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.     

Synthesis and Biological Activities of Nicotinaldehyde Based 1,4-Dihydropyridinedicarboxylates

Russian Journal of Bioorganic Chemistry - 1,4-Dihydropyridinecarboxylates were prepared by the reaction of nicotinaldehydes with aminocrotonoates in the presence of p-TsOH at room temperature. The...     

A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.     

Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Diversity of interferon gamma and granulocyte-macrophage colony-stimulating factor in restoring immune dysfunction of dendritic cells and macrophages during polymicrobial sepsis

The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) gamma, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor alpha, IFN-gamma, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-gamma and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-gamma and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-gamma/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response.     

Production of bio-ethanol from soybean molasses by Saccharomyces cerevisiae at laboratory, pilot and industrial scales

The aim of this work was to develop an economical bioprocess to produce the bio-ethanol from soybean molasses at laboratory, pilot and industrial scales. A strain of Saccharomyces cerevisiae (LPB-SC) was selected and fermentation conditions were defined at the laboratory scale, which included the medium with soluble solids concentration of 30% (w/v), without pH adjustment or supplementation with the mineral sources. The kinetic parameters - ethanol productivity of 8.08g/Lh, Y(P/S) 45.4%, Y(X/S) 0.815%, m 0.27h(-1) and mu(X) 0.0189h(-1) - were determined in a bench scale bioreactor. Ethanol production yields after the scale-up were satisfactory, with small decreases from 169.8L at the laboratory scale to 163.6 and 162.7L of absolute ethanol per ton of dry molasses, obtained at pilot and industrial scales, respectively.     

Metabolic relevance of selenium in the insectCorcyra cephalonica

Requirement, uptake, and subcellular distribution of Na₂ ⁷⁵SeO₃ in the larvae of the insectC. cephalonica was investigated. That Se is well tolerated byC. cephalonica upto an added level of 2 ppm in the diet is suggested by the observed increase in body weight, total protein, and succinate dehydrogenase levels. Significant increases in the State 3 respiration ensued with Se supplementation up to 2 ppm in the mitochondrial oxidation of D-glycerol 1-phosphate, succinate and NADH, along with concomitant unaltered State 4 respiration, leading to enhanced RCR values. Maximal uptake of⁷⁵Se was registered in the larvae maintained on basal diet when subjected to short-term exposure to 0.5 ppm⁷⁵Se level. When exposure level was further increased up to 20 ppm, the observed decrease in the uptake of⁷⁵Se suggested that Se status of larvae itself controlled the tissue uptake. Subcellular distribution pattern revealed maximal incorporation of⁷⁵Se (cpm/g tissue) in the supernatant fraction, whereas, maximal specific⁷⁵Se activity (cpm/mg protein) was associated with the mitochondrial fraction. Autoradiography of the soluble fractions indicated the presence of single selenoprotein in the larval group with short term 2 ppm⁷⁵Se exposure. Inherent Se controls both the extent and the nature of distribution of mitochondrial⁷⁵Se incorporation. Uptake of⁴⁵Ca by the insect mitochondria was enhanced by dietary Se up to 2 ppm but was unaffected by addition ofin vitro ⁷⁵Se in the medium. A more fundamental role for Se in the mitochondrial energy metabolism emerges from these studies.     

Variation in leaf photosynthetic characteristics in wild rice species

Variations in leaf gas-exchange characteristics, leaf pigment content, and other important leaf traits were investigated in seven wild Oryza species, five hybrids, and five improved varieties. The significant variations were observed in photosynthetic pigment contents amongst different species of Oryza. The mean chlorophyll (Chl) content was higher in O. sativa (varieties and hybrids), while O. eichengeri showed the lowest Chl content. The mean carotenoid (Car) content in O. sativa (varieties and hybrids) was higher than in other wild rice species. O. eichengeri and O. barthii had significantly lower Car contents than other rice species. Significant differences were noticed in the rate of photosynthesis (P _N), stomatal conductance (g _s), transpiration rate (E), internal CO₂ concentration (C _i), specific leaf mass (SLM), and leaf thickness amongst different Oryza species. The mean P _N was the highest in O. nivara followed by O. eichengeri. The mean P _N was the lowest in O. glumaepatula, which was lower than that of cultivated varieties and hybrids of O. sativa. High rates of photosynthesis were observed in O. nivara (ACC. No. CR 100097), O. rufipogon (ACC.No. CR 100267), and O. nivara (ACC.No. CR 100008). The O. nivara and O. rufipogon genotypes with high P _N might be used in rice improvement programmes for an increase of leaf photosynthesis in rice. Multiple correlations performed between different gas-exchange characteristics and other physiological traits revealed that the rate of photosynthesis was not dependent on the leaf pigment content or the leaf thickness. A strong positive correlation between P _N and the P _N/C_i ratio, which represents the carboxylation efficiency, indicated that the observed variation in P _N was not based on pigment content or other leaf traits.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.