from Xenopus prophase-arrested oocyte. Our findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase, Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independently of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process. (+info)
DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding.
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In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA. (+info)
Nicotine infusion modulates immobilization stress-triggered induction of gene expression of rat catecholamine biosynthetic enzymes.
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The relationship between nicotine and stress is complex and paradoxical. Although people claim they smoke because it relaxes them, nicotine can trigger some of the effects observed with stress, including the release and synthesis of the catecholamines and their biosynthetic enzymes. This study examined one aspect of this confusing relationship between nicotine and stress. Multiple injections of nicotine bitartrate (5 mg/kg) elevated mRNA levels for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase, and of preproneuropeptide Y in rat adrenal medulla more than did 1 mg/kg of nicotine bitartrate. In the locus ceruleus, substantia nigra, and ventral tegmental area both doses equally induced TH mRNA levels. Nicotine infusion (15 mg/kg/day) did not affect adrenal mRNA levels for any of the genes of interest and did not increase plasma corticosterone levels. However, in rats pre-exposed to nicotinic infusions, the response to a single immobilization (IMO) stress was markedly attenuated with respect to changes in adrenomedullary TH, DBH, and phenylethanolamine N-methyltransferase mRNA levels and in c-Fos protein levels. In the central nervous system, the chronic infusion of nicotine prevented the induction of TH mRNA by repeated IMO stress in the ventral tegmental area (but not in substantia nigra) and of DBH mRNA by single IMO in the locus ceruleus. These findings may explain some of the complex interactions between stress and exposure to nicotine. (+info)
Prompt recovery of damaged adrenal medullae induced by salinomycin.
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The morphologic changes in the adrenal medullae of rats treated with an ionophore antibiotic, salinomycin, are described. Male rats of approximately 7 wk of age were treated orally with a single dose of salinomycin at 80 mg/kg body weight. Following this treatment, the adrenal glands were examined, using immunohistochemistry, for neurofilament, laminin, fibronectin, and S-100 protein; the glands were also examined using transmission electron microscopy. One hour after the treatment, a karyopyknosis was observed in the clusters of affected chromaffin cells in which the neurofilament, laminin, and fibronectin were present. The lesions became progressively conspicuous between hours 5 and 10. Ultimately, the outcome was cell lysis. Five hours after salinomycin treatment, unaffected chromaffin cells strongly stained to tyrosine hydroxylase. At 10 hr, new chromaffin cells, which were irregular in shape with electron-dense cytoplasm (dark cell), that were strongly stained for tyrosine hydroxylase appeared at the basement membrane site of the necrotic clusters, and these cells contained very few immature catecholamine granules of less than 80 nm. At 17 hr, the catecholamine granules increased in number and size to about 200 nm. The newly formed chromaffin cells grew within the clusters to fill in the medulla by 24 hr, and cytoplasmic granules progressively increased in number and size. The interstitial tissue was seen to be edematous at 5 hr. New capillaries were found in the adrenal medullae of both control and salinomycin-treated rats. The protruding chromaffin cells (protruding cells), which we previously described in normal rats, were also observed in salinomycin-treated rats, which suggests that holocrine secretion is performed in the adrenal medullae. The results indicated that the rat adrenal medullae have the ability to make a rapid recovery after an insult by salinomycin. (+info)
Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut.
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Cells of the enteric nervous system are derived from the neural crest. Probes to a number of molecules identify neural crest-derived cells within the gastrointestinal tract of embryonic mice prior to their differentiation into neurons and glial cells. However, it is unclear whether the different markers are identifying all neural crest-derived cells. In this study the distribution of p75(NTR)-immunoreactivity was compared with that of Ret-, Phox2a-, Phox2b-, and tyrosine hydroxylase (TH) in undifferentiated neural crest-derived cells in the E10.5-E13.5 mouse intestine. Neural crest-derived cells colonise the embryonic mouse gut in a rostral-to-caudal wave between E9.5-E14, and differentiation into enteric neurons also occurs in a rostral-to-caudal wave. Thus, the most caudal neural crest-derived cells within the gut are undifferentiated. These most caudal neural crest-derived cells co-expressed p75(NTR)-, Phox2b- and Ret-immunoreactivity; at E10.5 a sub-population was also TH-positive. The most caudal cells did not show Phox2a-immunoreactivity at any stage. However, a sub-population of cells, which was rostral to the undifferentiated neural crest-derived cells, was Phox2a-positive, and these are likely to be cells beginning to differentiate along a neuronal lineage. The expression of Ret-, Phox2a-, Phox2b- and p75(NTR)-immunoreactivity by two classes of enteric neurons that differentiate prior to birth was also examined. Nitric oxide synthase (NOS) neurons showed Phox2b and Ret immunoreactivity at all ages, and Phox2a and p75(NTR) immunoreactivity only transiently. Calcitonin gene-related peptide (CGRP) neurons showed Phox2b and Ret-immunoreactivity, but not Phox2a immunoreactivity. It is concluded that all undifferentiated neural crest-derived cells initially express Phox2b, Ret, and p75(NTR); a sub-population of these cells also expresses TH transiently. Those cells that are beginning to differentiate along a neuronal lineage maintain their expression of Phox2b and Ret, and they start to express Phox2a, but down-regulate p75(NTR); those cells that differentiate along a glial lineage down-regulate Ret and maintain their expression of p75(NTR). Dev Dyn 1999;216:137-152. (+info)
Increased levels of epsilon and gamma isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with Creutzfeldt-Jakob disease.
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We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that epsilon and gamma isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were. (+info)
Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae.
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14-3-3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting. (+info)