Comparative pathobiology of Heterobasidion annosum during challenge on Pinus sylvestris and Arabidopsis roots: an analysis of defensin gene expression in two pathosystems

Heterobasidion annosum is widely known as a major root and butt rot pathogen of conifer trees, but little information is available on its interaction with the roots of herbaceous angiosperm plants. We investigated the infection biology of H. annosum during challenge with the angiosperm model Arabidopsis and monitored the host response after exposure to different hormone elicitors, chemicals (chitin, glucan and chitosan) and fungal species that represent diverse basidiomycete life strategies [e.g., pathogen (H. annosum), saprotroph (Stereum sanguinolentum) and mutualist (Lactarius rufus)]. The results revealed that the tree pathogen (H. annosum) and the saprotroph (S. sanguinolentum) could infect the Col-8 (Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. Germinated H. annosum spores had appressorium-like penetration structures attached to the surface of the Arabidopsis roots. Subsequent invasive fungal growth led to the disintegration of the vascular region of the root tissues. Progression of root rot symptoms in Arabidopsis was similar to the infection development that was previously documented in Scots pine seedlings. Scots pine PsDef1 and Arabidopsis DEFLs (AT5G44973.1) and PDF1.2 were induced at the initial stage of the infection. However, differences in the expression patterns of the defensin gene homologs from the two plant groups were observed under various conditions, suggesting functional differences in their regulation. The potential use of the H. annosum–Arabidopsis pathosystem as a model for studying forest tree diseases is discussed.     

In vitro propagation and characterization of phenolic content along with antioxidant and antimicrobial activities of Cichorium pumilum Jacq.

Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb. An efficient protocol for callus formation and whole plant propagation has been developed. Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 1.5?mg?l^?1 6-Benzyladenine (BA) and 0.5?mg?l^?1 Naphthalene acetic acid (NAA). Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 1.5?mg?l^?1 BA or Kinetin (Kin). The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid. In the present study, the antibacterial activity of C. pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species. The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively. Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts. This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds. The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants. The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C. pumilum.     

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia

Background

Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations.

Methodology/Principal Findings

Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied.

Conclusions/Significance

As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.     

Survey of Flea Infestation in Dogs in Different Geographical Regions of Iran

Medically important arthropods, including fleas, play an important role in causing clinical disorders and disease in man and domestic animals. This study was conducted to determine the seasonal flea infestations for domestic dogs from different geographic regions of Iran. A total of 407 fleas, belonging to 5 different species, were recovered from 83 domestic dogs from 3 regions. There was a distinctive pattern of species distribution and infestations with the highest infestation rates observed in a temperate climate and higher rainfall. Additionally, fleas were observed over all seasons, except February and March, with the highest infestation rate observed in August (24.7%) and the lowest rate in January (1.7%). They also parasitize dogs with a different spectrum of species. The cat flea, Ctenocephalides felis (67.5%), exhibited the highest prevalence among all flea species found on dogs. Thus, climatic conditions and seasonal patterns impact on flea infestation and must be considered in developing control programs.     

Protein modification and replicative senescence of WI‐38 human embryonic fibroblasts

Oxidized proteins as well as proteins modified by the lipid peroxidation product 4‐hydroxy‐2‐nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI‐38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis‐based proteomic approach coupled with immunodetection of HNE‐, AGE‐modified and carbonylated proteins. Thirty‐seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE‐modified proteins could be explained by the senescence‐associated decreased activity of glyoxalase‐I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age‐related build‐up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP‐stimulated Lon‐like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins.     

Protective effects of antioxidants on age-related changes in the electrophoretic patterns of cardiac LDH,hepatic ALP and serum proteins in male golden hamster

Basal and antioxidant-induced changes in the isoenzyme and isoform patterns of cardiac lactate dehydrogenase (EC 1.1.1.27) and hepatic alkaline phosphatase (EC 3.1.3.1), respectively, as well as the electrophoretic patterns of serum proteins in different age groups of male golden hamster were compared. This is to test whether age-induced changes could be corrected by long-term administration of antioxidants. Data indicated that aging causes no remarkable change in the total activity of either cardiac LDH or hepatic ALP, however a significant increase in the fractional activity of some cardiac LDH isoenzymes and a significant reduction in the fractional activity of some hepatic ALP isoforms were induced by aging. On the other hand, long-term administration of antioxidants appeared to manifest a clear counteracting effect on the age-related changes in old age. This effect was indicated in the fractional activity of cardiac LDH isoenzymes and of hepatic ALP isoforms. The present study has also shown a wide-range variation in serum protein patterns due to aging and/or antioxidant administration, which indirectly reflect a parallel variation in the process of gene expression and/or proteolytic activity.     

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.