The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae.
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Mutants of Saccharomyces cerevisiae bearing lesions in the ergosterol biosynthetic pathway exhibit a pleiotropic drug-sensitive phenotype. This has been reported to result from an increased permeability of the membranes of the mutant strains to different drugs. As disruption of the yeast multidrug resistance protein, Pdr5p, results in a similar pleiotropic drug-sensitive phenotype, the possibility that Pdr5p may be functioning with a reduced efficiency in these altered sterol backgrounds was examined. To do this, the function of Pdr5p in isogenic strains of S. cerevisiae that have disruptions in the late stages of the ergosterol biosynthesis pathway (ERG6, ERG2, ERG3, ERG4) was studied. A reduced ability of Pdr5p to confer resistance to different drugs in these strains was observed, which did not appear to be dependent solely on the permeability of the membrane towards the drug. A simultaneous examination was made of how the lipid composition might be altering the efficiency of Pdr5p by similar studies in strains lacking phosphatidylserine synthase (encoded by CHO1). The results indicated that the drug sensitivity of the erg strains is, to a significant extent, a result of the reduced efficiency of the Pdr5p efflux pump, and that the membrane environment plays an important role in determining the drug resistance conferred by Pdr5p. (+info)
Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.
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The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis. (+info)
Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone.
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In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis. (+info)
Role of endogenous cortisol in basal liquid clearance from distal air spaces in adult guinea-pigs.
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1. We investigated the role of endogenous cortisol in the modulation of distal air space liquid clearance in adult guinea-pigs. Cortisol synthesis was inhibited with the 11-beta-hydroxylase inhibitor metyrapone (0-7 days pretreatment). After cortisol synthesis inhibition, distal air space liquid clearance was measured by the increase in concentration of an instilled 5 % albumin solution after 1 h. 2. Two days of metyrapone pretreatment resulted in a 46+/-19 % decrease in plasma cortisol levels compared with control, which was paralleled by a 60+/-13 % decrease in distal air space liquid clearance. The Na+ channel inhibitor amiloride inhibited 40+/-22 % of distal air space liquid clearance in control animals but did not inhibit distal air space liquid clearance in the metyrapone-pretreated group. Co-injection of dexamethasone prevented the inhibition by metyrapone and the amiloride sensitivity of distal air space liquid clearance was greater than in control animals. After 7 days of metyrapone pretreatment, plasma cortisol levels and distal air space liquid clearance were not significantly different from normal, but amiloride sensitivity was greater than in control animals (91+/-37%). 3. Pretreatment with emetine, a protein synthesis inhibitor, reduced distal air space liquid clearance in control animals and in dexamethasone-co-injected animals, but failed to inhibit distal air space liquid clearance after metyrapone pretreatment. Expression of the epithelial sodium channel alpha-subunit (alphaENaC) mRNA in lung tissue was decreased after 2 days of metyrapone pretreatment and after 7 days pretreatment or after co-injection with dexamethasone, alphaENaC mRNA expression was restored towards control levels. 4. Thus, endogenous cortisol is important for maintaining normal liquid balance in the adult guinea-pig lung and a critical regulatory pathway is by modulation of ENaC expression and/or function. (+info)
A novel system for assigning the mode of inheritance in mitochondrial disorders using cybrids and rhodamine 6G.
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When normal human cultured skin fibroblasts were treated with the fluorescent dye rhodamine 6G (R6G), there was a drastic reduction in numbers of intact mitochondria and electron transport chain enzyme activities, despite the fact that mitochondrial DNA (mtDNA) was still present in treated cells. We used this observation to develop a novel system for generating cybrids. When cultured skin fibroblast cells from a patient with the mitochondrial encephalopathy and ragged-red fibers (MERRF) syndrome harboring the A8344G mtDNA mutation and which showed a severe reduction in cytochrome c oxidase activity were treated with R6G and fused to enucleated HeLaCOT cells, the resulting cybrid clones showed recovery of cytochrome c oxidase activity, and were shown to have mtDNA derived solely from the HeLaCOT cell line. R6G has significant advantages over ethidium bromide in removing the mitochondrial elements from cultured cells, and the results reported here demonstrate that this strategy can be used to determine the origin of the genetic defect in patients with electron transport chain abnormalities. (+info)
Coincident induction of long-term facilitation in Aplysia: cooperativity between cell bodies and remote synapses.
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Induction of long-term synaptic changes at one synapse can facilitate the induction of long-term plasticity at another synapse. Evidence is presented here that if Aplysia sensory neuron somata and their remote motor neuron synapses are simultaneously exposed to serotonin pulses insufficient to induce long-term facilitation (LTF) at either site alone, processes activated at these sites interact to induce LTF. This coincident induction of LTF requires that (i) the synaptic pulse occur within a brief temporal window of the somatic pulse, and (ii) local protein synthesis occur immediately at the synapse, followed by delayed protein synthesis at the soma. (+info)
Possible relationship of poly(A) shortening to mRNA turnover.
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Whereas the original size of poly(A) in HeLa cells is about 200 nucleotides, at steady state most of the poly(A) in mRNA contains less than 50 nucleotides. An endonucleolytic attack on poly(A) is suggested as the most likely method to accumulate short pieces of poly(A). Both poly(A) shortening and mRNA turnover appear to be inhibited by emetine, a drug that stops translation. It is possible that a random endonucleolytic attack leads to scission of poly(A) to a size below which the mRNA is unstable. (+info)
Corticotropin-releasing factor produces a protein synthesis--dependent long-lasting potentiation in dentate gyrus neurons.
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Corticotropin-releasing factor (CRF) was shown to produce a long-lasting potentiation of synaptic efficacy in dentate gyrus neurons of the rat hippocampus in vivo. This potentiation was shown to share some similarities with tetanization-induced long-term potentiation (LTP). In the present study, we further examined the mechanism underlying CRF-induced long-lasting potentiation in rat hippocampus in vivo. Results indicated that the RNA synthesis inhibitor actinomycin-D, at a concentration that did not change basal synaptic transmission alone (5 microgram), significantly decreased CRF-induced potentiation. Similarly, the protein synthesis inhibitor emetine, at a concentration that did not affect hippocampal synaptic transmission alone (5 microgram), also markedly inhibited CRF-induced potentiation. These results suggest that like the late phase of LTP, CRF-induced long-lasting potentiation also critically depend on protein synthesis. Further, prior maximum excitation of dentate gyrus neurons with tetanization occluded further potentiation of these neurons produced by CRF and vise versa. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed that CRF mRNA level in the dentate gyrus was significantly increased 1 h after LTP recording. Together with our previous findings that CRF antagonist dose-dependently diminishes tetanization-induced LTP, these results suggest that both CRF-induced long-lasting potentiation and tetanization-induced LTP require protein synthesis and that CRF neurons are possibly involved in the neural circuits underlying LTP. (+info)