Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H₂O₂ concentration. In contrast, an early and high accumulation of H₂O₂ was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.     

Akt contributes to activation of the TRIF-dependent signaling pathways of TLRs by interacting with TANK-binding kinase 1

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.     

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii

IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.     

QTL analysis for grain quality traits in 2 BC2F2 populations derived from crosses between Oryza sativa cv Swarna and 2 accessions of O. nivara

The appearance and cooking quality of rice determine its acceptability and price to a large extent. Quantitative trait loci (QTLs) for 12 grain quality traits were mapped in 2 mapping populations derived from Oryza sativa cv Swarna × O. nivara. The BC(2)F(2) population of the cross Swarna × O. nivara IRGC81848 (population 1) was evaluated during 2005 and that from Swarna × O. nivara IRGC81832 (population 2) was evaluated during 2006. Linkage maps were constructed using 100 simple sequence repeat (SSR) markers in population 1 and 75 SSR markers in population 2. In all, 21 QTLs were identified in population 1 (43% from O. nivara) and 37 in population 2 (38% QTLs from O. nivara). The location of O. nivara-derived QTLs mp1.2 for milling percent, kw6.1 for kernel width, and klac12.1 for kernel length after cooking coincided in the 2 populations and appear to be useful for Marker Assisted Selection (MAS). Four QTLs for milling percent, 1 QTL each for amylose content, water uptake, elongation ratio, 2 QTLs for kernel width, and 3 QTLs for gel consistency, each explained more than 20% phenotypic variance. Three QTL clusters for grain quality traits were close to the genes/QTLs for shattering and seed dormancy. QTLs for 4 quality traits were associated with 5 of the 7 major yield QTLs reported in the same 2 mapping populations. Useful introgression lines have been developed for several agronomic traits. It emerges that 40% O. nivara alleles were trait enhancing in both populations, and QTLs for grain quality overlapped with yield meta-QTLs and QTLs for dormancy and seed shattering.     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

Insights into archaeal chaperone machinery: a network-based approach

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.     

Oxygen uptake rate as a tool for on-line estimation of cell biomass and bed temperature in a novel solid-state fermentation bioreactor

Bioprocess and Biosystems Engineering - Direct measurement of cell biomass is difficult in a solid-state fermentation (SSF) process involving filamentous fungi since the mycelium and the solid...     

Tackling <Emphasis Type="Italic">Salmonella</Emphasis> Persister Cells by Antibiotic–Nisin Combination via Mannitol

Bacterial persisters (defined as dormant, non-dividing cells with globally reduced metabolism) are the major cause of recurrent infections. As they neither grow nor die in presence of antibiotics, it is difficult to eradicate these cells using antibiotics, even at higher concentrations. Reports of metabolites (which help in waking up of these inactive cells) enabled eradication of bacterial persistence by aminoglycosides, suggest the new potential strategy to improve antibiotic therapy. Here we propose, mannitol enabled elimination of Salmonella persister cells by the nisin–antibiotic combination. For this, persister cells were developed and characterized for their typical properties such as non-replicative state and metabolic dormancy. Different carbon sources viz. glucose, glycerol, and mannitol were used, each as an adjunct to ampicillin for the eradication of persister cells. The maximum (but not complete) killing was observed with mannitol–ampicillin, out of all the combinations used. However, significant elimination (about 78%) could be observed, when nisin (an antimicrobial peptide) was used with ampicillin in presence of mannitol, which might have mediated the transfer of antibiotic–nisin combination at the same time when the cells tried to grab the carbon molecule. Further, the effectiveness of the trio was confirmed by flow cytometry. Overall, our findings highlight the potential of this trio-combination for developing it as an option for tackling Salmonella persister cells.     

Identification of salt-induced transcripts by suppression subtractive hybridization and their expression analysis under the combination of salt and elevated CO<Subscript>2</Subscript> conditions in <Emphasis Type="Italic">Salicornia brachiata</Emphasis>

Soil salinity is a major abiotic stress that affects global agricultural productivity. Exploring the mechanisms that halophytes employ to thrive and flourish under saline environments is essential to increase the salt tolerance in sensitive crop species. Of the three halophytes used in this study Salicornia brachiata and Suaeda maritima belong to the same family Chenopodiaceae, while Sesuvium portulacastrum, a mangrove-associated halophyte, belongs to the family Aizoaceae. Assuming that halophytes of same family share similar salt tolerance mechanisms, we generated a suppression subtractive hybridization (SSH1) cDNA library from salt-treated leaf tissues of S. brachiata as tester and that of S. maritima as driver to identify salt-responsive genes unique to S. brachiata. To elucidate the difference in salt-tolerance mechanisms, and to identify salt-tolerance mechanisms amongst different families of halophytes, SSH2 library was generated from salt-treated leaf tissue of S. brachiata as tester and that of S. portulacastrum as driver. Totally, 87 and 49 EST clones representing unique genes were obtained from SSH1 and SSH2 libraries, respectively. Examination of the expression patterns of 17 (SSH1) and 15 (SSH2) differentially expressed genes using semi-quantitative RT-PCR confirmed up-regulation of these genes in shoots in response to salt treatment and elevated CO₂ condition, but to a different extent. This study has provided insights into the molecular responses of S. brachiata to salt stress and elevated CO₂ conditions.     

Evidence for altered structure and impaired mitochondrial electron transport function in selenium deficiency

Selenium (Se) deficiency in the experimental models,Coturnix coturnix japonica andCorcyra cephalonica, resulted in impaired mitochondrial substrate oxidations and lowered thiol levels. Studies with respiratory inhibitors confirmed reduced mitochondrial electron transport enzyme activities, especially at cytochromec oxidase (COX), the terminal segment. Enhanced mitochondrial lipid peroxidation in Se deficiency was more pronounced in the heart tissue of the quail compared to other tissues. Glutathione peroxidase (GSH-Px) activity toward H₂O₂ and cumene hydroperoxide were generally low in the insect muscle tissue and activity toward H₂O₂ was maximal in the quail heart mitochondria that was not very sensitive to Se status. Lowered COX activity in Se deficiency was more directly correlated with the increased level of lipid peroxidation than with the GSH-Px activity measured, suggestive of Se mediated protective mechanisms independent of GSH-Px. Electron microscopic observations revealed structural changes such as loss of cristae with proliferative and degenerative changes of the mitochondria in Se deficiency. Involvement of Se in maintaining structure and functional efficiency of mitochondria is evident from the present study.