Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

Background

Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins G??_q, G??₁₃ and G??_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.

Results

Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by G??_i-triggered signalling as well as by G?|?-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NF?B-pathway and thereby the production of TNF-??, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by G??_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by G?|?-mediated activation of PI3kinase and JNK activation.

Conclusions

On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host??s immune response.     

Estrogen sensitive cell lines: Establishment and characterization of new cell lines from estrogen-induced rat pituitary tumors

Rat pituitary tumors were induced by the monthly injection of high doses of estradiol valerate. Of 12 animals which received the estrogen, 10 developed tumors. From these 10 tumors, 4 cell lines were successfully established and they have been maintained in culture for over 18 months. Several clones have been isolated from these established cell lines. Three of the 4 cell lines produce tumors in females considerably faster than in males or castrated females. Tumor-bearing animals have significantly increased amounts of rat growth hormone and prolactin in their serum. The 4 established cell lines, as well as clones derived from them, and tumors obtained in situ by injection of these cells growing in culture or successive transplants have shown the presence of an estrogen-binding protein of high affinity and low capacity. This estrogen-binding protein is similar to that described in other target organs (uterus, mammary glands, etc). These cell lines will be used to study the mechanism of action of estrogen in target cells as well as the synthesis and secretion of pituitary hormones.     

The Legionella pneumophila F‐box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication

The environmental pathogen Legionella pneumophila encodes three proteins containing F‐box domains and additional protein–protein interaction domains, reminiscent of eukaryotic SCF ubiquitin–protein ligases. Here we show that the F‐box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella‐containing vacuole. Single, double and triple mutants of the F‐box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP‐1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo , and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two‐hybrid screen and co‐immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein–protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.     

A Model of Binge‐Like Eating Behavior in Mice That Does Not Require Food Deprivation or Stress

Binge eating disorder (BED) is characterized by excessive food intake during a short period of time and is often associated with obesity. Mouse models of binge‐like eating behavior are lacking making it difficult to employ genetic models in the identification of mechanisms regulating excessive eating. We report a rapid and simple model to induce binge‐like eating behavior in mice that does not require food deprivation or exogenous stressors. Weekly 24 h access to a nutritionally complete high energy diet (HED), along with continuous access to standard chow, resulted in a significant increase in HED intake following its presentation compared to mice that had continuous access to both diets. Mice exhibiting binge‐like eating consumed one‐third of their normal total daily caloric intake within 2.5 h of HED presentation. Moreover, total 24‐h caloric intakes were increased by 50% in mice exhibiting binge‐like eating. Following repeated cycles, binge‐like eating of the HED was maintained over several weeks with no evidence of habituation or significant alterations in body weight and adiposity. Pharmacological evaluation of binge‐like eating behavior was performed using clinically employed compounds. Interestingly, binge‐like eating was dose‐dependently decreased by fluoxetine, but not baclofen or topiramate. These data support clinical validation of this mouse model of binge‐like eating behavior, as fluoxetine has been shown to reduce binge frequency in human subjects with BED. The availability of transgenic and knockout mice will allow for the determination of genes that are involved in the initiation and maintenance of binge‐like eating behavior.     

Germination of <i>Eryngium regnellii</i>: a major species for ecological restoration of plant-pollinator interactions in the Southern Pampas (Buenos Aires,Argentina)

Eryngium regnellii Malme belongs to the largest genera in the Apiaceae family, with 250 species worldwide and 65 represented in South America. It is a herbaceous species typical of hill plant communities, which, along with remnant grassland patches, are the most relevant natural habitats for the maintenance of diversity in the Southern Pampas. Eryngium regnellii is key to the maintenance of pollination mutualisms, being a generalist (displaying a diverse assemblage of pollinators) and ubiquitous species (present in all studied sierras). However, fragmentation of the Pampean landscape due to agricultural intensification has led to the loss of natural environments. Therefore, the reintroduction of E. regnellii in strategic places would facilitate the occurrence of wild pollinators, while favoring pollination services in the agroecosystem. The germination requirements of E. regnellii were studied because a better knowledge of the reproductive biology of this species would provide information relevant to its reproduction and reintroduction into degraded areas. Germination percentages and mean time to germination were evaluated, using one control and two pre-germination treatments: chemical scarification with sulfuric acid, and mechanical scarification with sand paper. Chemical scarified seeds did not germinate. Mechanically scarified and control seed groups showed no significant differences either in germination percentages (49% and 59% respectively) or in mean germination time (13 and 14 days, respectively). Results indicate that E. regnellii shows no physical dormancy, and does not require specific pre-germination treatments for germination under the studied laboratory conditions. The high germination capacity of E. regnellii, along with its ecological attributes, make it a potential species for restoring plant-pollinator interactions in the fragmented landscapes of the Southern Pampas.     

Nitrate reductase activity,biomass, yield,and quality in cotton in response to nitrogen fertilization

In the production of cotton (Gossypium hirsutum L.), nitrogen fertilization is one of the most costly crop practices, but important to reach high yields. However, high nitrogen (N) content in plants does not always translate into a high fibre production. One way of assessing the efficiency of the N fertilizer is through the enzymatic activity of the nitrate reductase (NR). This is a key enzyme in N assimilation, whose activity is regulated by a number of endogenous and exogenous factors that determine yield. The aim of this study was to assess the effect of N fertilization on yield, fibre quality, biomass, and NR enzymatic activity in vivo in the cotton variety Fiber Max 989. The evaluated application rates were 0, 50, 100, and 150 kg/ha of N, using urea as a source (46% N) in a randomizedblock design with three replicates. At harvest, the maximum yield of seed cotton and the greatest accumulation of total foliar biomass through time was reached after applying 150 kg N/ha. The different N-application rates did not affect the components of cotton-fibre quality. The activity of endogenous NR was greater on plants where 150 kg N/ha were applied. The highest cotton yield and N contents were obtained on these plants. Therefore, the NR activity in vivo could be used as a bioindicator of the N nutritional level in cotton.     

Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.