When fed on a diet containing a proteinaceous cysteine protease inhibitor from soybean (scN), cowpea bruchid larvae enhance their overall digestive capacity to counter the inhibitory effect. Elevated proteolytic activity is attributed not only to the major digestive cysteine proteases (CmCPs), but also to aspartic proteases, a minor midgut protease component. In this study, we isolated a CmCatD cDNA from cowpea bruchid midgut that shares substantial sequence similarity with cathepsin D-like aspartic proteases of other organisms. Its transcript profile was developmentally regulated and subject to alteration by dietary scN. CmCatD transcripts weremore abundant in scN-fed 3rd and 4th instarmidguts than in control. The bacterially expressed recombinant CmCatD proprotein was capable of autoprocessing under acidic conditions, and mature CmCatD also exhibited pH-dependent proteolytic activity which was inhibited specifically by pepstatin A, indicative of its aspartic protease nature. CmCatD trans-activated CmCPs and vice versa, suggesting a cooperation between the minor midgut CmCatD and major digestive CmCPs. Further, CmCatD was able to degrade scN after extensive incubation. This activity partially restored CmCP proteolytic activity otherwise inhibited by scN. Thus CmCatD could facilitate insects’ coping with the challenge of dietary scN by exerting its scN-insensitive and scN-degrading activity, freeing cysteine proteases for food degradation. Taken together, cowpea bruchids coordinate the functionality of the two classes of digestive proteases to fend off the negative effect of scN, and fulfill their nutrient requirements. When fed on a diet containing a proteinaceous cysteine protease inhibitor from soybean (scN), cowpea bruchid larvae enhance their overall digestive capacity to counter the inhibitory effect. Elevated proteolytic activity is attributed not only to the major digestive cysteine proteases (CmCPs), but also to aspartic proteases, a minor midgut protease component. In this study, we isolated a CmCatD cDNA from cowpea bruchid midgut that shares substantial sequence similarity with cathepsin D-like aspartic proteases of other organisms. Its transcript profile was developmentally regulated and subject to alteration by dietary scN. CmCatD transcripts weremore abundant in scN-fed 3rd and 4th instarmidguts than in control. The bacterially expressed recombinant CmCatD proprotein was capable of autoprocessing under acidic conditions, and mature CmCatD also exhibited pH-dependent proteolytic activity which was inhibited specifically by pepstatin A, indicative of its aspartic protease nature. CmCatD trans-activated CmCPs and vice versa, suggesting a cooperation between the minor midgut CmCatD and major digestive CmCPs. Further, CmCatD was able to degrade scN after extensive incubation. This activity partially restored CmCP proteolytic activity otherwise inhibited by scN. Thus CmCatD could facilitate insects’ coping with the challenge of dietary scN by exerting its scN-insensitive and scN-degrading activity, freeing cysteine proteases for food degradation. Taken together, cowpea bruchids coordinate the functionality of the two classes of digestive proteases to fend off the negative effect of scN, and fulfill their nutrient requirements.

The genus Citrus contains several biologically-active limonoids that are uniquely distributed by only certain members of the Rutaceae family. Deacetylnomilin and other limonoids possess various biological activities, such as anti-proliferation of neuroblastoma cells, colon cancer and human breast cancer cell lines. Deacetylnomilin also has antifungal and antibacterial properties. In the present work, isolation of the limonoid aglycones from seeds of sour orange (Citrus aurantium L.), a widely-used rootstock species, was investigated. Seed powder was extracted in a Soxhlet extractor with hexane and ethyl acetate consecutively for 16h. The ethyl acetate extract was concentrated and loaded on a silica gel column and eluted with different mobile phases. All the fractions were analyzed for putative compounds of interest using a TLC and HPLC. Fractions containing similar spots were pooled and crystallized. The total yield of deacetylnomilin was 4.9 g per 6.9 kg dry weight. Purity of the isolated compound was assessed by an HPLC using a C18 column and detection at 210 nm. Furthermore, the structure of this compound was confirmed by mass spectrometry. This is the first report demonstrating significant yields of deacetylnomilin from late harvested sour orange seeds. Hence, seeds from such fruit may be a better source of deacetylnomilin for bioactivity assay studies.

Inflammatory bowel disease (IBD) patients are at high risk for developing folate deficiency and colon cancer. Since it is difficult to study the subtle global and gene-specific epigenetic mechanisms involved in folate-mediated tumor initiation and promotion, we have generated genetically modified mouse models by targeting the reduced folate carrier (RFC1) and folate-binding protein (Folbp1) genes. The transgenic mice were fed semi-purified diets for 8 weeks containing either normal (2 mg) or deficient (0.1 mg folate/kg diet) levels of folate. Compound heterozygous mice (Folbp1+/-; RFC1+/-) fed an adequate folate diet exhibited a reduction in plasma folate concentrations compared to heterozygous (Folbp1+/-) and littermate wild-type mice (P < 0.5). In contrast, no differences were observed in colonic mucosa. Consumption of a low folate diet significantly reduced (three- to fourfold) plasma and tissue folate levels in all animal models, although plasma homocysteine levels were not altered. In order to elucidate the relationship between folate status and inflammation-associated colon cancer, animals were injected with azoxymethane followed by destran sodium sulphate treatment in the drinking water. Mice were fed a normal folate diet and were terminated 5 weeks after carcinogen injection. The number of high multiplicity aberrant crypt foci per centimeter of colon was significantly elevated (P < .05) in compound Folbp1+/-; RFC1+/- (3.5 ± 0.4) mice as compared to Folbp1+/- (1.9 ± 0.3) and wild-type control mice (1.1 ± 0.1). These data demonstrate that the ablation of two receptor/carrier-mediated pathways for folate transport increases the risk for developing inflammation-associated colon cancer.

Naringin, hesperidin and narirutin are three predominant flavonoids in the citrus fruits. The biological activity of flavonoids has been demonstrated to include anti-inflammatory, anticarcinogenic, and anti-allergic properties. The variation of these compounds in processed citrus juice from various species is poorly understood. Quantification of these flavonoids in commercially available juices will enable us to understand the effects of processing on the bioavailability and health benefits of these compounds. The objectives of this study were to develop a rapid method for separating these compounds using HPLC technique and to further optimize the extraction procedure using various solvents from commercially available fruit juices using this method. Various solvents were used to optimize the extractability of solvents from grapefruit juice, orange juice, and cocktails. Flavonoids were separated using solvent gradient system of acetonitrile and 0.03% phosphoric acid. The flavonoids were detected at 280 nm.

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of
genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae ( Callosobruchus maculates ) fed on regular diet and diets ontaining
antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a B LAST search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

The color-forming ability of amino acids with thiosulfinate in crushed garlic was investigated. We developed reactions systems for generating pure blue pigments using extracted thiosulfinate from crushed garlic and onion and all 22 amino acids. Each amino acid was reacted with thiosulfinate solution and was then incubated at 60ºC for 3 h to generate pigments. Unknown blue pigments, responsible for discoloration in crushed garlic cloves (Allium sativum L.) were separated and tentatively characterized using high performance liquid chromatography (HPLC) and a diode array detector ranging between 200 and 700 nm. Blue pigment solutions exhibited 2 maximal absorbance peaks at 440 nm and 580nm, corresponding to blue and yellow, respectively, with different retention times. Our findings indicated that green discoloration is created by the combination of yellow and blue pigments. Eight naturally occurring blue pigments were separated from discolored garlic extracts using HPLC at 580 nm. This suggests that garlic discoloration is not caused by only 1 blue pigment, as reported earlier, but by as many as 8 pigments. Overall, free amino acids that formed blue pigment when reacted with thiosulfinate were lysine, argentine, lysine, serine, almandine, aspartic acid, asparagines, glutei acid, and tyrosine. Arginine, asparagines, and glutamine had spectra that were more similar to naturally greened garlic extract.

Background: Biotechnology to increase the nutrient content of fruits and vegetables is an innovative strategy to address insufficient mineral intakes. A novel biotechnologically modified carrot which has higher levels of calcium than control carrots has been developed. Objective: For dietary guidance, it is necessary to understand the relative servings of any specific product that would be needed to provide calcium compared to a standard source, such as milk. Methods: In a crossover study we used stable isotopes to measure calcium absorption from milk in 30 young adults and compared it to calcium absorption from both biotechnologically modified (MOD) and control (CON) carrots. Results: Using a total meal calcium of 300 mg of which 35-40 mg of the calcium is derived from the test product, fractional calcium absorption from milk was slightly higher than from the MOD carrot (50.1 ± 3.0% vs. 42.6 ± 2.8%, Mean ± SEM, p<0.05) but was similar to that from the CON carrot (50.1 ± 3.0% vs. 52.8 ± 3.3%; p=0.7). Conclusions: Biotechnologically-modified carrots have calcium bioavailability levels only slightly below that of milk. Serving sizes of enhanced carrots remain too large to be considered full substitutions for usual sources such as milk, but can supplement these sources effectively. Further biotechnological enhancements of a range of vegetable sources may lead to substantial benefits in nutritional status for minerals such as calcium with significant populationdeficient intakes.

In this study it was shown that wounding in combination with hormone stresses synergistically enhanced the accumulation of bioactive phenolic compounds in carrot tissue. Whole carrots and carrots cut in slices, pie-cuts and shreds were exposed to 250_L/L methyl jasmonate and 1000_L/L ethylene treatments. Results indicated that hormone stress-induced phenolic compounds and phenylalanine ammonia lyase enzyme activity were dependent upon wounding intensity. The synthesized phenolics correlated to an increase in antioxidant capacity (AOX). In addition, both stresses affected the phenolic profiles. The different proportions of chlorogenic acid, icaffeoylquinic acid and ferulic acid, for each stress and combinations, influenced the specific AOX of the phenolic profiles (the effectiveness of the phenolic compounds to neutralize free radicals). The bitter compound isocoumarin was mostly affected by the ethylene treatment compared to methyl jasmonate or wounding alone. Exposure to combined abiotic stresses could provide more AOX to regular diets and be a less expensive alternative to genetic modifications and breeding programs.

The effects of six foliar K sources: potassium chloride (KCl), potassium nitrate (KNO3), monopotassium phosphate (KH2PO4), potassium sulfate (K2SO4), potassium thiosulfate (K2S2O3), and a glycine amino acid-complexed K (Potassium Metalosate, KM) were evaluated on field-grown muskmelon (Cucumis melo L. cv. ‘Cruiser’) fruit quality parameters. Weekly foliar K treatments were established starting at fruit set and continuing to fruit maturity. Tissue K concentrations, soluble solids concentrations (SSC), total sugars, and the human wellness compounds ascorbic acid and β-carotene were generally higher in plants receiving supplemental foliar K than in the control plants. Significant differences were observed among K sources, with KNO3 consistently resulting in poor fruit quality compared to fruit treated with the other K sources. Although there were no consistent trends among the other foliar K sources, the data is consistent with previous controlled environment research findings that supplementing soil K supply with foliar K applications during fruit development and maturation can improve the marketable and human health quality attributes of muskmelon fruit.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions as a scaffold for RNA processing machineries that recognize differentially phosphorylated conserved (YSPTSPS)n repeats. Evidence indicates that proteins that regulate the phosphorylation status of the CTD are determinants of growth, development, and stress responses of plants; however, little is known about the mechanisms that translate the CTD phosphoarray into physiological outputs. We report the bioinformatic identification of a family of three phospho-CTD-associated proteins (PCAPs) in Arabidopsis and the characterization of the AtPRP40 (Arabidopsis thaliana PRE-mRNA-PROCESSING PROTEIN 40) family as PCAPs. AtPRP40s-CTD/CTD-PO4 interactions were confirmed using the yeast two-hybrid assay and far-Western blotting. WW domains at the N-terminus of AtPRP40b mediate the AtPRP40b-CTD/CTD-PO4 interaction. Although AtPRP40s interact with both phosphorylated and unphosphorylated CTD in vitro, there is a strong preference for the phosphorylated form in Arabidopsis cell extract. AtPRP40s are ubiquitously expressed and localize to the nucleus. These results establish that AtPRP40s are specific PCAPs, which is consistent with the predicted function of the AtPRP40 family in pre-mRNA splicing.

A novel chimeric gene with a 5” end containing the nearly complete sequence of the coxI gene and a 3” end showing homology with chive orfA501 was isolated by genome walking from two cytoplasm types: CMS-S and CMS-T, both of which induce male-sterility in onion (Allium cepa L.). In addition, the normal active and variant inactive coxI genes were also isolated from onions containing the normal and CMS-S cytoplasms, respectively. The chimeric gene, designated as orf725, was nearly undetectable in normal cytoplasm, and the copy number of the normal coxI gene was significantly reduced in CMS-S cytoplasm. RT-PCR results showed that orf725 was not transcribed in normal cytoplasm. Meanwhile, the normal coxI gene, which is essential for normal mitochondrial function, was not expressed in CMS-S cytoplasm. However, both orf725 and coxI were transcribed in CMS-T cytoplasm. The expression of orf725, a putative male-sterility-inducing gene, was not affected by the presence of nuclear restorer-of-fertility gene(s) in male-fertility segregating populations originating from the cross between a male-sterile plant containing either CMS-T or CMS-S and a male-fertile plant whose genotypes of nuclear restorer gene(s) might be heterozygous. The specific stoichiometry of orf725 and coxI in the mDNA of the three cytoplasm types was consistent among diverse germplasm. Therefore, a molecular market based on the relative copy numbers of orf725 and coxI was designed for distinguishing among the three cytoplasm types by one simple PCR. The reliability and applicability of the molecular marker was shown by testing diverse onion germplasm.

Phytochemicals may reduce chronic inflammation and cancer risk in part by modulating T-cell nuclear factor-KB (NF-KB) activation. Therefore, we examined the effects of curcumin (Cur) and limonin (Lim) feeding on NF-KB-dependent CD4+ T-cell proliferation. DO11.310 transgenic mice (n = 5-7) were fed diets containing 1% Cur or 0.02% Lim combined with either (n-6) PUFA [5% corn oil (COO)] or (n-3) PUFA [4% fish oil + 1T corn oil (FO)] for 2 wk, followed by splenic CD4+ T-cell isolation and stimulation with ovalbumin peptide 323-339 (OVA) and antigen-presenting cells from mice fed a conventional nonpurified rodent diet. Both Cur and Lim diets suppressed (P < 0.05) NF-KB p 65 nuclear translocation in activated CD4+ T-cells. In contrast, activator protein-1 (c-Jun) and nuclear factor of activated T-cells c1 were not affected compared with the CO control diet (no Cur or Lim). CD4+ T-cell in response to either mitogenic anti-CD3/28 monoclonal antibodies (mAb) or antigenic stimulation by OVA was also suppressed (P < 0.05) by Cur as assessed by carboxyfluorescein succinimidyl ester staining. In contrast, interlelukin-2 production was not directly associated with NF-KB status. Interestingly, dietary combination with FO enhanced the suppressive effects (P < 0.05) of Cur or Lime with respect to CD4+ T-cell proliferation in response to anti-CD3/28 mAb. These results suggest that combination chemotherapy (FO+Cur or Lim) may favorably modulate CD4+ T-cell-mediated inflammation.

Over the past several decades, data from both experimental animals studies and human clinical trials have shown that dietary n-3 polyunsaturated fatty acids (PUFA) exhibit anti-inflammatory bioactive properties, compared to n-6 PUFA. Collectively, these studies have identified multiple mechanisms by which n-3 PUFA affect immune cell responses. In this review, we discuss the putative targets of anti-inflammatory n-3 PUFA, specifically, cytokine protein acylation. In addition, we investigate the effect of n-3 PUFA on the coalescence of lipid rafts, specialized signaling platforms in the plasma membrane.

To assess the impact of enhanced root vacuole cadmium (Cd) sequestration on leaf Cd accumulation under a low Cd dose, as generally occurs in agriculture, leaf Cd accumulation was examined in field-grown tobacco plants expressing genes encoding the high-capacity-Cd, tonoplast-localized, divalent cation/H antiporters AtCAX4 and AtCAX2 (AtCAX, Arabidopsis cation exchanger). It has been shown previously that root tonoplast vesicles isolated from plants expressing these genes, directed by root-selective promoters, show enhanced Cd transport activity, and young plants show enhanced root Cd accumulation when grown in solution culture containing 0.02 M Cd, a moderate Cd dose. In this article, we present results which show that the lower leaves of mature plants expressing AtCAX2 or AtCAX4 , under the control of two different root-selective promoters, accumulate 15%–25% less lamina Cd than control plants when grown in the field (3 years, three different collection methods). Reciprocal grafting experiments of AtCAX2 shoots onto control roots (and vice versa), grown in solution culture with 0.005 M Cd, indicated that the root controls Cd translocation and accumulation in the shoot in control and AtCAX2 and AtCAX4 tobacco plants exposed to low Cd concentration. The results are consistent with a model in which supplementation of Cd/H antiporter activity in root cell tonoplasts enhances root Cd sequestration, resulting in decreased translocation of Cd to the shoot of field-grown plants. These results suggest that human Cd intake from food and tobacco use could be reduced via the enhancement of root vacuolar sequestration of this pollutant.

Sulfur (S) nutrition has a strong influence on onion pungency, and the production of sweet onions in the high-sulfur soil of Texas is a challenge to growers. This study was performed to determine the effects of S applications to fields already having sufficient levels of soil S on pungency and related compounds in four shortday onion cultivars. The onion cultivars ‘Cougar’, ‘Legend’, ‘Texas Early White’, and ‘Texas Grano 1015Y’ were grown in three commercial fields at Edinburg, Mercedes, and Weslaco of the Rio Grande Valley of South Texas with applications of sulfur at 0, 13, and 26 kg S/ha. Pyruvic acid, bulb weight, extractable S in soil and total S in bulb, soluble solids content (SSC), flavor precursor compounds [also called S-alk(en)yl-l-cysteine-sulfoxides (ACSOs)], and total sugars were measured. The pyruvic acid level of onions was not increased by the S applications and showed no significant correlation with soil S levels in any of the cultivars at locations having 30 or 235 ppm S. The SSC, total S in bulbs, total sugars, and total ACSO content also showed no correlation with the S applications and soil S levels. Onion pungency and content of flavor precursor compounds were significantly influenced by cultivar and cultivar × location interaction. We conclude that onion pungency is not increased by application of extra S fertilizer to commercial fields already containing sufficient levels of soil sulfur.

This study was conducted to characterize shortday onions of 3 pungency levels with regard to the composition of flavor related compounds. A total of 9 onion breeding lines/cultivars were selected, per each of low, medium, and high pungency level, with pyruvic acid contents of 1.9 to 2.8, 4.8 to 5.4, and 7.2 to 8.3 µmoles/ml., respectively. The contents of flavor precursors (S-1-propeny)-L-cysteine sulfoxide [1-PeCSO] and S-mythyl-L-cysteinesulfoxide [MCSO]), free amino acids, free sugars, soluble solids (SSC), and total sulfur (S) in onion bulbs were measured. The flavor precursor contents ranged from 0.03 to 0.16 mg/g fresh weight (FW) to MCSO, 0.07 to 0.65 mg for 1-PeCSO and 0.12 to 0.77 Mg in total, and precursor contents increased with the pungency levels. Onions of different pungency levels did not differ in the contents of individual or total free amino acids, and the most abundant amino acids were glutamine and arginine. The total sugar contents ranged from 50 to 75 mg/g FW, and total S contents (3.5 to 5.1 mg/g dry weight) were not correlated with the pungency levels. However, pungency levels were correlated inversely with bulb weight and positively with SSC, presumably by the effect of dilution. This study indicates that onion pungency is primarily determined by the content of flavor precursor compounds and not by total S, total sugars, or individual/total free amino acids in shortday bulbs..

Vegetables synthesize and accumulate phytochemicals that are often involved in signaling and stress protection. The synthesis and degradation of vitamins, minerals, dietary fiber, and antioxidants is regulated by the interaction of genetic and environmental factors. Agronomic strategies may significantly modify and/or improve phytochemical concentrations and profiles in vegetable crops. For example, high nitrogen fertilization has been shown to decrease ascorbic acid, and either decrease or increase carotenoids. Precision deficit irrigation and moderate to high salinity stress may enhance carotenoid levels, such as lycopene in watermelon and tomato, but at the risk of lowered productivity. The complex and interactive effects of the environment and agronomic practices on plant phytochemicals may be direct or indirect, cultivar and crop specific, and short-lived or sustained. This paper highlights agronomic crop strategies, with emphasis on nitrogen, irrigation and salinity, impacting health-promoting compounds and quality in vegetable species.

Muskmelons (Cucumis melo L.) are well-known as excellent sources of several vitamins, minerals and nonenzymatic antioxidant phytochemicals such as vitamin C and pro-vitamin A. Less well-studied is their potential role as sources of enzymatic antioxidants such as superoxide dismutase (SOD), which have been associated with enhanced reactive oxygen species scavenging capacity in some muskmelon fruits. In this study, we investigated the variability in SOD activities among diverse advanced breeding lines and commercial muskmelon cultivars grown in two different soil types—clay or sandy loam. Specific and total SOD activities varied significantly among the genotypes (P≤0.001), compared to soil type (P≤0.055). Netted (cantaloupe) genotypes generally had the lowest SOD activities compared to the green- and orange-fleshed honey dew types. Casaba type fruit had average SOD activities that were ∼ 1.6-fold greater than those of honey dew types, and ∼ 9.0-fold greater than those of cantaloupe types. These data indicate there is useful genetic diversity among commercial melon varieties and in exotic genotypes that could be used to develop C. melo as a functional food with enhanced SOD content.

Background: We reported that citrus consumption improves bone quality in orchidectomized male rats. In the present study, effects of feeding citrus bioactive compounds and crude extract on bone quality in orchidectomized rats were evaluated. Methods: Seventy 90-day-old male rats were randomly assigned to five groups for 60 days of feeding study. The treatment groups were SHAM-control, orchidectomy (ORX), ORX + crude extract, ORX + Limonin, and ORX + naringin. At termination, animals were euthanized, blood was collected for the plasma antioxidant status. Bone resorption and bone formation markers in the blood and urine were evaluated. Bone quality in the femur and the 5th lumbar and the total calcium concnetration in the bones and excreta were evaluated. Results: Orchidectomy lowered (p < 0.05) plasma antioxidant capacity, bone quality, and bone calcium; elevated (p < 0.05) TRAP, deoxypridinoline (DPD), and calcium excretion, and did not change the plasma IGF-1 in comparison to the SHAM group. The citrus crude extract or the purified bioactive compounds increased (p < 0.05) the plasma antioxidant status, plasma IGF-1, and bone density, preserved (p < 0.05) the concentration of calcium in the femur and in the 5th lumbar, and numerically improved bone strength. The crude extract and the bioactive compounds decreased (p < 0.05) focal excretion of calcium, numericall66 lowered the urinary excretion of calium, and suppressed (p < 0.05) the plasma TRAP activity without affect (p > 0.1) urinary excretion of DPD in comparison to the ORX group. Conclusions: Potential benefit of the citrus crude extract and its bioactive compounds on bone quality appears to preserve bone calcium concentration and increase antioxidant status.

The Arabidopsis vacuolar CAtion eXchangers (CAXs) play a key role in mediating cation influx into the vacuole. In Arabidopsis, there are six CAX genes. However, some members are yet to be characterized fully. • In this study, we show that CAX4 is expressed in the root apex and lateral root primordia, and that expression is increased when Ni2or Mn2levels are elevated or Ca2is depleted. • Transgenic plants expressing increased levels of CAX4 display symptoms consistent with increased sequestration of Ca2and Cd2into the vacuole. When CAX4 is highly expressed in an Arabidopsis cax1 mutant line with weak vacuolar Ca2/Hantiport activity, a 29% increase in Ca2/Hantiport is measured. A cax4 loss-offunction mutant and CAX4 RNA interference lines display altered root growth in response to Cd2, Mn2and auxin. The DR5::GUS auxin reporter detected reduces auxin responses in the cax4 lines. • These results indicate that CAX4 is a cation/Hantiporter that plays an important function in root growth under heavy metal stress conditions.

Phytochemicals present in citrus fruits, such as triterpenoids and related bitter compounds were of great concern to citrus industries during last decade due to their unpleasant taste. These same compounds are gaining importance during recent years, due to their proven benefits in prevention of cancer and other chronic diseases. Both limonin and its glucoside isolated and purified (purity of >99%) from Citrus aurantium L. seeds, were subjected to proliferation inhibitory activity of cultured human colon adenocarcinoma cells (SW480). Limonin has shown significant inhibitory activity at 25 M concentration, with inhibition of cancer cell proliferation by 42, 58 and 67% after 24, 48 and 72 Hrs of incubation, respectively. However the activity was not increased proportionately at 50 and 100 M (67.8 and 68.9% after 72 hrs). On the other hand, inhibitory effect of Limonin glucoside (LG) was significant at 6.25M concentration (inhibition of 61% after 24 hrs). Inhibition of proliferatation by 77% was observed in cells incubated with 100 M LG for 72 Hrs. These results were also confirmed by content of lactate dehydrogenase (LDH) leached by cells following treatment with compounds to medium indicating cytotoxicity. Compounds treated at 100M have shown DNA fragmentation of 200bp indicating apoptosis induction to cytotoxicity. The results of the current study indicates oxygenated terpenoids limonin and its glucoside may help in prevention of colon cancer. Further investigations are necessary to understand the possible mechanism of colon cancer prevention.

Our objectives were to construct a randomly amplified polymorphic DNA (RAPD) marker-based linkage map using an F2 population derived from the melon (Cucumis melo) cross of ‘Deltex’ × TGR 1551, and map quantitative trait loci (QTL) for sucrose, total soluble solids (TSS), ratio of sucrose to total sugars (RSTS), and ascorbic acid as well as the ms-3 locus for male sterility previously reported in other muskmelon crosses. Due to the dominant character of RAPD markers, we scored 192 ‘Deltex’ – and 158 TGR 1551-derived markers. One hundred eighty (94%) of the 192 markers fit the expected 3:1 ratio. On the basis of the 180 markers, we constructed a ‘Deltex’ linkage map of 171 markers distributed on 12 linkage groups (LGs) with a total map distance of 1182 cM. One hundred fifty (95%) of the 158 markers were identified to be nondistorted. We developed a TGR 1551 linkage map of 138 markers distributed on 12 LGs with a total distance of 1163 cM. A combined map of 12 LGs with a total map distance of 1394 cM was made from 82 marker pairs expressing codominance. Nine LGs were integrated into those of the existing composite map by 17 anchor markers. We mapped the ms-3 locus for male sterility of LG 9, which corresponds to LG10 of the classical map and LG VII of the composite map. Six QTL for sucrose were located on LGs 2, 3, 4, 6, and 11. Three on LGs 3, 4, and 6 and four on LGs 2, 3, 6, and 11 of the six QTL for sucrose were also noted to be QTL for TSS and RSTS, respectively. A single QTL for ascorbic acid was placed on LG 5. This map will also be used to identify QTL for fruit sweetness, quality, size, and shape traits, as well as disease resistance.

Vegetables represent an attractive means of providing increased calcium nutrition to the public. In this study, it was demonstrated that lettuce expressing the deregulated Arabidopsis H+/Ca2+ transporter sCAX1 (cation exchanger 1) contained 25%-32% more calcium than controls. These biofortified lettuce lines were fertile and demonstrated robust growth in glasshouse growth conditions. Using a panel of highly trained descriptive panelists, biofortified lettuce plants were evaluated and no significant differences were detected in flavour, bitterness or crispness when compared with controls. Sensory analysis studies are critical if claims are to be made regarding the efficacy of bioforified foods, and may be an important component in the public acceptance of genetically modified foods.

Mom’s conventional wisdom of eating fruits and vegetables to lead a healthy life has evolved with scientific, fact-finding research during the past four decades due to advances in science of “Foods for Health”. Epidemiological and prospective studies have demonstrated the vital role of fruits, vegetables, and nuts in reducing the risk of cancer and cardiovascular diseases. In recent years, several meta-analyses strongly suggested that by adding one serving of fruits and vegetables to daily diet, the risk of cardiovascular diseases will be decreased up to 7%. The multidisciplinary and partnership efforts of agriculture and medical scientists across the globe stimulated interest in establishing certain interdisciplinary centers and institutes focusing on “Foods for Health”. While the consumption of various healthy foods continues, several questions about toxicity, bioavailability, and food-during interactions of bioactive compounds are yet to be fully understood on the basis of scientific evidence. Recent research on elucidation of the molecular mechanisms to understanding the “proof of the concept” will provide the perfect answer when consumers are ready for a “consumer-to-farm” rather than the current “arm-to-consumer” approach. The multidisciplinary research and education efforts will address the role of healthy foods to improve eye, brain, and heart health while reducing the risk of cancer. Through this connection, this review is an attempt to provide insight and historical perspectives on some of the bioactive compounds from the day of discovery to their current status. The bioactive compounds discussed in this review are flavonoids, carotenoids, curcumin, ascorbic acid, and citrus limonoids..

Fruits of Citrus aurantifolia were subjected to hydro-distillation using Clevenger type apparatus to obtain volatile oil. Chemical composition of volatile oil. Chemical composition of volatile oil was analysed by CC-MS. Twenty-two compounds representing more than 89.5% of the volatile oil were identified. D-limonene (30.13%) and D-dihydrocarvone (309.47%) were found to be the major compounds in the lime volatile oil. This oil showed 78% inhibition of human colon cancer cells (SW-480) with 100 µg/ml concentration at 48 h. Lime volatile oil showed DNA fragmentation and induction of caspase-3 up to 1.8 and 2-folds after 24 h and 48 h, respectively, which may be due to the involvement of apoptosis. Analysis of apopotosis-related protein expression further confirmed apoptosis induction by lime volatile oil. The above results suggested that lime volatile oil has potential benefits in colon concer prevention. This is the first report, showing the possible mechanism of antiproliferative effect of lime volatile oil for the prevention of colon cancer in cell culture models.

Previous studies have indicated the chemopreventive potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), and deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of phase II enzymes in female A/J mice. Our results indicated the highest induction of glutathione S-transferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroguinoline 1-oxide (4NQO), while the same compound in the stomach induced GST by 51% compared to the control. The DNAG treated group induced 55% in stomach homogenates. Another phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 27% in liver and lung homogenates, respectively. Defun limonin induced QR in lung homogenates by 45%. Our results indicated that modification of limonin has differential induction of phase II enzyme. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in the detoxification of xenobiotics.

Weighing lysimeters are used to measure crop water use during the growing season. By relating the water use of a specific crop to a well-watered reference crop such as grass, crop coefficients (KC) can be developed to assist in predicting crop needs using meteorological data available from weather stations. This research was conducted to determine growth stage-specific KC and crop water use for onions (Allium cepa L.) and spinach (Spinacia oleracea L.) grown under south Texas conditions. Seven lysimeters, consisting of undisturbed 1.5 · 2.0 · 2.2-m deep soil monoliths, comprise the Texas AgriLife Research–Uvalde lysimeter facility. Six lysimeters, weighing ’14 Mg, have been placed each in the middle of a 1-ha field beneath a linear low-energy precision application irrigation system. A seventh lysimeter was established to measure reference grass reference evapotranspiration. Daily water use for onion and spinach was measured at 5-min intervals. Crop water requirements, KC determination, and comparison with existing Food and Agricultural Organization (FAO)KC values were determined over a 2- year period for each crop. The KC values determined over the growing seasons varied from 0.2 to 1.3 for onion and 0.2 to 1.5 for spinach with some of the values in agreement with those from FAO. It is assumed that the application of growth stage-specific KC will assist in irrigation management and provide precise water applications for a region of interest.

The present research focused on investigating cell growth inhibition and total and differential NFkB serine residue phosphorylations on estrogen insensitive MDAMB 231 breast cancer cells by various citrus limonoids and camptothecin. A cell growth assay was carried out after a 24 hr incubation with various concentrations of limonin glucoside, limonin, obacunone and obacunone glucoside (1, 5 and 10 μM) and camptothecin (10 μM), as a standard chemotherapy drug, along with DMSO solvent control. NFkB phosphorylations were assayed using a commercially available NFkB Elisa profiler kit (Active motif, Carlsbad CA) on these cells. The specific serine residue phosphorylation was studied using respective anti-phosphoserine antibodies (anti-phospho ser-468 and ser-536, respectively) supplied in the kit. The WST-1 cell growth assay showed that limonoids, at all tested concentrations, did not show a significant decrease in MDA-MB 231 cell growth when compared to camptothecin (37-40% growth inhibition). The NFkB p65 profiler Elisa assay with 10 uM concentration of three types of limonoids (limonin glucoside, obacunone and obacunone glucoside) exhibited a significant increase (50%) and limonin showed only a 10% increase in ser-468 residue phosphorylations than DMSO treated cells. However, camptothecin treated hormone insensitive cells exhibited a 50% increase in the phosphorylations of both ser-536 and ser-468 residues than DMSO treated cells and inhibiting the cell growth. The total constitutive NFkB phosphorylation activity of limonoids treated, show a decrease than DMSO treated control cells. On the other hand, camptothecin treated cells showed a significant increase in total constitutive NFkB phosphorylation activity. These results may suggest that hormone insensitive MDA-MB 231 cell growth were not inhibited by limonin glucoside, limonin, obacunone and obacunone glucoside, but perhaps was due to differential phosphorylations of serine residues of NFkB.

The flavonoid quercetin suppresses cell proliferation and enhances apoptosis in vitro. In this study, we determined whether quercetin protects against colon cancer by regulating the protein level of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt or by suppressing the expression of proinflammatory mediators [cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS)] during the aberrant crypt (AC stage. Forty male rats were randomly assigned to receive diets containing quercetin (0 or 4.5 g/kg) and injected subcutaneously with saline or azoxymethane (AOM, 2 times during wk 3 and 4). The colon was resected 4 wk after the last AOM injection and samples were used to determine high multiplicity AC foci (HMACF; foci with > 4 AC) number, colonocyte proliferation and apoptosis by immunohistochemisty, expression of Pl 3-kinase (p85 and p85α subunits) and Akt by immunoblotting, and COX-1, COX-2, and iNOS expression by real time RT-PCR. Quercetin-fed rats had fewer (P – 0.033) HMACF. Relative to the control diet, quercetin lowered the proliferation index (P – 0.035) regardless of treatment and diminished the AOM-induced elevation in crypt column cell number (P – 0.044) and expansion of the proliferate zone (P – 0.021). The proportion of apoptotic colonists in AOM-injected rats increased with quercetin treatment (P = 0.014). Levels of p85 and p85α subunits of PI 3-kinase and total AKT were unaffected by dietary quercetin. However, quercetin tended to suppress (P < 0.06) the expression of COX-1 and COX-2. Expression of iNOS was elevated by AOM inject (P = 0.0001). In conclusion, quercetin suppresses the formation of early preneoplastic lesions in colon carcinogenesis, which occurred in concert with reductions in proliferation and increases in apoptosis. It is possible the effects of proliferation and apoptosis resulted from the tendency for quercetin to suppress the expression of proinflammatory mediators.

In plants, high capacity tonoplast cation/H_ antiport is mediated in part by a family of CAX (cation exchanger) transporters. Functional association between CAX1 and CAX3 has previously been inferred; however, the nature of this interaction has not been established. Here we analyze the formation of “hetero-CAX” complexes and their transport properties. Co-expressing both CAX1 and CAX3 mediated lithium and salt tolerance in yeast, and these phenotypes could not be recapitulated by expression of deregulated versions of either transporter. Coincident expression of Arabidopsis CAX1 and CAX3 occurs during particular stress responses, flowering, and seedling growth. Analysis of cax1, cax3, and cax1/3 seedlings demonstrated similar stress sensitivities. When plants expressed high levels of both CAXs, alterations in transport properties were evident that could not be recapitulated by high level expression of either transporter individually. In planta coimmunoprecipitation suggested that a protein-protein interaction occurred between CAX1 and CAX3. In vivo interaction between the CAX proteins was shown using a split ubiquitin yeast two-hybrid system and gel shift assays. These findings demonstrate cation exchange plasticity through hetero-CAX interactions.