Molecular cloning, expression and Insilco analysis of drought stress inducible MYB transcription factor encoding gene from C4 plant Eleusine coracana

Drought is one of the key abiotic stresses that critically influences the crops by restraining their growth and yield potential. Being sessile, plant tackle the detrimental effects of drought stress by modulating the cellular state by changing the gene expression. The transcriptional syndicate essentially drives such alteration of gene expression. Transcription factors (TF) are the key regulatory protein that controls the expression of their target gene by binding to the cis-regulatory elements present in the promoter region. Myb-TF, ubiquitously present in all eukaryotes belong to one of the largest TF family, and play a wide array of biological functions in plants, including anthocyanin biosynthesis, vasculature system, cell signalling, seed maturation and abiotc stress responses. The present performed isolation and molecular cloning of full length Myb TF from Eleusine corocana. The isolated full-length coding sequence has 1053 bp and 350 aa was submitted to NCBI (Accession number MT312253). The transcript level of EcMYB increases under different abiotic stress treatments including dehydration, salinity, and high-temperature stress. The promoter region of EcMyb1 was found to be enriched in stress-responsive cis-regulatory elements such as DRE, HSE, ABRE etc. In phylogenetic analysis, EcMyb1 appeared to have high homology with its monocot orthologs particularly Sateria italica, Hordeum vulgare, Saccharum barberi and Oryza sativa. The three-dimension protein structure was generated based on homology modeling and structural aspects were discussed. Further, Insilco analysis was conducted to explore the physiological properties, subcellular localization, potential posttranslational modification sites (phosphorylation and glycosylation sites), and molecular and biological function of the full-length protein. Overall, the expression profiling and Insilco analysis of EcMyb1 strongly indicated its potential role in abiotic stress response in Eleusine corocana.

Behavioral and Biochemical Consequences of Simulated Vehicle Exhaust Exposure in Rats

Air pollution is one of the most serious environmental threats to mankind. According to a report by the World Health Organization, urban air pollutants, specifically gasoline and diesel emissions from internal combustion engines of automobiles, trucks, locomotives and ships, contribute to a wide range of adverse health effects including cardiovascular, respiratory and neurological complications. While there has been a significant amount of research on the effect of vehicle exhaust exposure on heart and lungs, and some surveys have reported occurrence of mental co-morbidities with air pollution; adverse effects of exhaust emissions on the brain and its psychological impact, has been largely ignored. Interestingly, gaseous constituents of vehicle exhaust, namely, carbon dioxide (CO2), carbon monoxide (CO) and nitrogen dioxide (NO2), are pro-oxidants in nature. This is important considering the recently established causal link between oxidative stress in the brain and behavioral and cognitive impairments. Our postulation is that pro-oxidants in vehicle exhaust potentially increase oxidative stress in the brain damaging neuronal circuits and networks that modulate behavior and cognition. Therefore, we believe that it is critical to examine whether chronic exposure to vehicle exhaust emissions elevates oxidative stress and compromises our psychological health. In this project, we examined the neurobiological effect of prolonged exposure to pro-oxidant constituents of vehicle exhaust in rats using a simulated vehicle exhaust exposure (SVEE) model. We hypothesized that sub-chronic and chronic exposure to simulated exhaust leads to increased oxidative stress in the brain eventually resulting in behavioral and cognitive deficits. Our second hypothesis was that interventions that attenuate oxidative stress such as mitochondria-targeted antioxidant, Mito-Q prevent SVEE-induced increase in oxidative stress and consequently prevent occurrence of behavioral and cognitive deficits. First, we developed a model using adult male Sprague Dawley (SD) rats and utilized a simulated mixture of vehicle exhaust comprising of pro-oxidant exhaust constituents, namely, carbon dioxide (13%), carbon monoxide (0.68%) and nitrogen dioxide (1000 ppm) in air. Rats were exposed either to a high dose with brief duration (1:10 dilution {CO2: 1.3%, CO: 0.068%, NO2: 100 ppm}, 30 min/day) or low dose with prolonged duration (~1:1000 dilution {CO2: 0.04%, CO: 3 ppm, NO2: 0.9 ppm}, 5 h/day) of simulated exhaust in separate experiments for a duration of 2 weeks (sub-chronic) or 6 weeks (chronic). Durations of exposures were comparable to daily exposure of exhaust levels in areas of high traffic. Control rats were exposed to normal air for the same duration. Following SVEE, a comprehensive behavioral and cognitive analysis was performed to assess anxiety- and depression-like behavior, as well as cognitive function and intelligence quotient (IQ) in rats. Later, rats were sacrificed for biochemical analysis to determine the effect of SVEE on oxidative stress and oxidative stress-associated pathways such as mitochondrial impairment. It was revealed that at both sub-chronic and chronic level of high dose-brief and low dose-prolonged duration SVEE, exposed rats exhibited an increased anxiety-like, depression-like behavior, impaired memory and lowered IQ. At the biochemical level, SVEE led to increase in oxidative stress in specific regions of the brain including pre-frontal cortex, hippocampus and amygdala as indicated by increased level of oxidative stress markers and reduced antioxidant function. SVEE-induced oxidative stress also led to mitochondrial impairment in the form of lowered oxygen consumption, suppressed ATP synthesis and alteration in mitochondrial fission/fusion. In order to test the second hypothesis that antioxidant interventions have a protective role in preventing SVEE-induced impairments, SD rats were pre-treated with Mito-Q/ drinking water for 4 weeks prior to SVEE following which behavioral and biochemical analysis were performed. It was revealed that Mito-Q treated rats were protected from SVEE-induced increased anxiety- and depression-like behavior. Mito-Q also prevented learning-memory and IQ impairment. This protective effect of Mito-Q was extended at the molecular level where it prevented SVEE-induced increased oxidative stress levels in the three brain regions. Mito-Q pre-treated rats were also protected from SVEE-induced mitochondrial impairment. In summary, our study suggests that prolonged exposure to pro-oxidant constituents of vehicle exhaust emissions is associated with behavioral and cognitive deficits. Targeted antioxidant intervention such as Mito-Q seem to be protective against vehicle exhaust-induced deficits at behavior, cognitive as well as at molecular level. This suggests that exhaust emissions most likely cause excessive generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the emotion and cognition regulating brain regions. This buildup of oxidative stress adversely affects neuronal circuitry leading to the development of an altered behavioral and cognitive phenotype. This is the first preclinical evidence suggesting negative neurobiological impact of pro-oxidant constituents of vehicle exhaust emissions, a finding highly relevant to human populations exposed to high vehicular traffic. Furthermore, our study provides a novel and innovative model that can not only be used to study behavioral and psychological effects of a variety of laboratory simulations but it can also be used to probe underlying neurobiological mechanisms.Pharmacological and Pharmaceutical Sciences, Department o

Simulated vehicle exhaust exposure (SVEE) in rats impairs renal mitochondrial function

Purpose  Vehicle exhaust emissions primarily comprise of nitrogen, oxygen, water, CO2, NO2, CO, hydrocarbons and particulate matter. While adverse effects of hydrocarbon and particulate matter on cardiovascular functions are known, the effect of pro-oxidants CO2, NO2 and CO are not clear. Methods  Here, using an animal model of a simulated mixture of pro-oxidants (0.04% CO2, 0.9 ppm NO2 and 3 ppm CO with air as a base), we examined the effect of simulated vehicle exhaust exposure (SVEE) on various cardiovascular parameters. Male Sprague-Dawley rats were exposed to SVEE or ambient air (Control: CON) for 30 min/day for 2 weeks. Thereafter, systolic and diastolic blood pressure, heart rate and glomerular filtration rate were measured. Later, rats were sacrificed, blood plasma and kidneys were collected. Results  The systolic and diastolic blood pressure, heart rate and glomerular filtration rate remained unchanged. Plasma corticosterone increased in SVEE rats when compared to CON group. Plasma 8-isoprostane, a systemic marker of oxidative stress, increased while total antioxidant capacity decreased in SVEE but not in CON. Kidney cortical tissue homogenates exhibited increase in superoxide, hydrogen peroxide and protein carbonylation in SVEE but not CON, all indicative of heightened oxidative stress. Renal cortical mitochondrial SOD activity was significantly reduced in SVEE than CON. Conclusion  Significant decline in mitochondrial respiration and oxygen consumption was observed, in addition to low ATP, reduced ATP synthase and cytochrome C oxidase levels, as well as accelerated mitochondrial fission, and reduced fusion processes, were observed in SVEE than CON rats, all indicative of renal mitochondrial impairment

Morpho-physiological and biochemical responses in wheat foliar sprayed with zinc-chitosan-salicylic acid nanoparticles during drought stress

Drought is considered as a significant stress that hampers growth, development as well as productivity of wheat crop around the globe. The present investigation was performed to determine the changes induced by drought on morpho-physiology, antioxidative system, metabolism and yield parameters in wheat crop. The study also focussed on evaluating the effect of zinc-chitosan-salicylic acid (ZCS) nanoparticles in alleviating physiological and biochemical alterations and overcoming yield losses caused by drought. Drought was provided during the vegetative stage on four different varieties (two drought tolerant viz. C-306 and PBW-644 and two drought susceptible viz. HUW-322 and HUW-843) by withholding irrigation and maintaining moisture capacity of soil at 40%. ZCS nanoparticles were foliar sprayed on wheat at concentrations of 100, 200 and 400 mg L−1. Application of ZCS nanoparticles at 100 mg L−1 significantly (p < 0.05) enhanced relative water content (RWC), alleviated levels of antioxidative enzymes like superoxide dismutase, ascorbate peroxidase, glutathione reductase and guaiacol peroxidase and metabolites like proline, ascorbate, malondialdehyde and flavanoid in wheat leaves subjected to drought. Drought recovery was noteworthy in tolerant as well as sensitive varieties. Water stress reduced grain yield / plant by nearly 45% in tolerant varieties and nearly 50% in sensitive varieties. Spray of the nanoparticles on wheat foliage incremented the yield to 63% and 41% in tolerant varieties and 50% and 46% in sensitive varieties. This study suggests an outstanding role of ZCS nanoparticles at a concentration of 100 mg L−1 in mitigation of ill effects of drought. These nanoparticles have the ability to improve osmotic status of plant, enhance synthesis of osmoprotectants, activate ROS scavenging enzymes for maintaining membrane integrity and cellular protection and promote yield increment during drought stress. This implicates its role in ensuring food security and sustainable agriculture with reduction in environmental pollution due to limited use of fertilizers

Tempol treatment reduces anxiety-like behaviors induced by multiple anxiogenic drugs in rats.

We have published that pharmacological induction of oxidative stress (OS) causes anxiety-like behavior in rats. Using animal models, we also have established that psychological stress induces OS and leads to anxiety-like behaviors. All evidence points towards the causal role of OS in anxiety-like behaviors. To fully ascertain the role of OS in anxiety-like behaviors, it is reasonable to test whether the pro-anxiety effects of anxiogenic drugs caffeine or N-methyl-beta-carboline-3-carboxamide (FG-7142) can be mitigated using agents that minimize OS. In this study, osmotic pumps were either filled with antioxidant tempol or saline. The pumps were attached to the catheter leading to the brain cannula and inserted into the subcutaneous pocket in the back pocket of the rat. Continuous i.c.v. infusion of saline or tempol in the lateral ventricle of the brain (4.3 mmol/day) was maintained for 1 week. Rats were intraperitoneally injected either with saline or an anxiogenic drug one at a time. Two hours later all groups were subjected to behavioral assessments. Anxiety-like behavior tests (open-field, light-dark and elevated plus maze) suggested that tempol prevented anxiogenic drug-induced anxiety-like behavior in rats. Furthermore, anxiogenic drug-induced increase in stress examined via plasma corticosterone and increased oxidative stress levels assessed via plasma 8-isoprostane were prevented with tempol treatment. Protein carbonylation assay also suggested preventive effect of tempol in the prefrontal cortex brain region of rats. Antioxidant protein expression and pro-inflammatory cytokine levels indicate compromised antioxidant defense as well as an imbalance of inflammatory response

Effect of hydrogen sulfide donors on intraocular pressure in rabbits

Purpose: In this study, we investigated the effect of a slow-releasing hydrogen sulfide (H2S) donor, GYY 4137, on intraocular pressure (IOP) in normotensive rabbits. Furthermore, we compared the IOP-lowering action of GYY 4137 with those elicited by other H2S-producing compounds, l-cysteine and ACS67 (a hybrid compound of latanoprost with an H2S-releasing moiety). Methods: IOP was measured in New Zealand normotensive male albino rabbits using a pneumatonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY). At 0 h, 50 μL of test compounds were applied topically to 1 eye of each animal, while the contralateral eye received the same quantity of vehicle (saline). IOP was measured hourly until baseline IOP readings were attained and animal eyes monitored for potential side effects (i.e., tearing, hyperemia). Results: GYY 4137 (0.1%-2%) produced a dose-dependent decrease in IOP reaching a maximum of 27.8% ± 3.14% (n = 5) after 6 h. Interestingly, a significant contralateral effect was observed in vehicle-treated controls eyes at all doses tested. l-cysteine (5%) and ACS67 (0.005%) also elicited a significant (P \u3c 0.01) decrease in IOP that achieved a maximum of 28.84% ± 1.53% (n = 5) and 23.27% ± 0.51% (n = 5), respectively, after 3 h. All 3 H2S-producing compounds also caused a significant contralateral effect in vehicle-treated control eyes. Conclusion: We conclude that GYY 4137 and other H2S-producing donors can reduce IOP in normotensive rabbits. However, the profile of IOP-lowering action of GYY 4137 was different from the other H2S donors affirming its ability to act as a slow-releasing gas donor

Effect of anxiogenic drugs on plasma corticosterone levels.

<p>Anxiogenic drugs significantly elevated the plasma corticosterone levels, while tempol prevented this increase as measured using a commercially available ELISA kit. (*) significantly different at p<0.05. Bars represent means ± S.E.M, n = 6–8 rats/group.</p

Examination of Cu-Zn SOD, Mn SOD, GLO-1 and GSR-1 protein levels in the prefrontal cortex, hippocampus and amygdala of rats.

<p>Protein levels of Cu-Zn SOD (A-C), Mn SOD (D-F), GLO-1 (G-I) and GSR-1 (J-L) were determined by western blotting. The upper panels are representative blots for Cu-Zn SOD, Mn SOD, GLO-1 and GSR-1 and the lower panels are protein loading control β-actin. Bar graphs are ratios of Cu-Zn SOD, Mn SOD, GLO-1 and GSR-1 to β-actin, respectively. (*) significantly different from control, p< 0.05. Bars represent means ± SEM, n = 3–5 rats/group.</p