Relationship of nitrate supply with growth rate, plasma membrane-bound and cytosolic nitrate reductase, and tissue nitrate content in tobacco plants

cNR, cytosolic nitrate reductase
PM-NR, plasma membrane-bound nitrate reductase

Activities of plasma membrane-bound nitrate reductase (PM-NR) and cytosolic nitrate reductase (cNR) in tobacco (Nicotiana tabacum L. cv. Samsun) are regulated differently, depending upon the nitrate supply to the culture medium (in sand culture). The cNR activity of roots was higher at low nitrate concentrations with the maximum at 5 mM nitrate supply and declined to low values beyond 5 mM . In contrast, the PM-NR activity of roots increased with higher nitrate concentrations with the maximum at 25 mM nitrate and clearly decreased only at 40 mM . This high PM-NR activity correlated with a low growth rate and might be one of the responses to excess nitrate. Internal nitrate and total nitrogen content of the tissues, however, showed a relative minimum in shoots and in roots of between 15 and 25 mM external nitrate. With declining PM-NR activities beyond 25 mM external nitrate, the nitrate content in the tissue increased indicating an inverse relationship between tissue nitrate content and root PM-NR activity. In leaves both NR activities (cNR and PM-NR) correlated with the internal nitrate content, but with a different response at low nitrate.     

Cell Surface Interactions between Bean Leaf Cells and Colletotrichum lindemuthianum: Cytochemical Aspects of Pectin Breakdown and Fungal Endopolygalacturonase Accumulation

After a brief period of biotrophic growth, the anthracnose fungus Colletotrichum lindemuthianum (Sacc. et Mgn.) Bri et Cav. develops extensively in bean leaf cells, causing severe wall alterations and death of the host protoplast. Aplysia gonad lectin, a polygalacturonic acid-binding agglutinin, was complexed to gold and used to study the extent of pectin breakdown during the necrotrophic phase of the infection process. In view of its specific binding properties for the endopolygalacturonase produced by C. lindemuthianum, a polygalacturonase-inhibiting protein isolated from bean cell walls was successfully tagged with gold particles and used for localizing the sites of enzyme accumulation in infected host tissues. The basal level of endopolygalacturonase produced by C. lindemuthianum grown in culture was found to increase severalfold when the fungus developed in host plant tissues. The enzyme was able to diffuse freely in the host cell wall, causing drastic degradation of the pectic material of primary walls and middle lamella matrices. The enzymatic alteration of plant cell walls was accompanied by the release of pectic fragments and by the accumulation of pectic molecules at specific sites, such as intercellular spaces and aggregated cytoplasm of infected host cells. The occurrence of pectic molecules at those sites where fungal growth is likely to be restricted is discussed in relation to their origin and their implication in the plant's defense system.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Classification of videocapsule endoscopy image patterns: comparative analysis between patients with celiac disease and normal individuals

Background  

Quantitative disease markers were developed to assess videocapsule images acquired from celiac disease patients with villous atrophy, and from control patients.     

Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought‐stress conditions

A transgenic gene‐silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down‐regulated ACC synthases (ACSs), enzymes that catalyse the rate‐limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought‐stress and rain‐fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype × transgene) × environment interaction existed among the events, highlighting the need to better understand the context in which the down‐regulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis‐silking interval and a concomitant increase in kernel number/ear in transgene‐positive events versus nulls. Selected events were also field tested under a low‐nitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/acre) yield increase. This set of extensive field evaluations demonstrated that down‐regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.     

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.     

Soluble and membrane-associated nitrate reductases in the dinoflagellate Peridinium gatunense

An improved method of cell fractionation allowed the extraction of soluble (sNR) and membrane-associated (mNR) forms of nitrate reductase (NR) from a dinoflagellate, even though in previous studies only mNR had been found in these algae. Both activities were assayed in cell-free extracts of Peridinium gatunense from Lake Kinneret, Israel, after disruption of the cells and differential centrifugation. In the cultures used, sNR showed much higher NO₃⁻-reducing activity. Only a low proportion, 2.5–3% of NR activity, was found to be associated with mNR. Moreover, mNR comprised two forms as indicated by protein solubilization: a tightly membrane-bound and a more weakly attached NR. Ascorbate inhibited all NR activities, but that of mNR recovered after its removal. Polyvinyl pyrrolidone (PVP) and DTT also diminished sNR and mNR activities. For both enzymes, pH optima (7.65) and temperature optima (13–25°C) were similar, and agreed with those for optimum growth of P. gatunense both in culture and in the lake. The most efficient electron donor was NADH, though NADPH sustained low NR activities. Carboxylic anions such as succinate and malate did not support any reduction of NO₃⁻, nor did they cause any stimulation of sNR or mNR activities. Both forms of NR showed a high affinity for their substrates: K _m was c. 10 μM for NO₃⁻ and c. 5 μM for NADH. The high efficiency of NO₃⁻ assimilation by Peridinium seems to be limited mainly by energy under otherwise optimal nutritional conditions and, at low nitrate concentrations, the low K _m may be one of the main reasons for the high competitivity of this alga in Lake Kinneret.     

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

Background

ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A₂ activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1).

Methods

Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection.

Results

In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively.

Conclusion

ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.     

Differential effects of age,cytomegalovirus-seropositivity and end-stage renal disease (ESRD) on circulating T lymphocyte subsets

The age- and cytomegalovirus (CMV)-seropositivity-related changes in subsets and differentiation of circulating T cells were investigated in end-stage renal disease (ESRD) patients (n = 139) and age-matched healthy individuals. The results show that CMV-seropositivity is associated with expansion of both CD4⁺ and CD8⁺ memory T cells which is already observed in young healthy individuals. In addition, CMV-seropositive healthy individuals have a more differentiated memory T cell profile. Only CMV-seropositive healthy individuals showed an age-dependent decrease in CD4⁺ naïve T cells. The age-related decrease in the number of CD8⁺ naïve T cells was CMV-independent. In contrast, all ESRD patients showed a profound naïve T-cell lymphopenia at every decade. CMV-seropositivity aggravated the contraction of CD4⁺ naïve T cells and increased the number of differentiated CD4⁺ and CD8⁺ memory T cells. In conclusion, CMV-seropositivity markedly alters the homeostasis of circulating T cells in healthy individuals and aggravates the T cell dysregulation observed in ESRD patients.