Divergence Involving Global Regulatory Gene Mutations in an Escherichia coli Population Evolving under Phosphate Limitation

Many of the important changes in evolution are regulatory in nature. Sequenced bacterial genomes point to flexibility in regulatory circuits but we do not know how regulation is remodeled in evolving bacteria. Here, we study the regulatory changes that emerge in populations evolving under controlled conditions during experimental evolution of Escherichia coli in a phosphate-limited chemostat culture. Genomes were sequenced from five clones with different combinations of phenotypic properties that coexisted in a population after 37 days. Each of the distinct isolates contained a different mutation in 1 of 3 highly pleiotropic regulatory genes (hfq, spoT, or rpoS). The mutations resulted in dissimilar proteomic changes, consistent with the documented effects of hfq, spoT, and rpoS mutations. The different mutations do share a common benefit, however, in that the mutations each redirect cellular resources away from stress responses that are redundant in a constant selection environment. The hfq mutation lowers several individual stress responses as well the small RNA-dependent activation of rpoS translation and hence general stress resistance. The spoT mutation reduces ppGpp levels, decreasing the stringent response as well as rpoS expression. The mutations in and upstream of rpoS resulted in partial or complete loss of general stress resistance. Our observations suggest that the degeneracy at the core of bacterial stress regulation provides alternative solutions to a common evolutionary challenge. These results can explain phenotypic divergence in a constant environment and also how evolutionary jumps and adaptive radiations involve altered gene regulation.

The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5`-untranslated region:beta-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Avaliação dos efeitos da adição de sal e da densidade no transporte de tambaqui

Os objetivos deste trabalho foram testar a eficiência do sal como redutor de estresse e verificar a melhor densidade de transporte de juvenis de tambaqui (Colossoma macropomun) em caixas de plástico adaptadas. No primeiro experimento foram testadas diferentes concentrações de sal de cozinha (NaCl) na água; no segundo, o transporte foi realizado por três horas em caixas de plástico de 200 L estocadas com diferentes densidades de peixe, com 8 g de sal/L de água. O cortisol plasmático dos peixes sofreu aumento significativo após o transporte no tratamento sem sal e com 2 g de sal/L de água, retornando para níveis normais após 96 horas. A glicose plasmática dos peixes sofreu aumento após o transporte em todas as concentrações de sal testadas, com exceção da com 8 g/L de água, retornando para níveis normais em 24 horas. Nos peixes transportados no segundo experimento, com 8 g de sal/L de água, não foi verificada mudança significativa no cortisol plasmático, mas a glicose aumentou significativamente em todas as densidades após o transporte, retornando para níveis normais em 24 horas. Houve mortalidade de 11% em uma das repetições da densidade de 200 kg/m³ de água. Para o transporte com 8 g de sal/L de água, a densidade máxima deve ser de 150 kg/m³ de água. Nesta densidade os parâmetros físico-químicos de qualidade de água se mantêm com características adequadas, as respostas ao estresse são mínimas e não há mortalidade.

Biochemical dissection of diageotropica and Never ripe tomato mutants to Cd-stressful conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Respostas fisiológicas de estresse no matrinxã (Brycon amazonicus) após exercício físico intenso durante a captura

Para determinar as respostas de estresse do matrinxã após perseguição com puçá, juvenis (26,7±6,7 g) foram aclimatados em caixas plásticas e submetidos aos tratamentos: Controle (sem perseguição), Perseguição por 2 minutos, Perseguição por 5 minutos, Perseguição por 10 minutos (quatro repetições, N=8/tratamento). Amostras de sangue foram coletadas 15, 30 e 60 minutos após a perseguição para determinação do cortisol, glicose, sódio, cloreto, potássio, hematócrito, hemoglobina, número total de eritrócitos e osmolaridade. O perfil das respostas após o exercício físico dos peixes não mostrou as alterações típicas do estresse. Até 60 minutos após o estímulo, não ocorreram alterações nos níveis sanguíneos de cortisol, glicose e potássio nos peixes dos diferentes tratamentos. Os níveis de cloreto foram reduzidos 15 minutos após a natação forçada, enquanto os níveis do sódio mais baixos foram registrados 60 minutos depois. Houve redução da osmolaridade a partir dos 30 minutos após o estímulo, independente do tempo de perseguição. A natação forçada não interferiu nos indicadores hematológicos, corroborando os outros indicadores usados. Dessa forma, o exercício intenso dos peixes por até 10 minutos não foi estímulo suficiente para gerar respostas de estresse, sugerindo que o matrinxã é bastante resistente ao manejo de criação.

Transcription profiling of signal transduction-related genes in sugarcane tissues

A collection of 237,954 sugarcane ESTs was examined in search of signal transduction genes. Over 3,500 components involved in several aspects of signal transduction, transcription, development, cell cycle, stress responses and pathogen interaction were compiled into the Sugarcane Signal Transduction (SUCAST) Catalogue. Sequence comparisons and protein domain analysis revealed 477 receptors, 510 protein kinases, 107 protein phosphatases, 75 small GTPases, 17 G-proteins, 114 calcium and inositol metabolism proteins, and over 600 transcription factors. The elements were distributed into 29 main categories subdivided into 409 sub-categories. Genes with no matches in the public databases and of unknown function were also catalogued. A cDNA microarray was constructed to profile individual variation of plants cultivated in the field and transcript abundance in six plant organs (flowers, roots, leaves, lateral buds, and 1(st) and 4(th) internodes). From 1280 distinct elements analyzed, 217 (17%) presented differential expression in two biological samples of at least one of the tissues tested. A total of 153 genes (12%) presented highly similar expression levels in all tissues. A virtual profile matrix was constructed and the expression profiles were validated by real-time PCR. The expression data presented can aid in assigning function for the sugarcane genes and be useful for promoter characterization of this and other economically important grasses.

Indicadores da resposta ao estresse agudo associados com a familiaridade ao ambiente e aos procedimentos de banho e tosa em cães (Canis familiaris) da raça poodle

The stress responses can be parameters used in order to identify the welfare of animals. Behavior parameters can also be regarded as means to identify their relation to stressing situations. In this study, adult male and female poodle dogs, accustomed (group 1) or not (group 2) to the environment of a bath and grooming salon were analyzed through the plasmatic concentration of cortisol, cell counting of the immune system (total leukocytes and percentage of neutrophils and lymphocytes) and through observation of changes in behavior before and after the bath and grooming service. After arriving at the kennel, the dogs were taken to the bath and grooming facility, where they had their blood samples taken and were observed for ten minutes. The subsequent procedures consisted of the bath and grooming services and the collection of another blood samples. The research results were analyzed through non-parametric statistic tests and p < 0.05. Accustomed and non-accustomed dogs presented different hormonal and behavior responses: accustomed dogs presented an increasing of the cortisol level at the second blood samples collection, thus indicating stress towards the aforementioned procedures and presenting behavior responses which can be described as moderated stress. Non-accustomed dogs presented the same cortisol levels in both first and second samples suggesting that they were already reacting to the new environment. Dogs also shown behavioral responses which can be described as acute stress. Both male and female dogs showed similar immunological changes as well as different endocrine and behavioral profiles. Concerning aggression, this study demonstrates that biting the owner can be a predicting behavior of aggression during the bath and grooming service, male dogs were more aggressive than the female, and that males were more reactive to unknown caretaker. The results of this research propose that the familiarization of the animal with the bath and grooming environment and caretaker is important to the welfare of poodle dogs, especially to that of male dogs

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST),program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.

Alternative pathways for catecholamine action in oral motor control

Orofacial movement is a complex function performed by facial and jaw muscles. Jaw movement is enacted through the triggering of motoneurons located primarily in the trigeminal motor nucleus (Mo5). The Mo5 is located in the pontine reticular formation, which is encircled by premotor neurons. Previous studies using retrograde tracers have demonstrated that premotor neurons innervating the Mo5 are distributed in brainstem areas, and electrophysiological studies have suggested the existence of a subcortical relay in the corticofugal-Mo5 pathway. Various neurotransmitters have been implicated in oral movement. Dopamine is of special interest since its imbalance may produce changes in basal ganglia activity, which generates abnormal movements, including jaw motor dysfunction, as in oral dyskinesia and possibly in bruxism. However, the anatomical pathways connecting the dopaminergic systems with Mo5 motoneurons have not been studied systematically. After injecting retrograde tracer fluorogold into the Mo5, we observed retrograde-labeled neurons in brainstem areas and in a few forebrain nuclei, such as the central nucleus of the amygdala, and the parasubthalamic nucleus. By using dual-labeled immunohistochemistry, we found tyrosine hydroxylase (a catecholamine-processing enzyme) immunoreactive fibers in close apposition to retrograde-labeled neurons in brainstem nuclei, in the central nucleus of the amygdala and the parasubthalamic nucleus, suggesting the occurrence of synaptic contacts. Therefore, we suggested that catecholamines may regulate oralfacial movements through the premotor brainstem nuclei, which are related to masticatory control, and forebrain areas related to autonomic and stress responses. (C) 2005 Elsevier B.V.. All rights reserved.

Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Eucalyptus ESTs involved in the production of 9-cis epoxycarotenoid dioxygenase, a regulatory enzyme of abscisic acid production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ventilatory frequency of Nile tilapia subjected to different stressors

Ventilatory frequency (VF) was investigated in the fish Nile tilapia, Oreochromis niloticus, subjected to confinement, electroshock or social stressor. Fish were allowed to acclimatize to tank conditions for 10 days (1 fish/aquarium). VF baseline was determined 5 days after adjustment had been started. At the 10th day of isolation, stressor effects on VF were assessed. The stressors were imposed during 60 min: pairing with a larger resident animal (social stressor), or gentle electroshock (AC, 20 V, 15 mA, 100 Hz for 1 min every 4 min), or space restriction (confinement). VF was quantified immediately after the end of the stressor imposition. Baseline of VF was not statistically different among groups. Social stressor clearly induced VF to increase, while an increased or decreased VF can be observed for both confinement and electroshock. However, fish tend to increase their VF in response to confinement and decrease in the case of electroshock. These results suggest that VF is a sensitive behavioural indicator for distinguishing stress responses in the fish Nile tilapia among different stressors. © 2006 Elsevier GmbH. All rights reserved.

The Edinger-Westphal nucleus II: Hypothalamic afferents in the rat

Numerous functions have been attributed to the Edinger-Westphal nucleus (EW), including those related to feeding behavior, pain control, alcohol consumption and the stress response. The EW is thought to consist of two parts: one controls accommodation, choroidal blood flow and pupillary constriction, primarily comprising cholinergic cells and projecting to the ciliary ganglion; and the other would be involved in the non-ocular functions mentioned above, comprising peptide-producing neurons and projecting to the brainstem, spinal cord and prosencephalic regions. Despite the fact that the EW is well known, its connections have yet to be described in detail. The aim of this work was to produce a map of the hypothalamic sources of afferents to the EW in the rat. We injected the retrograde tracer Fluoro-Gold into the EW, and using biotinylated dextran amine, injected into afferent sources as the anterograde control. We found retrogradely labeled cells in the following regions: subfornical organ, paraventricular hypothalamic nucleus, arcuate nucleus, lateral hypothalamic area, zona incerta, posterior hypothalamic nucleus, medial vestibular nucleus and cerebellar interpositus nucleus. After injecting BDA into the paraventricular hypothalamic nucleus, lateral hypothalamic area and posterior hypothalamic nucleus, we found anterogradely labeled fibers in close apposition to and potential synaptic contact with urocortin 1-immunoreactive cells in the EW. On the basis of our findings, we can suggest that the connections between the EW and the hypothalamic nuclei are involved in controlling stress responses and feeding behavior. © 2013 The Authors.

Efeito do L-triptofano na mediação do estresse de transporte e social do matrinxã (Brycon amazonicus)

Pós-graduação em Aquicultura - FCAV