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C M Maragos

Publications and source records attributed to C M Maragos.

At least 19 recordsLinked to original sources
PubMed2002

Fluorescence polarization as a tool for the determination of deoxynivalenol in wheat.

The mould Fusarium graminearum is found worldwide as a pathogen of cereal grains, in particular of wheat and maize, and it produces a mycotoxin known as deoxynivalenol (DON or vomitoxin). Each year, the presence of this compound and related trichothecenes causes substantial losses to agricultural productivity. Rapid methods for the measurement of the toxin in grains are required to monitor and divert effectively contaminated grain from the food supply. A fluorescence polarization (FP) immunoassay using a previously described monoclonal antibody for DON was developed. The assay was based on the competition of unlabeled DON from a sample with a fluorescently tagged DON, DON-fluorescein (DON-FL), for a DON-specific monoclonal antibody in solution. The FP of the tagged DON was increased upon binding with the antibody. In the presence of free toxin, less of the DON-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of wheat with the DON-FL and antibody. The sensitivity of the assay was strongly dependent upon the time between mixing of the sample with the tracer and measurement of the fluorescence polarization, with midpoints for the competition curves ranging from 0.03 microg ml(-1) with a 15-s incubation to >1 microg ml(-1) with a 12-min incubation. Samples of wheat naturally contaminated with DON were evaluated by FP and by an HPLC-UV method, with a good correlation (r2 = 0.97). Although the FP method tended to overestimate DON slightly in the wheat samples, by approxiamtely 20%, the assay was easy to use and very useful for the screening of wheat.

Antibodies, Monoclonal↗
PubMed2001

Fluorescence polarization as a means for determination of fumonisins in maize.

Fumonisins, mycotoxins produced by certain species of Fusaria, are commonly found worldwide as contaminants in maize. This paper reports the development of a rapid, portable fluorescence polarization-based assay for fumonisins in maize. The assay was based on the competition of unlabeled fumonisin, from a sample, with a fluorescently tagged fumonisin (FB(1)-FL) for a fumonisin-specific monoclonal antibody in solution. The fluorescence polarization (FP) of the tagged fumonisin was increased upon binding with the antibody. In the presence of free toxin, less of the FB(1)-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of maize with the tagged fumonisin and antibody, and required <2 min per sample, excluding extraction time. Two permutations of the assay were tested, one with each sample matrix serving as its own blank, and the other with all of the samples compared relative to a PBS blank with normalization of the data similar to an ELISA. The limit of detection, defined as the toxin content associated with a fluorescence polarization signal 5 standard deviations from that of a fumonisin-free control, was 0.5 microg of FB(1)/g in spiked maize. Recoveries from spiked maize over the range of 0.5-20 ppm averaged 94.3 +/- 13.8%. Forty-eight samples of field-contaminated maize were tested by the FP and an established HPLC method, with a good correlation between the two (r(2) = 0.85-0.88). For these samples, the two variations of the FP assay also compared well to one another (r(2) = 0.97), suggesting the assay principle is very robust. The results, combined with the speed and ease of use for the assay, suggest that this technology has substantial potential as a screening tool for mycotoxins in foods.

Antibodies, Monoclonal↗
PubMed2000

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels.

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.

Beer↗
PubMed2000

Fusarium species from nepalese rice and production of mycotoxins and gibberellic acid by selected species.

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.

Carboxylic Acids↗
PubMed1999

Fiber-optic immunosensor for mycotoxins.

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.

Aflatoxin B1↗
PubMed1996

Analysis of fumonisin B1 in corn by capillary electrophoresis.

Intact fumonisins contain two tricarballylic acid groups and can therefore acquire a net negative charge. The anionic nature of the fumonisins is the basis behind the widely used method for cleanup of corn with strong anion exchange (SAX) columns. This property also enables the fumonisins to be separated by electrophoretic techniques which, until now, have not been applied to the analysis of fumonisins in corn. Fumonisin B1, extracted from corn with 80/20 (v/v) methanol/water and isolated with a commercially available affinity column, was derivatized with fluorescein isothiocyanate for analysis by capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). Recoveries from corn fortified with 0.25 to 5.0 ppm FB1 averaged 89% (range 71 to 102%). As little as 0.05 ppm FB1 could be detected in corn. For corn naturally contaminated with FB1, the CZE-LIF method compared favorably to established SAX/HPLC and C18/HPLC methods. Capillary electrophoresis can be used for quantitation of FB1 in corn, with minimal use of organic solvents and provides an additional tool for confirming fumonisin contamination.

Carcinogens, Environmental↗
PubMed1996

Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg culture material.

Fumonisins, a group of mycotoxins produced by the ubiquitous fungi Fusarium moniliforme and F. proliferatum, were first identified about eight years ago. They have been shown to cause a variety of health effects in animals, including epidemiological evidence of esophageal cancer in humans. Cattle are less sensitive to ill effects than horses and swine. Fumonisins are common contaminants of low quality grain fed to cattle. Culture material containing fumonisins (FB1, FB2, and FB3) was mixed into the total diet and fed for 14 days to two midlactation Jersey cows to determine if fumonisins are excreted in milk. The dietary equivalent of fumonisin was approximately 75 ppm and the two cows consumed an average of 3 mg fumonisin B1/kg body weight (bwt)/day. Fumonisins were not detected in any of the milk samples by two analytical laboratories using methods with a sensitivity of 5 ng/ml. Except for transient diarrhea at the beginning of the contaminant feeding period and an increase in serum cholesterol, clinical and hematologic changes were not observed in the animals. The appearance or carry over of fumonisins from feed to milk in dairy cows does not appear to be significant and likely not a hazard or food safety concern for humans.

Animal Feed↗
PubMed1996

Determination of hydrolysed fumonisin B1 (HFB1) in corn by competitive direct enzyme-linked immunosorbent assay.

Fumonisin B1, a mycotoxin produced by certain Fusarium moulds, consists of two tricarballyic acid groups esterified to a 20-carbon backbone. Under alkaline conditions, or through metabolism, the aminopentol backbone, also known as hydrolysed Fumonisin B1 (HFB1) can be formed and is itself cytotoxic. Although the occurrence of HFB1 in corn-based foods is suspected, because of the ubiquitous nature of FB1 in corn, analytical methods for its detection are difficult. In the present report we describe a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the rapid analysis of HFB1 in corn. The concentration required to inhibit enzyme conjugate binding by 50% (IC50) was 36 ng/ml. The limit of detection of HFB, by the CD-ELISA was 2ng/ml, when HFB1 was added in bovine serum albumin-phosphate buffered saline. The antibody also cross-reacted with the hydrolysis products of FB2, FB3, and FB4, having IC50 values of 331, 174, and 1700 ng/ml respectively. The antibody did not react with the intact fumonisins, sphingosine, sphinganine, or tricarballylic acid. Samples of corn spiked with HFB1 over the range of 5-1000 ng/g indicated the CD-ELISA has a limit of detection of 5 ng/g and an IC50 of 41 ng/g in the matrix. The CD-ELISA provides a sensitive and rapid tool for the analysis of corn-based foods for HFB1.

Animals↗
PubMed1993

Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release.

Cell-mediated antitumor effects have, in part, been attributed to the production of NO. Compounds which generate NO might, therefore, be useful in attenuating the growth of tumor cells. Six nitric oxide/nucleophile adducts that release NO spontaneously in solution were tested for their effectiveness in inhibiting DNA synthesis in A375 human melanoma cells. The complexes of NO with spermine, 3-(n-propylamino)propylamine (PAPA/NO), and diethylamine reduced thymidine incorporation by 50% at concentrations of 24, 44, and 128 microM, respectively. The degree of inhibition was, in general, related to the rate and extent of NO release in solution. A melanoma cell clone sensitive to interleukin 1-mediated cytostasis (A375-C6) was no more sensitive to PAPA/NO than a clone resistant to interleukin 1 (A375-C5), suggesting that the differing inhibitory effects of interleukin 1 in the two A375 cell clones are not due to a differential sensitivity to nitric oxide. Oxymyoglobin (125 microM), a known scavenger of NO, restored the ability of A375-C6 cells to incorporate thymidine in the presence of up to 200 microM PAPA/NO. When PAPA/NO was added to a solution of oxymyoglobin, nitrosylmyoglobin was formed, indicating that the protective effect of myoglobin was due to scavenging of NO. The results are consistent with a nitric oxide-mediated mechanism for NO/nucleophile cytostasis and suggest that such compounds may be useful as tools for investigating the role of reactive nitrogen intermediates in cytostasis and cytotoxicity.

Antineoplastic Agents↗
PubMed1993

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium.

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.

Biotransformation↗
PubMed1993

Mechanism of vascular relaxation induced by the nitric oxide (NO)/nucleophile complexes, a new class of NO-based vasodilators.

Compounds formed by reacting nitric oxide (NO) with various nucleophiles have been shown to dilate aortic segments with a potency that correlates strongly with the amount of NO they release spontaneously in aqueous buffers. We performed experiments aimed at confirming their mechanism of action and using the data to design improvements in their pharmacologic properties. That the vasorelaxant action these agents induce is endothelium-independent was demonstrated by exposure of denuded versus intact aortic segments to the diethylamine/NO complex (DEA/NO); denudation had no significant effect on potency. Similarly, NG-monomethyl-L-arginine, an NO synthase inhibitor, did not affect the action of DEA/NO. However, both the vasorelaxant potency of DEA/NO and the amount of cyclic guanosine monophosphate it induced were significantly diminished by the guanylate cyclase inhibitor, methylene blue. The results support the view that the NO/nucleophile adducts induce vasodilation by spontaneously releasing NO, which then activates guanylate cyclase. This mechanistic conclusion suggests that not only potency but also duration of action, a clinically relevant parameter not studied in the previous investigation, might also be controllable by structural modification. We tested this hypothesis by comparing DEA/NO and the spermine/NO adduct (SPER/NO), whose half-lives (t1/2) are 2.1 and 39 min, respectively, for persistence of their dilatory effects. The response to DEA/NO rapidly peaked (maximum at 5 min) and receded during the 60-min observation period; SPER/NO required 15 min to reach peak relaxation but maintained this level throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

DNA deaminating ability and genotoxicity of nitric oxide and its progenitors.

Nitric oxide (NO), a multifaceted bioregulatory agent and an environmental pollutant, can also cause genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and intact DNA at physiological pH. That similar DNA damage can also occur in vivo was tested by treating Salmonella typhimurium strain TA1535 with three NO-releasing compounds, including nitroglycerin. All proved mutagenic. Observed DNA sequence changes were greater than 99% C----T transitions in the hisG46 (CCC) target codon, consistent with a cytosine-deamination mechanism. Because exposure to endogenously and exogenously produced NO is extensive, this mechanism may contribute to the incidence of deamination-related genetic disease and cancer.

Codon↗
PubMed1991

Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects.

Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.

Animals↗
PubMed1991

Gastric nitrate reduction and nitrosation of trimethylurea in swine treated with pentagastrin or cimetidine.

Swine were treated with cimetidine in order to quantify the reduction of nitrate to nitrite and the gastric formation of N-nitrosotrimethylurea (NTMU) under conditions similar to those in the achlorhydric human stomach. Gastric-fistulated swine were instilled with 6.0 mmol of nitrate in 50 ml water, after which gastric nitrate, nitrite and pH were monitored. Trimethylurea, 250 mumol in 50 ml water, was instilled via the fistula 10 min following the peak gastric nitrite concentration. Similar experiments were conducted with pentagastrin-stimulated animals, in order to quantitate the effect of gastric pH and microflora on the presence of nitrate, nitrite and NTMU formation. The stomachs of cimetidine-treated pigs (elevated pH) were colonized by nitrate reductase organisms to levels of 10(4)-10(7)/ml gastric fluid. Gastric nitrite concentration in cimetidine-treated animals reached a maximum of 370-2085 microM, 60 min following the nitrate dose. Trimethylurea was only marginally nitrosated (less than 0.1 mumol/l gastric fluid) in cimetidine- or pentagastrin-stimulated animals. The low yield of NTMU at elevated pH, in the presence of substantial nitrite, suggests that the nitrate-reducing bacteria present in the porcine stomach did not catalyze trimethylurea nitrosation in vivo.

Animals↗
PubMed1991

Formation of N-trimethyl-N-nitrosourea in the gastric lumen of the fistulated pig.

The formation of N-trimethyl-N-nitrosourea (TMNU) in the stomach of full-sized gastric-fistulated pigs was determined. Nitrite was added to the stomach in amounts that have been reported to occur in the human stomach (25-375 mumol). The vehicle was an 'artificial meal' made up of synthetic gastric juice. The total amount of TMNU formed (not concentration) was estimated by determining the concentration and gastric volume using multiple additions of a nonabsorbable marker (polyethylene glycol). At an initial pH of 3, 23-1000 micrograms of TMNU were found after 10-25 min. The rate of loss of polyethylene glycol and TMNU from the stomach were the same, indicating that TMNU was not directly absorbed. These data suggest that formation of TMNU under conditions similar to those found in the human stomach is possible.

Animals↗
PubMed1990

Quantitative estimates of N-nitrosotrimethylurea formation in the porcine stomach.

Gastric formation of N-nitrosotrimethylurea (NTMU) was quantified by repeated sampling of the gastric contents of full-sized fistulated pigs after the addition of 250 mumol trimethylurea (TMU) and 25-125 mumol nitrite. Maximum gastric nitrite concentration was 100-500 microM based on the gastric volume and amount of added nitrite, which is within the range reported in human gastric contents. The total amount of NTMU formed over time was quantified by estimating both the NTMU concentration and gastric volume using multiple additions of polyethylene glycol. Administration of 25 mumol of nitrite resulted in the formation of 320 nmol of NTMU. The total amount of NTMU formed was linearly related to nitrite dose. Conversion of nitrite to NTMU was of the order of 1-3%. Potassium thiocyanate (200-2000 mumol) had no effect on NTMU formation when given immediately preceding 75 mumol nitrite. Ascorbic acid at 225 and 341 mumol (0.66 and 1.0 of the recommended dietary allowance) inhibited nitrosation of TMU by an average of 54 and 84%. A pig with an average gastric pH of 1.9 formed 4.5-fold more NTMU than did a second pig at pH 4.8. These results suggest that low micromolar amounts of N-nitrosoureas can be formed in the normal stomach when nitrite is consumed in amounts to which humans are commonly exposed.

Animals↗