Browsing by Subject "Phytochemicals"
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Item Inducible plant responses triggered by phytochemical and bacterial elicitors(Texas Tech University, 2004-05) Farag, Mohamed Ali AliPlants release elevated levels of volatile organic compounds (VOCs) in response to insect or mechanical damage with some volatile components serving as chemical signals for attracting or repelling other organisms. Plants exposed to biogenic VOCs present in the environment can also modulate plant growth and development, although the nature of VOC-triggered plant responses has yet to be characterized for most plant species. The overall goal of this research has been to examine how certain environmental VOCs can trigger primary and secondary responses in plants. Specifically we examined (1) metabolic changes triggered by Ce alcohols and aldehydes ubiquitously released from damaged plants focusing on the model systems tomato and maize, as well as (2) C4 alcohols emitted from plant growth promoting rhizobacteria in the classic model plant Arabidopsis. Metabolic changes were monitored at genomic levels by reverse transcriptase-PCR (RT-PCR), slot blot analysis and GUS fusion assays. Changes in metabolic levels were monitored by GC, HPLC and Western analyses. The Ce-volatile (£)-2-hexenal triggered the release of local and systemic mono-and sesquiterpenes in tomato which was shown to be mediated through the jasmonic acid (JA) signaling pathway. Release of VOCs triggered with Ce-volatile treatment did not affect the accumulation of proteinase inhibitor enzymes (PI) or stored phytochemicals. In maize, Ce-com volafile (Z)-3-hexenoI resulted in an increase in transcript level for a series of defense genes including pal (phenyl alanine ammonium lyase), lox (lipoxygenase), igl (indole-3-glycerol phosphate lyase) and mpi (maize-proteinase inhibitor). The induction of transcripts were compared with metabolites generated from the respective pathways. Structure activity relationships has established that a series of Cft- alcohols were more active than tested aldehydes in triggering VOC emissions in both model systems. Biochemical and plant assays showed that the bacterial volatile elicitor 2,3- butanediol triggered growth promotion and induced disease resistance in Arabidopsis. Using transgenic and mutant lines of Arabidopsis, the signal pathway activated by bacterial volatiles was found to be dependent on cytokinin activation for growth promotion and dependent on ethylene signaling for induced pathogen resistance. These data provide new insight into the role of short chain alcohols and aldehydes as signaling molecules mediating plant-plant or plant-microbial interactions.Item Induction of apoptosis and cell cycle arrest in renal carcinoma cells by phenethyl isothiocyanate and the mechanisms involved(2011-05) Khan, Maruf; DeGraffenried, Linda; Ciolino, Henry P.; Sanders, Bob G.; Nunez, Nomeli P.; Fischer, Susan M.Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cells and the underlying mechanisms. We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell cycle was studied using Propidium Iodide staining. DNA damage was determined using phospho [gamma]-H2AX antibody. Protein expression and phosphorylation was determined using immunoblotting. PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 [mu]M PEITC induced apoptosis in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage as well as upregulation of the death receptors Fas and DR5. Lower doses (up to 10 [mu]M) arrested Caki-1 cells in G2/M phase, and this was associated with increased p38 and MK2 (Thr334) phosphorylation. The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. 15 and 20 [mu]M PEITC treatment resulted in increased [gamma]-H2AX phosphorylation suggesting DNA damage, but this was completely blocked by caspase inhibitor. In summary, our study shows that PEITC induces apoptosis in Caki-1 and Caki-2 cells by upregulating Fas and DR5 and activating the downstream apoptosis cascade. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase inhibitor. PEITC induces G2/M arrest in Caki-1 cells and the mechanism involves p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC.Item Production Systems and Processing Effect on Phytochemicals in Citrus Fruits and Their Analytical and Isolation Methods(2012-12-13) Uckoo, Ram 1980-The emerging scientific evidences on the role of food components in prevention of several chronic diseases are the momentum for shifting from a traditional focus on production to enhancement of nutritional quality. To further understand the role of these phytochemicals this dissertation describes the development of rapid analytical and isolation methods, and the effect of production systems and processing techniques on the levels of phytochemicals in citrus fruits. In the first study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid analysis of amines and organic acids was developed. The simultaneous extraction and analysis of samples provides an economical method for analyzing a large number of samples. In the second study, rapid separation method of potent health beneficial phytochemicals such as polymethoxyflavones from citrus peels using flash chromatography was developed. Using the developed method, five polymethoxyflavones were separated and isolated with high purity in gram level quantity. In the third study, the levels of phytochemicals in organically and conventionally grown lemons and their storage at market simulated conditions were determined. Results suggest that organically produced citrus fruits have higher content of organic acids and flavonoids than conventionally produced. The fourth and fifth study determined the influence of household processing (blending, juicing, hand squeezing techniques) and emerging processing (high pressure processing [HPP], thermal processing) on the phytochemicals content of ?Rio Red? grapefruits. Fruits processed by blending had significantly higher levels of flavonoids, furocoumarins and limonin compared to juicing and hand squeezing, while HPP enabled in extending the shelf life of the processed juice without any adverse effects. Therefore, consuming grapefruit juice processed by blending may provide higher levels of health beneficial phytochemicals. The sixth study describes a rapid flash chromatography method for isolation of PMFs and furocoumarins from citrus industrial by products such as peel oil. In the seventh study the developed method was applied to isolate 10 different phytochemicals from an unexplored citrus species, Miaray mandarin (Citrus miaray TAN.). Among them, the 5,7,8,3',4' pentamethoxyflavone was isolated for the first time from the genus Citrus.Item Relevance of tolC and expression of acrAB and emrAB in Erwinia chrysanthemi(Texas Tech University, 2003-12) Barabote, Ravi DamodarPlants produce a repertoire of antimicrobial chemicals, some of which are produced in response to an infection. Although much is understood about the virulence capabilities of many bacterial plant pathogens, little is known about the mechanisms employed by phytopathogens to survive die onslaught of the plant chemical environment. Here, die first report of the role of TolC in phytopathogenesis is presented. TolC is the outer membrane component of several multi-drug resistance (MDR) efflux pumps, such as AcrAB and EmrAB in E. coli, and plays an important role in the survival and virulence of many bacterial animal pathogens. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective m the efflux of berberine, a model antimicrobial plant chemical. These results suggest that the E. chrysanthemi to/C plays an important role in the survival and colonization of the pathogen in plant tissue by conferring resistance to the antimicrobial compounds produced by plants. Therefore, in order to assess the plausible implication of MDR during plant disease, the expression of the E. chrysanthemi acrAB and emrAB homologs was investigated using their respective promoter-fusions to reporter genes. Both pumps appear to be expressed in planta and in vitro in the presence of several plant-derived molecules. Plant-derived molecules, such as salicylic acid, hydrogen peroxide, paraquat, and genistein have previously been shown to stimulate antibiotic resistance in other bacteria. Interestingly, jasmonate, which is produced in plants as part of the plant defense response to pathogen invasion, was found to stimulate expression of both the pumps in E. chrysanthemi. This is the first report of jasmonate-dependent expression of bacterial efflux pump genes. Avirulence of the E. chrysanthemi tolC mutant and expression of acrAB and emrAB pumps in planta as well as in vitro in the presence of plant-defense related molecules suggest that MDR efflux pumps may play an important role in pathogenesis of this bacterium.