Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240?h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18?h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8?h PI. Hypoferremia peak was observed at 12?h and returned to normal levels at 30?h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6?h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1?μg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2?h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.     

G protein–coupled receptor kinase 2 modifies the ability of Caenorhabditis elegans to survive oxidative stress

Survival and adaptation to oxidative stress is important for many organisms, and these occur through the activation of many different signaling pathways. In this report, we showed that Caenorhabditis (C.) elegans G protein–coupled receptor kinases modified the ability of the organism to resist oxidative stress. In acute oxidative stress studies using juglone, loss-of-function grk-2 mutants were more resistant to oxidative stress compared with loss-of-function grk-1 mutants and the wild-type N2 animals. This effect was Ce-AKT-1 dependent, suggesting that Ce-GRK2 adjusted C. elegans oxidative stress resistance through the IGF/insulin-like signaling (IIS) pathway. Treating C. elegans with a GRK2 inhibitor, the selective serotonin reuptake inhibitor paroxetine, resulted in increased acute oxidative stress resistance compared with another selective serotonin reuptake inhibitor, fluoxetine. In chronic oxidative stress studies with paraquat, both grk-1 and grk-2 mutants had longer lifespan compared with the wild-type N2 animals in stress. In summary, this research showed the importance of both GRKs, especially GRK2, in modifying oxidative stress resistance.     

Mapping quantitative trait loci associated with spot blotch and net blotch resistance in a doubled-haploid barley population

Spot blotch and net blotch are important foliar barley (Hordeum vulgare L.) diseases in Canada and elsewhere. These diseases result in significant yield reduction and, more importantly, loss of grain quality, downgrading barley from malt to feed. Combining resistance to these diseases is a breeding priority but is a significant challenge using conventional breeding methodology. In the present investigation, an evaluation of the inheritance of resistance to spot and net blotch was conducted in a doubled-haploid barley population from the cross CDC Bold (susceptible)?×?TR251 (resistant). The population was screened at the seedling stage in the Phytotron and at the adult-plant stage in the field for several years. Chi-squared analysis indicated one- to four-gene segregation depending on disease, isolate, plant development stage, location and year. A major seedling and adult-plant resistance quantitative trait locus (QTL), designated QRpt6, was re-confirmed for net-form net blotch resistance, explaining 32?C61% of phenotypic variation in different experiments. Additional QTL for seedling and adult-plant resistance to net blotch were identified. For spot blotch resistance, a major seedling resistance QTL (QRcss1) was detected on chromosome 1H for isolate WRS1909, explaining 79% of the phenotypic variation. A highly significant QTL on 3H (QRcs3) was identified for seedling resistance to isolate WRS1908 and adult-plant resistance at Brandon, MB, Canada in 2008. The identification of QTL at only one location or from 1?year suggests spot blotch resistance is complex and highly influenced by the environment. Efforts are being made to combine spot and net blotch resistance in elite barley lines using molecular marker-assisted selection.     

Validation of molecular markers for covered smut resistance and marker-assisted introgression of loose and covered smut resistance into hulless barley

Inheritance of covered smut resistance was investigated in three hulless × hulled barley populations (CDC Candle/Q21861, CDC McGwire/Q21861, and CDC McGwire/TR640). Greenhouse and/or field screening indicated resistance was controlled by a single major gene from Q21861 and TR640. Three molecular markers (UhR 450, aHor 2 and OPO6₇₈₀) linked to the covered smut resistance gene (Ruhq) in the hulled line Q21861 were assessed for their usefulness in selecting resistant hulless barley. All markers were linked to covered smut resistance in the three populations evaluated, although aHor 2 was only polymorphic in CDC Candle/Q21861. Two strategies, doubled haploidy (DH) and marker-assisted backcrossing (MAB), were used to simultaneously introgress Ruhq and loose smut resistance (Run8) into the hulless barley cultivar CDC McGwire. Thirty-five DH lines were developed from a cross of hulless loose smut resistant line SH00752 (CDC McGwire/TR251) by hulless covered smut resistant line SH01470 (CDC McGwire/Q21861). By screening the 35 DH lines for each of the markers, 14 were identified as positive for both. Following three rounds of screening by artificial inoculation, 12 of those were identified as resistant to both diseases. In the MAB program, “blind” selection based solely on markers was conducted through the BC₃F₂ generation; lines resistant to both diseases were obtained. One line, designated HB390, is being advanced to 2nd year of the Western Canadian Hulless Barley Co-operative yield trials, the final step to release of a cultivar for commercial production in Canada. These results confirm that molecular markers can be used in either DH or MAB programs to assist in the rapid introgression of simply inherited disease resistance genes into elite lines, with considerable time and cost savings.     

Extraction,profiling and bioactivity analysis of volatile glucosinolates present in oil extract of <Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">raya</Emphasis>

Cruciferous vegetables are rich source of glucosinolates (GSLs), which in presence of myrosinase enzyme cause hydrolytic cleavage and result in different hydrolytic products like isothiocyanates, thiocyanates, nitriles and epinitriles. The GSLs hydrolytic products are volatile compounds, which are known to exhibit bioactivities like antioxidant, fungicidal, bioherbicidal and anticancer. Among the Brassicaceae family, Brassica juncea is very well known for high content of GSLs. In the present study, the isolation of volatile oil of B. juncea var. raya was done by hydrodistillation method using clevenger apparatus and further there extraction was done by solvents ethyl acetate and dichloromethane. The volatile compounds present in the extract were analysed by gas chromatography/gas chromatography–mass spectrometry (GC/GC–MS). Fatty acid esters, sulphur and/or nitrogen compounds, carbonyl compounds and some other volatile compounds were also identified. Besides the analytical studies, the extracts were analysed for their bioactivities including radical scavenging activity by using DNA nicking assay and cytotoxic effect using different human cancer cell lines viz. breast (MCF-7 and MDA-MB-231), prostate (PC-3), lung (A-549), cervix (HeLa) and colon (HCT116) by MTT assay. The oil extracts were efficiently able to reduce the increase of cancer cells in a dose-dependent manner. Among all cell lines, the most effective anticancer activity was observed in case of breast (MCF-7) cancer cell line. So, MCF-7 cells were used for further mechanistic studies for analysing the mechanism of anticancer activity. Confocal microscopy was done for analysing morphological changes in the cells and the images confirmed the features typical of apoptosis. For evaluating the mode of cell death, spectrofluorometric determination of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) was done. The volatile oil extract treated MCF-7 cells had a significant increase in number of ROS, also there was a rise in percentage of cells with increased disruption of MMP. So, the present study marks necessary indication that B. juncea (raya) oil extracts significantly induces apoptosis in all the above mentioned cancer cells lines through a ROS-mediated mitochondrial pathway and thus play a remarkable role in death of cancer cells.