The differential effects of PGE on the immune response in normal and immunosuppressed mice

The effect of 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) on humoral and cellular immunoresponsiveness has been compared in normal mice and in mice immunosuppressed by splenectomy and thymectomy plus antithymocyte serum (ATS). Splenectomy resulted in immunosuppression manifested by augmentation of B-16 melanoma growth; this stimulatory effect was reversed by di-M-PGE₂. In animals immunosuppressed by thymectomy plus ATS, di-M-PGE₂ augmented the humoral and cellular immune responses; this was manifested by slowing of the growth of B-16 melanoma and by stimulating the number of plaque-forming cells, hemagglutinin titers, and delayed-hypersensitivity reactions to sheep erythrocytes. In contrast, in normal (nonthymectomized) mice, di-M-PGE₂ was mildly immunosuppressive. Finally, adriamycin-immunosuppressed normal mice and this suppression were reversed by the addition of di-M-PGE₂ to the treatment regimen.     

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis-CK. Since no isomerization of (trans)[²H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.     

PGE restores the immune response in chemotherapy-treated,tumor-bearing mice

Two hundred seventy-one B-16 melanoma-bearing mice were randomized and treated for 4 days with either control diluent, 10 μg of 16, 16-dimethyl-PGE₂-methyl-ester (di-M-PGE₂), chemotherapy, or chemotherapy plus di-M-PGE₂. The chemotherapeutic regimens included adriamycin (7.5 mg/kg), 5-fluorouracil (250 mg/kg), nitrogen mustard (5 mg/kg), and vincristine (0.5 mg/kg). The number of plaque-forming cells and hemagglutinin titers in response to sheep erythrocytes were used as measures of humoral immunity while cellular immunity was assessed by evaluation of delayed hypersensitivity. As we previously reported, the presence of subcutaneous B-16 tumors induced substantial immunosuppression and this suppression was reversed by treatment with di-M-PGE₂. Treatment with all four chemotherapeutic agents induced profound immunosuppression. Similarly, the addition of di-M-PGE₂ to the chemotherapy protocols resulted in significant augmentation of cellular and humoral immunity.     

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

Cotrimoxazole for prenatal treatment of congenital toxoplasmosis?

Congenital toxoplasmosis is still one of the most frequent causes of fetal death. Despite a significant improvement in diagnosis, particularly in utero diagnosis, maternal treatment is only partially effective in preventing transmission to the fetus and treating fetal infection. Maternal treatment is based on drugs developed 50 years ago, which may have limited efficacy (spiramycin) or serious side-effects (pyrimethamine). Data on the use of cotrimoxazole in mouse models of toxoplasmosis and for preventing toxoplasmic encephalitis in patients suffering from AIDS have led Francis Derouin and colleagues to consider the potential of cotrimoxazole for prenatal prevention and treatment of toxoplasmic fetal death.     

Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K(+)/Na(+) selectivity and proline accumulation

The eco-physiology of salt tolerance, with an emphasis on K⁺ nutrition and proline accumulation, was investigated in the halophyte Thellungiella halophila and in both wild type and eskimo-1 mutant of the glycophyte Arabidopsis thaliana, which differ in their proline accumulation capacity. Plants cultivated in inert sand were challenged for 3 weeks with up to 500 mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300 mM NaCl in T. halophila. Na⁺ content generally increased with the amount of salt added in the culture medium in both species, but T. halophila showed an ability to control Na⁺ accumulation in shoots. The analysis of the relationship between water and Na⁺ contents suggested an apoplastic sodium accumulation in both species; this trait was more pronounced in A. thaliana than in T. halophila. The better NaCl tolerance in the latter was associated with a better K⁺ supply, resulting in higher K⁺/Na⁺ ratios. It was also noteworthy that, despite highly accumulating proline, the A. thaliana eskimo-1 mutant was the most salt-sensitive species. Taken together, our findings indicate that salt tolerance may be partly linked to the plants’ ability to control Na⁺ influx and to ensure appropriate K⁺ nutrition, but is not linked to proline accumulation.     

Cutting edge: chemoattractant receptor-homologous molecule expressed on Th2 cells plays a restricting role on IL-5 production and eosinophil recruitment

PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.     

Structural Changes in Senescing Oilseed Rape Leaves at Tissue and Subcellular Levels Monitored by Nuclear Magnetic Resonance Relaxometry through Water Status

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.The main physiological outcome of leaf senescence is the recycling of organic resources and the provision of nutrients to sink organs such as storage and growing tissues (Buchanan-Wollaston, 1997; Hikosaka, 2005; Krupinska and Humbeck, 2008). In crop plants, senescence progresses from the lower older leaves to the younger top leaves. Macromolecular degradation and the mechanism of reallocation of breakdown products are mediated by the up-regulation of senescence-related genes (Lee et al., 2001) in close relationship with both developmental and environmental conditions (Gombert et al., 2006). This leads to remobilization of carbon and nitrogen (N) compounds mostly from plastidial compartments (Martínez et al., 2008; Guiboileau et al., 2012), involving proteolytic activity in plasts, vacuole, and cytosol (Adam and Clarke, 2002; Otegui et al., 2005), chlorophyll breakdown (Hoertensteiner, 2006), galactolipid recycling (Kaup et al., 2002) in the plastoglobules (Brehelin et al., 2007), and loading of Suc and amino acids into the phloem through appropriate transporters (Wingler et al., 2004; Masclaux-Daubresse et al., 2008). In terms of leaf senescence at the cell level, where chloroplasts are degraded sequentially, relative organelle volume does not seem to be greatly modified, the vacuole remains intact, and in darkness-induced senescence the number of chloroplasts per cell decreases only slightly (Keech et al., 2007). However, major changes in metabolic fluxes and cell water relationships are expected during the senescence program that may be associated with macromolecule catabolism, organic solute synthesis, transport and remobilization, and cell structure reconfiguration such as chloroplast evolution to gerontoplast (Hoertensteiner, 2006; Zhang et al., 2010) through the autophagy process (Wada et al., 2009), accumulation of senescence-associated vacuoles (Otegui et al., 2005), and cell wall degradation (Mohapatra et al., 2010).The senescent leaves of oilseed rape (Brassica napus), a major oleiferous crop, generally fall while still maintaining a high N content (about 2.5%–3% [w/w] of the dry matter; Malagoli et al., 2005). In addition to the environmental impact of this leaking of N out of the plant, the low capacity to remobilize foliar N is associated with a high requirement for N fertilization to meet the potential crop yield (Dreccer et al., 2000). In order to improve the nitrogen use efficiency (NUE), new genotypes are being selected for their ability to maintain high yields under limited N fertilization, mainly via the improvement of N uptake efficiency and N mobilization from the senescing leaves (Hirel et al., 2007). In Arabidopsis (Arabidopsis thaliana) and oilseed rape, N can be remobilized from old to expanding leaves at the vegetative stage during sequential senescence as well as from leaves to seeds at the reproductive stage during monocarpic senescence (Malagoli et al., 2005; Diaz et al., 2008; Lemaitre et al., 2008). Senescence can also be induced by environmental stress such as N starvation (Etienne et al., 2007) or water deficit (Reviron et al., 1992) and propagated from old to mature leaves and delayed in young leaves, suggesting finely tuned high regulation of metabolism at the whole-plant level with consequences for NUE (Desclos et al., 2008). One major challenge to understanding the efficiency of senescence-induced organic resource reallocation and to highlighting major molecular and mechanistic attributes of nutrient recycling is monitoring the kinetics of the structural reorganization of cell structures. This reorganization will provide nutrients remobilized through phloem loading. From a technological and phenotyping point of view, the measurement of N remobilization efficiency has already been addressed in crop species and oilseed rape, as it is a reliable trait to screen for the genetic variability of NUE (Franzaring et al., 2012). However, techniques such as stable isotope feeding are time consuming, destructive, and difficult to adapt to large genotype panels. Therefore, it is important to develop a technique for following changes in water distribution at the cell level in order to understand metabolic reconfigurations occurring throughout senescence.NMR relaxometry has been used in several studies to investigate plant cell structure and functioning (Hills and Duce, 1990; Van As, 1992). The ¹H-NMR signal originates almost entirely from water protons because other ¹H nuclei in the plant produce much less intense signals, as they correspond to molecules that are at a much lower concentration than water. The technique allows the measurement of longitudinal (T1) and transverse (T2) relaxation times and proton spin density. Water proton relaxation times are related to the rotational and translational mobility of water molecules (Van As, 2007). They are also modified by the mobility and structure of the surrounding macromolecules (i.e. starch, proteins, and polysaccharides) through proton exchange (chemical exchange). In plant cells, the water in different cell compartments has different chemical and physical properties and, therefore, different bulk T2 values. Moreover, relaxation times are affected by the exchange of molecules between different compartments that is determined by water diffusion and, therefore, by the compartment size and membrane permeability (Van der Weerd et al., 2002). The slow diffusion process between compartments results in multiexponential behavior of the relaxation signal. The multiexponential relaxation reflects water in cell compartments and, therefore, can be used to study changes in water distribution and properties at a subcellular level and, hence, can be used for the estimation of structural and volume transformations in cell compartments. The T2 relaxation time is more sensitive to small variations in water content and chemical exchange processes than T1 and, therefore, is usually preferred. Indeed, differences in T1 for the different compartments are relatively small, resulting in an averaging effect that results in poor discrimination between water compartments (Van As, 2007).To date, NMR relaxometry has mainly been used for the characterization of fruit and vegetable tissues and has been shown to be effective in providing valuable information about cell organization (Sibgatullin et al., 2007). However, although a number of studies have contributed to the interpretation of the NMR results (Snaar and Van As, 1992; Hills and Nott, 1999; Marigheto et al., 2009), this is still not always straightforward, as the NMR signal depends both on the nature of the plant tissue and on the NMR measurement protocol. The situation is even more complex in the case of leaves, because leaves contain different tissue types characterized by different cell sizes and structures (Teixeira et al., 2005), and only a few studies involving NMR relaxometry in leaves have been reported. Changes in T2 in response to high temperature were investigated in wheat (Triticum aestivum; Maheswari et al., 1999) in order to develop a method for the detection of heat injury. McCain (1995) measured the T2 relaxation time of chloroplast and nonchloroplast water in maple (Acer platanoides) leaves by separating corresponding peaks in an NMR spectrum without taking into account the compartmentalization of nonchloroplast water. Oshita et al. (2006) investigated cell membrane permeability to water in spinach (Spinacia oleracea) leaves by measuring the T1 relaxation time of the leaf protoplasts without consideration of the subcellular structure. Qiao et al. (2005) attempted to associate NMR signal components with different chive (Allium schoenoprasum) cells using combined transverse relaxation and restricted diffusion measurements. Finally, Capitani et al. (2009) recently used a portable unilateral NMR instrument to detect the water status of leaves of herbaceous crops, mesophyllous trees, and natural Mediterranean vegetation under field conditions. Further investigations are necessary to improve leaf characterization by NMR, especially in the attribution of NMR signal components to the tissue and subcellular compartments. Progress in this field would make it possible to use the full potential of noninvasive NMR relaxometry in plant research and phenotyping.Using NMR relaxometry, we describe here the differences in water status that occurred at tissue and cellular levels through different leaf ranks of well-irrigated oilseed rape plants, from the young leaves at the top of the canopy to the senescing older leaves at the bottom of the plant. The aim of the study was to show that changes that occur in the leaves while senescing can be related to changes in water distribution and cell structure. As these changes are directly linked to the modifications in cell compartment organization, especially those occurring in the chloroplast, vacuole, and cell wall due to macromolecule degradation and N and carbon reallocation processes, this study was designed to contribute to the understanding of these physiological processes.