Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Prey range of the predatory ladybird Cryptolaemus montrouzieri

The prey range of Cryptolaemus montrouzieri was studied in the laboratory to investigate whether the mealybug destroyer can contribute to the suppression of other pest insects besides mealybugs and to assess its potential impact on non-mealybug populations as part of an environmental risk assessment for its use in biological control. Prey tested in these experiments were: tobacco aphid Myzus persicae nicotianae (Sulzer)(Hemiptera: Aphididae), pea aphid Acyrthosiphon pisum (Harris)(Hemiptera: Aphididae), tobacco whitefly Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae), southern green stinkbug Nezara viridula (L.)(Hemiptera: Pentatomidae) eggs, western flower thrips Frankliniella occidentalis (Pergande)(Thysanoptera: Thripidae), two-spotted ladybird Adalia bipunctata (L.)(Coleoptera: Coccinellidae) eggs and eggs of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae). Larval survival was high to moderate when C. montrouzieri was provided with hemipteran prey and poor to zero when the ladybirds were provided with non-hemipteran prey. Females reared on M. persicae and A. pisum produced similar numbers of eggs as their counterparts fed the citrus mealybug Planococcus citri (Risso)(Hemiptera: Pseudococcidae), but fecundity was significantly lower when the ladybirds were reared on B. tabaci nymphs or on A. bipunctata eggs. Prey species that were found to be less suitable for immature development of C. montrouzieri could still be an adequate food source for reproduction and survival of adult ladybirds. For example, only 8 % of the predator larvae reached the adult stage when provided with A. bipunctata eggs, but females that had developed on eggs of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and that were supplied with A. bipunctata eggs from adult emergence on, were only 35 % less fecund than females provided with mealybugs in their adult life. The results are discussed in relation to the development of a suitable methodology for prey/host range testing in the framework of an environmental risk assessment for arthropod natural enemies.     

Immunohistochemical localization of a gastrin-like peptide in the brain of an amphibian,Xenopus laevis Daud.

Summary The indirect immunofluorescence technique was used to demonstrate a substance reacting with gastrin antisera in the brain of Xenopus laevis.Immunoreactive material was found in two sites: (1) In the caudal hypothalamus more precisely in the nucleus infundibularis ventralis, (NIV) of the pars ventralis of the tuber cinereum, (PVTC). The fluorescent axons of the reactive parikarya of the NIV give rise to two symmetrical tracts which run rostro-ventrally and join, in the infundibular floor, the preoptico-hypophysial tract, where they form an uneven median tract coursing caudally and running along the medio-tuberal area before entering the external zone of the median eminence. (2) In the anterior preoptic area (APOA), where numerous nerve fibers and endings form a dense network near the preoptic recess. The exact origin of these terminals has not yet been determined.Control of immunohistochemical specificity shows that the labeling by gastrin antisera is suppressed by gastrin (2–17), but also by cholecystokinin (CCK) and pentagastrin (Peptavlon). These results indicate that the immunoreactive substance revealed belongs to the gastrin group and has an antigenic determinant composed of the amino acid sequence or a portion thereof common to gastrin, CCK and Peptavlon (Trp-Met-Asp-Phe).It should be emphasized that, in the brain of Xenopus laevis, both gastrinimmunoreactive sites correspond to the sites of uptake of steroid hormones (Kelley et al., 1975; Morrell et al., 1975).Supported by the D.G.R.S.T., Contrat n 77.7.0648     

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.     

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra).

Methodology/Principal Findings

Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia.

Conclusions/Significance

GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.     

Nitrate-dependent control of root architecture and N nutrition are altered by a plant growth-promoting Phyllobacterium sp

Both root architecture and plant N nutrition are altered by inoculation with the plant growth-promoting rhizobacteria (PGPR) Phyllobacterium strain STM196. It is known that NO₃⁻ and N metabolites can act as regulatory signals on root development and N transporters. In this study, we investigate the possible interrelated effects on root development and N transport. We show that the inhibition of Arabidopsis lateral root growth by high external NO₃⁻ is overridden by Phyllobacterium inoculation. However, the leaf NO₃⁻ pool remained unchanged in inoculated plants. By contrast, the Gln root pool was reduced in inoculated plants. Unexpectedly, NO₃⁻ influx and the expression levels of AtNRT1.1 and AtNRT2.1 genes coding for root NO₃⁻ transporters were also decreased after 8 days of Phyllobacterium inoculation. Although the mechanisms by which PGPR exert their positive effects remain unknown, our data show that they can optimize plant development independently from N supply, thus alleviating the regulatory mechanisms that operate in axenic conditions. In addition, we found that Phyllobacterium sp. elicited a very strong induction of AtNRT2.5 and AtNRT2.6, both genes preferentially expressed in the shoots whose functions are unknown.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.     

Contribution of the endoplasmic reticulum to the glucose-induced [Ca(2+)](c) response in mouse pancreatic islets

Thapsigargin (TG), a blocker of Ca(2+) uptake by the endoplasmic reticulum (ER), was used to evaluate the contribution of the organelle to the oscillations of cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by repetitive Ca(2+) influx in mouse pancreatic beta-cells. Because TG depolarized the plasma membrane in the presence of glucose alone, extracellular K(+) was alternated between 10 and 30 mM in the presence of diazoxide to impose membrane potential (MP) oscillations. In control islets, pulses of K(+), mimicking regular MP oscillations elicited by 10 mM glucose, induced [Ca(2+)](c) oscillations whose nadir remained higher than basal [Ca(2+)](c). Increasing the depolarization phase of the pulses while keeping their frequency constant (to mimic the effects of a further rise of the glucose concentration on MP) caused an upward shift of the nadir of [Ca(2+)](c) oscillations that was reproduced by raising extracellular Ca(2+) (to increase Ca(2+) influx) without changing the pulse protocol. In TG-pretreated islets, the imposed [Ca(2+)](c) oscillations were of much larger amplitude than in control islets and occurred on basal levels. During intermittent trains of depolarizations, control islets displayed mixed [Ca(2+)](c) oscillations characterized by a summation of fast oscillations on top of slow ones, whereas no progressive summation of the fast oscillations was observed in TG-pretreated islets. In conclusion, the buffering capacity of the ER in pancreatic beta-cells limits the amplitude of [Ca(2+)](c) oscillations and may explain how the nadir between oscillations remains above baseline during regular oscillations or gradually increases during mixed [Ca(2+)](c) oscillations, two types of response observed during glucose stimulation.