Insights into the role of phytohormones regulating pAtNIP5;1 activity and boron transport in Arabidopsis thaliana.

Aiming to counteract B deficiency impacts, plants have developed different strategies in order to reach an optimal growth in soils with limited B availability. These include B transport mechanisms that involves a facilitated transport, via channel proteins, and a high-affinity active transport driven by borate transporters. The AtNIP5;1 channel protein is a member of Major Intrinsic Protein family which facilitates B influx into the roots under low B supply. In order to explore the phytohormone-dependent regulation of AtNIP5;1, the effects of abscisic acid (ABA), ethylene, auxins and cytokinins on the activity of AtNIP5;1 promoter were evaluated using the reporter line pNIP5;1-GUS. The results show that ABA treatment increased pAtNIP5;1 activity. Besides, a larger B uptake was found following ABA treatment under B deficiency suggesting a role of ABA inducing B uptake. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) caused an induction of AtNIP5;1 expression although did not correlate with higher B concentrations nor with an improvement in root growth. On the contrary, auxins and cytokinins caused slight changes in pAtNIP5;1 induction. Altogether, these results show a regulatory role of phytohormones in AtNIP5;1 promoter what may affect B transport. The herein provided information may contribute to better understand the regulation of B transport in plants towards minimizing B deficiency impacts on agriculture.

[In vitro activities of colistin combinations against Pseudomonas aeruginosa isolated from the intensive care unit]. / Actividad in vitro de terapia combinada con colistina frente a Pseudomonas aeruginosa aisladas en Unidad de Cuidados Intensivos.

Introduction. As the number of multidrug-resistant strains of Pseudomonas aeruginosa has risen in the intensive care unit (ICU) of the San Carlos Clinic Hospital, 12 consecutive isolates from different patients were collected to determine the possibility of an epidemic outbreak caused by the spread of a single strain. We determined the antimicrobial susceptibility to the most common agents used in the treatment of infections caused by this bacteria. The results of susceptibility studies suggest that different strains of P. aeruginosa are responsible for the respiratory tract infections in ICU. Methods. The clonal relationship between the isolates using was determined using BOX and ERIC primers by means of repetitive sequence-based polymerase chain reaction (rep-PCR). The in vitro activity of these strains against colistin, rifampicin, doxicycline and azythromycin was studied to determine in which cases the combination of colistin with any of the other three antibiotics was synergistic. Results. Sensitivity studies point out the presence of several strains of P. aeruginosa as the causal agents of respiratory infections produced by this microorganism in the ICU. Combinations of colistin with doxycicline and colistin with azithromycin were synergistic for some isolates in the synergy studies. Discussion. Clonal studies reveal the presence of five different clones among our isolates. Therefore we can conclude that there was no outbreak of P. aeruginosa in the ICU. Synergistic activity of combinations of colistin plus azithromycin, colistin plus doxicycline and colistin plus rifampicin was less than expected and a high percentage of indifferent results was observed.

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development.

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.

Essentiality of Boron for Symbiotic Dinitrogen Fixation in Pea (Pisum sativum) Rhizobium Nodules.

The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined. The absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity. Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures. These results indicate that boron is a requirement for normal nodule development and functionality.

Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron-deficient pea nodules.

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

MucR is necessary for galactoglucan production in Sinorhizobium meliloti EFB1.

Sinorhizobium meliloti can produce two types of acidic exopolysaccharides, succinoglycan and galactoglucan, that are interchangeable for infection of alfalfa nodules. Strain SU47 and derivatives produce only succinoglycan, unless it grows under phosphate limitation or carries a mutation in either of two regulatory loci, mucR or expR. It has been proposed that MucR acts as a transcriptional repressor that blocks the expression of the exp genes responsible for galactoglucan production. Strain EFB1 simultaneously produces both exopolysaccharides. Heterologous expression of lacZ transcriptional fusions of the expE promoters has shown that genetic background is more important that promoter sequence for exp gene expression, since expE promoters from both strains are expressed at high level in EFB1 and not in SU47. We have found that mucR is present in mucoid and nonmucoid strains, and in EFB1 differs from SU47 in only one conservative amino acid change. MucR proteins from both strains are interchangeable. An mucR mutant of EFB1 cannot produce galactoglucan and does not express mucS.

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH.

The role of external Ca2+ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca2+ in the medium. The observed Ca2+ -mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca2+ in the medium. Thus, the ability of Ca2+ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca2+ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca2+ channel blockers tested, such as verapamil and LaCl3 , inhibited the observed Ca2+ -mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m-chlorophenyl hydrazone, inhibited this Ca2+ -mediated response, whereas monensin, an inhibitor of the Na+ /H+ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca2+ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.

Protection by CO(2) against Sodium Deficiency in a Mutant of Nostoc muscorum.

The effects of Na-deficient culture were studied on a mutant of Nostoc muscorum unable to fix N(2). Na deficiency made the cells sensitive to photooxidation, thus at a light intensity of lO W m(-2) and a low concentration of CO(2) (0.03 %), Na deficiency caused chlorosis and cell lysis. At a lower light intensity, cell damages did not take place. Higher levels of CO(2) prevented photooxidation, so that only a partial inhibition of photosynthetic oxygen evolution was found under these conditions.

[A rare case of silent cholinesterase genetic variant (author's transl)]. / Un caso excepcional de variante genética colinesterásica silente.

Some patients present prolonged apnea following the administration of succinylcholine because of substantial portion of plasma cholinesterase (the enzyme responsible for the hydrolysis of this muscle relaxant) is present as an atypical variant. Up to the present four types of cholinesterase genes are known which combination can cause apnea to a greater or less extent. Silent homozygous EsEs present apnea of greatest duration due to the complete absence of cholinesterase activity. The case reported belongs to a family of four members and is an example of this extremely uncommon genetic variant, with a frequency of 1/170.000. To determine total cholinesterase activity genetic typing of the whole family was carried out utilizing the spectrophotometric technique of Szasz and Knedel with butirylthiocholine iodide as substrate. Inhibition studies were performed using benzoylcholine and dibucaine, sodium fluoride, scholine, and sodium chloride as inhibitors.

Validez y confiabilidad de los instrumentos de percepción de barreras y de beneficios para el ejercicio en adolescentes colombianos / Validity and reliability of the perceiving barriers and benefits of exercising instruments for Colombian adolescents / Validação e confiabilidade dos instrumento para perceber barreiras e benefícios do exercício em adolescentes colombianos

Objetivo: validar en el contexto colombiano los instrumentos de percepción de barreras y de beneficios asociados a la realización\r\nde ejercicio en adolescentes basados en el modelo de promoción de la salud de Nola J. Pender. Metodología: los instrumentos\r\nseleccionados fueron sometidos a estos procedimientos: permisos, traducción y pruebas de validez facial, de contenido, de constructo\r\ny de confiabilidad. Resultados: el instrumento de percepción de barreras para el ejercicio tiene un índice de Lawshe\r\nmodificado de 0,83 y un kappa de Fleiss de 0,59 para relevancia y de 0,53 para pertinencia. El instrumento de percepción de\r\nbeneficios por el ejercicio presenta un índice de Lawshe modificado de 0,93 y un kappa de Fleiss para relevancia 0,70 y de 0,91\r\npara pertinencia. El análisis factorial identificó en cada uno de los instrumentos dos factores: barreras para la salud física y afectivas o emocionales; en cuanto a los beneficios se relacionaron con las condiciones ambientales y el tiempo disponible para\r\nrealizar el ejercicio, y la confiabilidad se encontró en 0,82 y 0,83 para la percepción de barreras y de beneficios, respectivamente.\r\nConclusiones: los instrumentos de percepción de barreras y de beneficios para la realización de ejercicio adaptados al contexto\r\ncolombiano son válidos y confiables. Por lo anterior, se consideran apropiados para evaluar y diseñar estrategias que contribuyan\r\na mejorar la realización de actividad física en los adolescentes.

Objective: To validate the instruments for perceiving barriers\r\nand benefits of exercising for Colombian adolescents based on\r\nthe health promotion model of Nola Pender. Methodology:\r\nThe instruments for perceiving barriers and benefits of exercising\r\nwere subject to the following procedures: permits, translation,\r\nback translation, and validity tests: facial, content, and construction.\r\nResults: The instruments for perceiving barriers and\r\nbenefits of exercising have a modified Lawshe index of 0.83 and\r\nFleiss Kappa of 0.59 for relevance and pertinence of 0.53. The\r\ninstruments for perceiving barriers and benefits of exercising\r\nalso present a modified Lawshe index of 0.93 and Fleiss Kappa\r\nfor relevance 0.70 and for pertinence 0.91. The factor analysis\r\nidentified in each of the instruments 2 factors: barriers to physical\r\nand affective or emotional health; in terms of benefits, they\r\nwere related to environmental conditions and the time available\r\nto perform the exercise, and reliability was found in 0.82 and 0.83\r\nfor the barrier and benefits perceptions, respectively. Conclusions:\r\nThe instruments for perceiving barriers and benefits of\r\nexercising adapted to the Colombian context are valid and reliable.\r\nTherefore, they are considered appropriate for evaluating\r\nand designing strategies that contribute to improving the\r\nperformance of physical activity in adolescents.

Objetivo: validar no contexto colombiano os instrumentos de\r\npercepção de barreiras e benefícios para a realização de exercício\r\nem adolescentes com base no modelo de promoção da saúde\r\nde Nola Pender. Metodologia: os instrumentos de percepção\r\nde barreiras e benefícios para o exercício foram submetidos aos\r\nprocedimentos: autorizações, tradução, e testes de validade\r\nfacial, conteúdo e construto e confiabilidade. Resultados: o\r\ninstrumento de Percepção de Barreiras para o exercício possui\r\num índice de Lawshe modificado de 0,83 e Kappa de Fleiss de\r\n0,59 para relevância e pertinência de 0,53. O instrumento de\r\npercepção de benefícios para o exercício também apresenta\r\num índice de Lawshe modificado de 0,93 e Kappa de Fleiss para\r\nrelevância de 0,70 e para pertinência de 0,91. A análise fatorial\r\nidentificou em cada um dos instrumentos 2 fatores: barreiras à\r\nsaúde física e afetiva ou emocional; em termos de benefícios,\r\neles foram relacionados às condições ambientais e ao tempo\r\ndisponível para a realização do exercício, e a confiabilidade\r\nse encontrou em 0,83 e 0,83 para a percepção de barreiras e\r\nbenefícios respectivamente. Conclusões: os instrumentos\r\nde percepção de barreiras e benefícios para a realização de\r\nexercícios adaptados ao contexto colombiano são válidos e\r\nconfiáveis. Por isso, são considerados adequados para avaliar e\r\ndesenhar estratégias que contribuam para melhorar o desempenho\r\nda atividade física em adolescentes.

Closed-loop control of renal perfusion pressure in physiological experiments.

This paper presents the design, experimental modeling, and control of a pump-driven renal perfusion pressure (RPP)-regulatory system to implement precise and relatively fast RPP regulation in rats. The mechatronic system is a simple, low-cost, and reliable device to automate the RPP regulation process based on flow-mediated occlusion. Hence, the regulated signal is the RPP measured in the left femoral artery of the rat, and the manipulated variable is the voltage applied to a dc motor that controls the occlusion of the aorta. The control system is implemented in a PC through the LabView software, and a data acquisition board NI USB-6210. A simple first-order linear system is proposed to approximate the dynamics in the experiment. The parameters of the model are chosen to minimize the error between the predicted and experimental output averaged from eight input/output datasets at different RPP operating conditions. A closed-loop servocontrol system based on a pole-placement PD controller plus dead-zone compensation was proposed for this purpose. First, the feedback structure was validated in simulation by considering parameter uncertainty, and constant and time-varying references. Several experimental tests were also conducted to validate in real time the closed-loop performance for stepwise and fast switching references, and the results show the effectiveness of the proposed automatic system to regulate the RPP in the rat, in a precise, accurate (mean error less than 2 mmHg) and relatively fast mode (10-15 s of response time).

Boron Protection for O(2) Diffusion in Heterocysts of Anabaena sp. PCC 7119.

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N(2) fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.

A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120.

The impact of calcium signals in virtually all cells has led to the study of their role in prokaryotic organisms as stress response modulators. Cell differentiation in adverse conditions is a common Ca(2+)-requiring response. Nitrogen starvation induces the differentiation of N(2)-fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. This paper reports the use of a recombinant strain of this organism expressing the photoprotein aequorin to monitor the intracellular free-calcium concentration during the course of heterocyst differentiation. A specific calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and generated by intracellular calcium stores was identified. The intracellular calcium signal was manipulated by treatment with specific calcium drugs, and the effect of such manipulation on the process of heterocyst differentiation was subsequently assessed. Suppression, magnification or poor regulation of this signal prevented the process of heterocyst differentiation, thereby suggesting that a calcium signal with a defined set of kinetic parameters may be required for differentiation. A hetR mutant of Anabaena sp. PCC7120 that cannot differentiate into heterocysts retains, however, the capacity to generate the calcium transient in response to nitrogen deprivation, strongly suggesting that Ca(2+) may be involved in a very early step of the differentiation process.

Effect of Boron Deficiency on Photosynthesis and Reductant Sources and Their Relationship with Nitrogenase Activity in Anabaena PCC 7119.

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O(2) evolution and in CO(2) fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N(2) fixation is independent of its effects on photosynthesis and reductant supply.

Boron requirement in cyanobacteria : its possible role in the early evolution of photosynthetic organisms.

The effect of boron on heterocystous and nonheterocystous dinitrogen fixing Cyanobacteria was examined. The absence of boron in culture media inhibited growth and nitrogenase activity in Nodularia sp., Chlorogloeopsis sp., and Nostoc sp. cultures. Examinations of boron-deficient cultures showed changes in heterocyst morphology. However, cultures of nonheterocystous Cyanobacteria, Gloeothece sp. and Plectonema sp., grown in the absence of boron did not show any alteration in growth or nitrogenase activity. These results suggest a requirement of boron only by heterocystous Cyanobacteria. A possible role for this element in the early evolution of photosynthetic organisms is proposed.

Binding of cadmium by cyanobacterial growth media: free ion concentration as a toxicity index to the cyanobacterium Nostoc UAM 208.

A quantitative study of cadmium binding to three different growth media for nitrogen-fixing cyanobacteria was done with the aid of a solid state ion-specific electrode. Kratz and Myers modified medium and Arnon medium bound large amounts of Cd2+, BG11o medium had less binding capacity. Of the media components, phosphate ion showed the greatest ability to bind Cd2+. Different pHs, the size of cell inoculum and two buffers (Tricine and HEPES, 25 mM) also changed the availability of free cadmium ion in solution. The effect of free Cd2+ ion towards the cyanobacterium Nostoc UAM 208, isolated from a heavy metal polluted environment, also was tested. The effective concentration affecting 50% of population (EC50), at 120 h of exposure, was less for nitrogenase activity (0.26 microgram/mL) than for growth (0.55 microgram/mL), suggesting that this enzyme activity is more sensitive to cadmium than growth. Furthermore, cadmium toxicity was influenced by the addition of buffers to the growth medium. In the presence of buffer, Tricine (25 mM), growth and nitrogenase activity was reduced by 50% at a total cadmium concentration of about 115 micrograms/mL, although no free ion was detected in this case. These results suggest that although generally cadmium toxicity is a function of free metal ion concentration, this can also vary in the presence of complexing agents.

Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria.

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.