RESUMO
The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.
Assuntos
Fator de Iniciação 1 em Eucariotos , Higromicina B , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Núcleo Celular/metabolismo , Núcleo Celular/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Higromicina B/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismoRESUMO
Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.
Assuntos
Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Benomilo/farmacologia , Viabilidade Microbiana , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Domínios Proteicos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Moduladores de Tubulina/farmacologia , Vacúolos/metabolismoRESUMO
3-nitropropionic acid (3-NPA) is a nitrogenated compound produced by plants and fungi and has been associated with poisoning episodes in humans, animals, and to induction of Huntington disease symptoms in rats. The production of 3-NPA by endophytes has been reported, but the function and biosynthesis are not well-defined. The specie of endophytic strain G-01 was confirmed as Diaporthe citri using a multilocus sequence analysis, and was verified different concentrations of 3-NPA produced at different initial pHs by these strain. The chemical analysis indicated that 3-NPA was the majority compound present in the crude extracts. The better extraction condition was at an initial pH of 7.0 for 22 d, yielding about 80 % of 3-NPA per mg of extract. It was observed that the concentration of 3-NPA increased after the initial consumption of reduction sugars, indicating that the compound is produced after the high energetic production phase of the fungus. These and other studies demonstrate the production of this compound by plants and endophytic fungi, indicating that 3-NPA may be involved in defence and nutrition systems of endophytes and host plants, and they also might participate in the biogeochemical nitrogen cycle.
Assuntos
Ascomicetos/classificação , Ascomicetos/metabolismo , Endófitos/classificação , Endófitos/metabolismo , Nitrocompostos/metabolismo , Filogenia , Propionatos/metabolismo , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Meios de Cultura/química , Endófitos/genética , Endófitos/isolamento & purificação , Concentração de Íons de Hidrogênio , Tipagem de Sequências Multilocus , Plantas/microbiologiaRESUMO
The pathological findings and the immunohistochemical and molecular diagnosis of two natural cases of pulmonary adenocarcinoma in goats are described. Ovine pulmonary adenocarcinoma (OPA), a contagious lung tumor, caused by an exogenous Jaagsiekte Sheep Retrovirus (JSRV), is reported commonly in sheep and rarely in goats. The affected lungs had a focally extensive and firm nodule or multiple nodules on the cranioventral region. On the cut surface, the nodules were greyish, granular and moist, resembling classical form of OPA. Microscopically, the lung sections showed unencapsulated nodules of neoplastic epithelial cells, from alveoli and bronchioles, forming papillary projections or acini. On the immunohistochemical analysis, JSRV capsid protein was demonstrated in the neoplastic epithelial cells. Genomic DNA was extracted from the lung tumor tissues and was subjected to U3-hn-PCR that further confirmed the presence of JSRV. The pathological findings in goats were similar to that of OPA affecting lungs of sheep and, to the authors knowledge, this is the first report of the disease in goats with immunohistochemical and PCR confirmation of JSRV.
Assuntos
Animais , Adenocarcinoma/diagnóstico , Adenocarcinoma/veterinária , Adenomatose Pulmonar Ovina/diagnóstico , Pulmão/patologia , Retrovirus Jaagsiekte de Ovinos , Ruminantes , Imuno-Histoquímica/veterináriaRESUMO
The pathological findings and the immunohistochemical and molecular diagnosis of two natural cases of pulmonary adenocarcinoma in goats are described. Ovine pulmonary adenocarcinoma (OPA), a contagious lung tumor, caused by an exogenous Jaagsiekte Sheep Retrovirus (JSRV), is reported commonly in sheep and rarely in goats. The affected lungs had a focally extensive and firm nodule or multiple nodules on the cranioventral region. On the cut surface, the nodules were greyish, granular and moist, resembling classical form of OPA. Microscopically, the lung sections showed unencapsulated nodules of neoplastic epithelial cells, from alveoli and bronchioles, forming papillary projections or acini. On the immunohistochemical analysis, JSRV capsid protein was demonstrated in the neoplastic epithelial cells. Genomic DNA was extracted from the lung tumor tissues and was subjected to U3-hn-PCR that further confirmed the presence of JSRV. The pathological findings in goats were similar to that of OPA affecting lungs of sheep and, to the authors knowledge, this is the first report of the disease in goats with immunohistochemical and PCR confirmation of JSRV.(AU)
Assuntos
Animais , Ruminantes , Adenocarcinoma/diagnóstico , Adenocarcinoma/veterinária , Adenomatose Pulmonar Ovina/diagnóstico , Retrovirus Jaagsiekte de Ovinos , Pulmão/patologia , /veterinária , Imuno-Histoquímica/veterináriaRESUMO
Modulation of phytohormones homeostasis is one of the proposed mechanisms to explain plant growth promotion induced by beneficial rhizobacteria (PGPR). However, there is still limited knowledge about the molecular signals and pathways underlying these beneficial interactions. Even less is known concerning the interplay between phytohormones in plants inoculated with PGPR. Auxin and ethylene are crucial hormones in the control of plant growth and development, and recent studies report an important and complex crosstalk between them in the regulation of different plant developmental processes. The objective of this work was to study the role of both hormones in the growth promotion of Arabidopsis thaliana plants induced by the well-known PGPR Burkholderia phytofirmans PsJN. For this, the spatiotemporal expression patterns of several genes related to auxin biosynthesis, perception and response and ethylene biosynthesis were studied, finding that most of these genes showed specific transcriptional regulations after inoculation in roots and shoots. PsJN-growth promotion was not observed in Arabidopsis mutants with an impaired ethylene (ein2-1) or auxin (axr1-5) signaling. Even, PsJN did not promote growth in an ethylene overproducer (eto2), indicating that a fine regulation of both hormones signaling and homeostasis is necessary to induce growth of the aerial and root tissues. Auxin polar transport is also involved in growth promotion, since PsJN did not promote primary root growth in the pin2 mutant or under chemical inhibition of transport in wild type plants. Finally, a key role for ethylene biosynthesis was found in the PsJN-mediated increase in root hair number. These results not only give new insights of PGPR regulation of plant growth but also are also useful to understand key aspects of Arabidopsis growth control.
RESUMO
The flowers of the species belonging to the genus Passiflorashow a range of features that are thought to have arisen as adaptations to different pollinators. Some Passiflora species belonging to the subgenus Decaloba sect. Xerogona, show touch-sensitive motile androgynophores. We tested the role of auxin polar transport in the modulation of the androgynophore movement by applying auxin (IAA) or an inhibitor of auxin polar transport (NPA) in the flowers. We recorded the movement of the androgynophore during mechano-stimulation and analyzed the duration, speed, and the angle formed by the androgynophore before and after the movement, and found that both IAA and NPA increase the amplitude of the movement in P. sanguinolenta. We hypothesize that auxin might have a role in modulating the fitness of these Decaloba species to different pollination syndromes and demonstrate that an interspecific hybrid between insect- and hummingbird-pollinated Xerogona species present a heterosis effect on the speed of the androgynophore movement.
RESUMO
In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.