Distribution and Pathogenicity of Colletotrichum Species Associated With Mango Anthracnose in Mexico.

Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango (Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides. The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2/MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum, C. asianum, C. fructicola, C. siamense, and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum, C. fructicola, C. siamense, and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

Direct analysis of aluminum alloys by CSigma laser-induced breakdown spectroscopy.

We report the application of CSigma laser-induced breakdown spectroscopy (Cσ-LIBS) to quantitative analysis of aluminum alloys without sample preparation. Cσ-LIBS simplifies strongly the conventional calibration procedure of LIBS, replacing it with a characterization stage performed from the spectrum of a single standard sample. The aim of this work has been to provide a complete evaluation of the use of Cσ-LIBS for direct analysis by obtaining its figures of merit, including precision and limits of detection. Ten elements (Si, Fe, Cu, Mn, Mg, Cr, Ni, Zn, Ti and Ca) are determined in a set of six certified samples with a wide range of concentrations, from percent down to µg/g levels. The average precision is 8.0% for concentrations higher than 0.1 wt% and 13% for concentrations between 0.1 wt% and 0.01 wt%. The limits of detection are in the range 1.4-9.7 µg/g.

The level and source of free-methionine affect body composition and breast muscle traits in growing broilers.

Although dietary Met, as the first limiting amino acid (AA), has been extensively studied for poultry, little is known about how the supply and source of free Met affect tissue composition. The purpose of this study was to investigate the effect of feeding young broiler chickens with a deficient or sufficient TSAA (Met+Cys) supply, using either dl-Met (dl-Met+ and dl-Met-, for respectively diets sufficient and deficient in TSAA) or dl-2-hydroxy-4-methylthiobutyric acid (HMTBA+ and HMTBA-, for respectively diets sufficient and deficient in TSAA) as a Met source on tissue composition and breast muscle traits. For both Met sources, the deficient diets were formulated to provide true digestible Met:Lys and TSAA:Lys respectively 45% and 30% below that of the sufficient diets. Performance and tissue weights were affected by the Met supply but not by the Met source. In TSAA-deficient chickens, ADG and FCR, and protein content in empty body and pectoralis major muscles (PM) were lower than in TSAA-sufficient chickens (P < 0.05). Reducing the Met content of the diet increased the redness value of PM (a*) and the hue angle (H°; P < 0.01). The source of Met affected body AA composition and the partitioning of body Cys among tissues (P < 0.05). In TSAA-deficient birds, body Cys mass decreased in the commercial carcass and PM, but increased in the rest of the body (P < 0.01). The Met source also had an impact on the Cys mass, which was reduced in the commercial carcass and PM of dl-Met birds, but higher in the rest, especially in the feathers of TSAA-deficient birds (P < 0.05). The Met source, supply, or both altered the AA composition of the empty body, mostly in the commercial carcass. In conclusion, a dietary TSAA deficiency altered performance, tissue composition and quality traits of PM of broilers. There was no impact between dietary dl-Met and dl-HMTBA on performance or muscle weight, although the Met source affected the partitioning of Cys among tissues.

The amino acid composition of tissue protein is affected by the total sulfur amino acid supply in growing pigs.

The factorial approach to assess the amino acid (AA) requirements of pigs is based on the assumption that the AA composition of body protein is constant. However, there are indications that this assumption may not be valid because the AA composition of body protein can be affected by the AA supply. The extent to which different tissues are affected by an AA deficiency is unknown. The objective of this study was to investigate the effect of feeding pig diets with a deficient or sufficient total sulfur AA supply (TSAA; Met+Cys) from 6 to 23 weeks of age on tissue composition and meat quality. The deficient diet (TSAA-) provided 24% Met : Lys and 51% TSAA : Lys on a standardized ileal digestible basis, which are 19% and 16% below the recommended requirements, respectively. The sufficient diet (TSAA+) provided 33% Met : Lys and 60% TSAA : Lys. Diets were offered slightly below the ad libitum feed intake capacity of the pigs. Pigs offered diet TSAA- had a lower average daily gain, lower weights of the longissimus dorsi (LM) and rhomboideus muscles (RM), and of selected skin sections (P<0.05). The weight of different sections of the small intestine and the liver was not affected by the diet. The protein content of the LM and RM decreased in pigs offered diet TSAA- (P<0.05), whereas the protein content of other tissues was not affected. The TSAA supply affected the AA composition (g/16 g N) of protein in all tissues, but the Met content was changed only in the liver (P<0.05). Pigs receiving diet TSAA- had a lower Cys content in the RM and in the distal jejunum and ileum (P<0.01). The deficient TSAA supply resulted in a lower carcass weight and higher muscle glycogen stores (P<0.05), but did not affect other meat quality traits. The results of this study indicate that the muscles, jejunum and ileum respond more to a prolonged AA deficiency than the liver. In addition, the observed changes in AA composition of tissue protein question the use of a constant AA profile of retained protein to assess AA requirements.

Growth of body components and carcass composition of Iberian pigs of 10 to 150 kg body weight as affected by the level of feeding and dietary protein concentration.

A total of 211 growing-finishing Iberian (IB) pigs from 4 separate and independent sets of trials were slaughtered at several stages of growth from 10 to 150 kg BW to determine growth and development of chemical and physical components of the cold eviscerated carcass (CC; without head, feet, and tail). Within each set of trials, a factorial arrangement of treatments, involving several concentrations of ideal protein in the diets as 1 factor and 2 or 3 levels of feed intake as the other, was used. The main objective of the present study was to provide information on the relative growth of physical and chemical components of the CC of IB pigs, which differed because of the dietary treatment imposed, involving a wide range of protein-to-energy ratios and feeding levels. Allometric relationships (P < 0.001) were established between the weight of a chemical component in the CC and empty BW or CC weight. Irrespective of the adequacy of the dietary protein-to-energy ratio, the growth coefficient for CC weight relative to empty BW was >1 (P < 0.001), whereas those for protein, water, and ash relative to empty BW or CC weight were <1 (P < 0.001). In contrast, relative growth coefficients >1 (P < 0.001) were obtained for fat mass and total energy, reflecting the increase in fat relative content that occurs with increasing weight. Multiple-regression equations (P < 0.001) were developed using a stepwise procedure, which estimates the chemical (g/kg) or energy (MJ/kg) composition of CC as a function of empty BW, dietary protein-to-energy ratio, and feeding level, expressed as a multiple of the ME required for maintenance. It is concluded that even if the pattern of developmental growth for the IB pig may show some similarities (increased fat content or decreased proportional weight of some primal cuts with BW or age) with that observed for pigs of different genetic background, relevant differences were detected. They are related to a much smaller relative size of the IB pig lean tissues and cuts, their slower rates of growth, and the increased total body fat, with marked changes in its distribution among depots. Consequently, relationships obtained for lean or conventional genotypes are not applicable to the IB pig.

Changes in body composition in broilers by a sulfur amino acid deficiency during growth.

In the factorial approach, amino acid (AA) requirements are determined using the AA composition of retained protein, which is assumed to be constant. However, this hypothesis may not be valid because the AA composition of body protein can be affected by the diet. The objective of this study was to quantify the changes in chemical body composition of broilers receiving diets either deficient (TSAA-) or sufficient (TSAA+) in TSAA. Diet TSAA+ was formulated according to the Ross recommendation. Diet TSAA- provided 36% true digestible Met:Lys and 64% true digestible TSAA:Lys, which were, respectively, 34 and 22% lower compared with diet TSAA+. Performance and tissue weight gain between 7 and 42 d of age were not affected by the TSAA supply. In TSAA- chickens, protein gain was lower in the carcass (P < 0.01) and tended to be lower in the empty body (P = 0.06) and pectoralis major muscle (P = 0.10). Compared with TSAA+ chickens, lipid gain in TSAA- chickens was 78% greater in the pectoralis muscle (P < 0.001), 28% greater in abdominal fat (P < 0.05), and 10% greater in the carcass (P = 0.10). In the pectoralis muscle, there was a tendency for an increase in the redness value (a*; P = 0.10). The TSAA supply affected the AA composition of tissues and tissue gain, but the Met and Cys concentrations were changed only in the offal (P = 0.08). The deficient TSAA supply resulted in an increase in the Ser concentration in the empty body, carcass, and pectoralis muscle (P < 0.05). In contrast, it resulted in a decrease in the concentrations of Lys and Glu in the empty body, of Phe, Tyr, Gly, and Glu in the pectoralis muscle, and of Ala in the offal (P < 0.05). This indicates that although chickens cope with a TSAA deficiency predominantly by changing the protein and lipid concentration in the body, the AA composition is also affected. This calls into question the use of a constant ideal AA profile in poultry nutrition.

Peripheral tumor necrosis factor α regulation of adipose tissue metabolism and adipokine gene expression in neonatal pigs.

The neonatal pig is susceptible to stress and infection, conditions which favor tumor necrosis factor α (TNFα) secretion. This study examined whether TNFα can alter metabolic activity and cytokine gene expression within neonatal pig adipose tissue. Cell cultures were prepared from neonatal subcutaneous adipose tissue using standard procedures. Cultures (5 experiments) were incubated with medium containing (14)C-glucose for 4 h to measure glucose conversion to lipid in the presence of combinations of TNFα (10 ng), insulin (10 nM) and an anti-pig TNFα antibody (5 µg). Basal lipogenesis was not affected by TNFα treatment (P > 0.05). However, insulin stimulated lipogenesis was reduced by TNFα (P < 0.02). For gene expression studies, cultures were incubated with 0, 2.5, 5.0 or 10 ng TNFα for 2, 4 or 24 h (n = 4 experiments). Interleukin 6 and TNFα gene expression were acutely (2-4 h) stimulated by exogenous TNFα treatment (P < 0.05), as analyzed by real-time PCR. Adiponectin mRNA abundance was reduced (P < 0.001) while monocyte chemotactic gene expression was increased by TNFα treatment at all time points (P < 0.001). Chronic treatment (24 h) was required to increase monocyte multiplication inhibitory factor or suppress lipoprotein lipase gene expression (P < 0.02). These data suggest conditions which increase serum TNFα, like sepsis, could suppress lipid accumulation within adipose tissue at a time of critical need in the neonate and induce a variety of adipose derived cytokines which may function to alter adipose physiology.

Response analysis of the Iberian pig growing from birth to 150 kg body weight to changes in protein and energy supply.

A total of 251 growing-finishing Iberian (IB) pigs, 32 of which were suckling piglets, were used in 5 separate sets of trials. The comparative slaughter procedure was used to determine nutrient and energy retention at several stages of growth from birth to 150 kg BW. A factorial arrangement was used within each set of trials, involving several concentrations of ideal protein in the diets as 1 factor and 2 or 3 levels of feed intake as the other. The main objective of these studies was to derive the optimal protein-to-energy ratio in the diet to allow for the expression of maximum protein deposition rates. The effect of feed restriction on growth performance, protein deposition, and fat deposition was also assessed. According to allometric equations, empty BW (EBW) was related to whole body components or total chemical constituents of empty body mass (P < 0.001). For pigs receiving solid feed, highly statistically significant multiple regression equations were constructed, which derived nutrient (g/kg) or energy (MJ/kg) composition as a function of EBW, dietary protein-to-energy ratio, and level of feeding (P < 0.001). In pigs offered adequate protein-to-energy diets, ADG at each stage of production was predicted as a function of the average BW and feeding level (P < 0.001). It was observed that the estimates of ME required for maintenance and net efficiency of utilization of ME for growth change were within rather narrow ranges throughout the growth stages studied. Preferred values (413 kJ/kg BW(0.75) × d(-1) and 0.593 for ME(m) and k(g), respectively) were obtained by regressing total energy retention (kJ/kg BW(0.75) × d(-1)) against ME intake (kJ/kg BW(0.75) × d(-1)). A multiple-regression approach revealed that in the IB pig, ME costs for protein deposition and fat deposition reach 60 and 62 kJ/g, which is considerably greater than in conventional or lean pig genotypes. In the IB pig, the maximum daily rate of protein deposition (PD(max), g) seemed to follow a linear-plateau shape with a breaking point at 32.5 kg BW, beyond which PD(max) remained at an average rate of 75 g × d(-1). The marginal efficiency of body protein deposition was estimated at each growth stage. In pigs fed on optimal or suboptimal protein-to-energy diets, the relationship between PD and ME intake declined, following a curvilinear pattern with increasing BW; thus, implying relative increases in lipid gain as BW increased.

Metabolic regulation of fatty acid esterification and effects of conjugated linoleic acid on glucose homeostasis in pig hepatocytes.

Conjugated linoleic acids (CLAs) are geometric and positional isomers of linoleic acid (LA) that promote growth, alter glucose metabolism and decrease body fat in growing animals, although the mechanisms are poorly understood. A study was conducted to elucidate the effects of CLA on glucose metabolism, triglyceride (TG) synthesis and IGF-1 synthesis in primary culture of porcine hepatocytes. In addition, hormonal regulation of TG and IGF-1 synthesis was addressed. Hepatocytes were isolated from piglets (n = 5, 16.0 ± 1.98 kg average body weight) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Hepatocytes were cultured in William's E containing dexamethasone (10-8 and 10-7 M), insulin (10 and 100 ng/ml), glucagon (0 and 100 ng/ml) and CLA (1 : 1 mixture of cis-9, trans-11 and trans-10, cis-12 CLA, 0.05 and 0.10 mM) or LA (0.05 and 0.10 mM). Addition of CLA decreased gluconeogenesis (P < 0.05), whereas glycogen synthesis and degradation, TG synthesis and IGF-1 synthesis were not affected compared with LA. Increased concentration of fatty acids in the media decreased IGF-1 production (P < 0.001) and glycogen synthesis (P < 0.01), and increased gluconeogenesis (P < 0.001) and TG synthesis (P < 0.001). IGF-1 synthesis increased (P < 0.001) and TG synthesis decreased (P < 0.001) as dexamethasone concentration in the media rose. High insulin/glucagon increased TG synthesis. These results indicate that TG synthesis in porcine hepatocytes is hormonally regulated so that dexamethasone decreases and insulin/glucagon increases it. In addition, CLA decreases hepatic glucose production through decreased gluconeogenesis.

Nutrient and energy retention in weaned Iberian piglets fed diets with different protein concentrations.

Fifty-eight purebred castrated male Iberian (IB) piglets (initial BW 9.9 ± 0.1 kg) were used in an experiment to determine the effect of dietary protein content (PC) and feeding level (FL) on the rates of BW gain, whole body protein deposition (PD), and energy utilization between 10 and 25 kg of BW using the serial slaughter method. Treatments followed a 4 × 2 factorial arrangement with 4 PC (201, 176, 149, and 123 g of CP/kg of DM) and 2 FL (0.95 and 0.70 × ad libitum) and 6 or 7 piglets per combination of treatments. All diets were formulated to have an optimal AA pattern. Six piglets were slaughtered at the start of the trial to estimate initial body composition. The experimental pigs were individually housed in an environmentally controlled room (27 ± 2°C) until they reached 25 kg of BW, when they were slaughtered and analyzed for body composition. Positive linear effects of dietary PC on ADG, G:F, and gain:ME intake were observed (P < 0.001). Piglets fed at the highest FL showed greater ADG, G:F, and gain:ME intake (P < 0.001). An average increase was estimated to be 38.0 g of gain/MJ of ME intake. Protein deposition increased linearly from 35.6 to 50.9 g/d with increasing dietary PC (P < 0.001). A daily increase was estimated to be 0.35 g of PD/g of CP intake. Although the maximal genetic potential for PD of the IB piglet was not attained, a maximal value of 59.9 g/d for whole-body PD was achieved when the diet provided 201 g of CP/kg of DM and was fed at 0.95 × ad libitum. Piglets on the highest FL deposited on average 39% more body protein (P < 0.001) than restricted piglets. An average value of 4.39 g increase in PD/MJ of ME intake was obtained for diets containing 201 and 176 g of CP/kg of DM. Maintenance energy requirements and net efficiency of utilization of ME for growth, calculated by linear regression of ME intake on body retained energy, were 427 kJ/kg of BW(0.75)·d(-1) and 0.552, respectively. The corresponding partial efficiencies of utilization of ME for protein and fat deposition were 0.378 and 0.672, respectively, considerably less than the accepted values for conventional pig breeds. Practical diets of the young IB piglet should contain at least 201 g of ideal CP/kg of DM.

A sulfur amino acid deficiency changes the amino acid composition of body protein in piglets.

Experiments carried out to determine the amino acid requirement in growing animals are often based on the premise that the amino acid composition of body protein is constant. However, there are indications that this assumption may not be correct. The objective of this study was to test the effect of feeding piglets a diet deficient or not in total sulfur amino acids (TSAA; Met + Cys) on nitrogen retention and amino acid composition of proteins in different body compartments. Six blocks of three pigs each were used in a combined comparative slaughter and nitrogen balance study. One piglet in each block was slaughtered at 42 days of age, whereas the other piglets received a diet deficient or not in TSAA for 19 days and were slaughtered thereafter. Two diets were formulated to provide either 0.20% Met and 0.45% TSAA (on a standardized ileal digestible basis) or 0.46% Met and 0.70% TSAA. Diets were offered approximately 25% below ad libitum intake. At slaughter, the whole animal was divided into carcass, blood, intestines, liver, and the combined head, tail, feet and other organs (HFTO), which were analyzed for nitrogen and amino acid contents. Samples of the longissimus muscle (LM) were analyzed for myosin heavy chain (MyHC) and actin contents. Nitrogen retention was 20% lower in piglets receiving the TSAA-deficient diet (P < 0.01). In these piglets, the nitrogen content in tissue gain was lower in the empty body, carcass, LM and blood (P < 0.05) or tended to be lower in HFTO (P < 0.10), but was not different in the intestines and liver. The Met content in retained protein was lower in the empty body, LM and blood (P < 0.05), and tended to be lower in the carcass (P < 0.10). The Cys content was lower in LM, but higher in blood of piglets receiving the TSAA-deficient diet (P < 0.05). Skeletal muscle appeared to be affected most by the TSAA deficiency. In LM, the Met content in retained protein was reduced by 12% and total Met retention by more than 60%. The MyHC and actin contents in LM were not affected by the TSAA content of the diet. These results show that a deficient TSAA supply affects the amino acid composition of different body proteins. This questions the use of a constant ideal amino acid profile to express dietary amino acid requirements, but also illustrates the plasticity of the animal to cope with nutritional challenges.

Synergistic effects of betaine and conjugated linoleic acid on the growth and carcass composition of growing Iberian pigs.

An experiment was conducted to determine the efficacy of dietary betaine, CLA, or both as growth promotants and carcass modifiers in growing Iberian pigs. Twenty gilts (20 kg of BW) were individually penned and fed barley- and soybean meal-based diets (12% CP, 0.81% Lys, and 14.8 MJ of ME/kg of DM) containing either no added betaine or CLA (control), 0.5% betaine, 1% CLA, or 0.5% betaine + 1% CLA, at 95% of ad libitum energy intake. An additional group of 5 pigs was slaughtered at the beginning of the experiment to obtain the initial body composition. At 30 kg of BW, a balance experiment was conducted. At 50 kg of BW, pigs were slaughtered and viscera was removed and weighed. Betaine or CLA alone did not affect growth performance. However, betaine + CLA increased ADG (601 vs. 558 g, P = 0.03) and gain relative to ME intake (25.4 vs. 22.2 g/MJ, P = 0.03) compared with control pigs. Digestibility of nutrients and metabolizability of energy did not differ among diets (P = 0.46 to 0.75). Carcass protein, water, and lean deposition (g/d) increased (19.8, 24.2, and 23.4%, respectively, P < 0.01) in pigs fed betaine + CLA compared with control pigs. Similarly, protein deposition relative to ME intake increased by 28% in betaine + CLA-supplemented pigs (P < 0.05). Fat and mineral deposition did not differ among treatments. Carcass protein, water, and lean content (g/kg of carcass) of pigs fed betaine + CLA-supplemented diets tended to increase (P = 0.07 to 0.09) and carcass fat content tended to decrease (P = 0.09). Similarly, estimated composition of carcass gain was affected, such that water and lean content tended to increase (P = 0.06 to 0.08), whereas fat tended to decrease (P = 0.08) in pigs fed betaine + CLA-supplemented diets. Longissimus muscle area was not altered by treatments (P = 0.49). The liver of pigs fed betaine + CLA diets had increased weight (19%, P < 0.05) compared with control pigs. Overall, dietary supplementation of betaine + CLA increased ADG, protein, water, and lean deposition in growing Iberian gilts. There appears to be a synergistic action when betaine and CLA are used together.

Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome.

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to gamma-irradiation, and the resulting formation of single strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ((•)OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for (•)OH, we are able to estimate the rate coefficient for the reaction of (•)OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10(9) - 2×10(9) L mol(-1) s(-1) at 10(7) s(-1), and increases with increasing scavenging capacity.

Spatial distributions of the number densities of neutral atoms and ions for the different elements in a laser induced plasma generated with a Ni-Fe-Al alloy.

Spatially-resolved emission spectroscopy, including spatial devonvolution of the spectra, has been used to determine the three-dimensional distributions of the relative number densities of neutral atoms and ions of the elements present in a laser-induced plasma generated with a Ni-Fe-Al alloy. The method is based on the precise measurement of the local electronic temperature from Saha-Boltzmann plots constructed with Fe I and Fe II lines. The plasma was generated in air at atmospheric pressure using a 1064-nm Nd:YAG laser, and the emission was detected in the time window 3.0-3.5 micros. The ionization fraction was very high (above 0.9) for the three elements in the sample, only decreasing behind the expanding plasma front. The relative number densities were obtained from the emissivities of selected elemental lines as well as the temperature. The error in this procedure was estimated, and it was found that it is largely due to the uncertainties in the transition probability values used. The spatial distributions of the total relative number densities of the three elements were shown to coincide within the error, a result which is relevant to the development of models of plasma emission used in analytical applications. The ratios of the total number densities of the elements in the plasma were compared to their concentration ratios in the sample; however, the relatively high errors in the relative number densities did not permit any definitive conclusions to be drawn about the stoichiometry of the laser ablation process.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions.

PURPOSE: Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation. The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss. This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation. The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them. MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with gamma-irradiation. These conditions result in the very efficient production of guanyl radicals in the plasmid. We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined. RESULTS: The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed. The additional presence of nitrite decreased this effect of doxorubicin. CONCLUSION: Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical. Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime. Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime. The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals. It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

One-electron oxidation of plasmid DNA by selenium(V) species.

PURPOSE: To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with 137Cs gamma-rays (662 keV). The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. RESULTS: In general, gamma-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after gamma-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. CONCLUSION: By comparing the yields of breaks produced after gamma-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength.

PURPOSE: Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN)2*-. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN)2*- is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of one-electron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137Cs gamma-irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. RESULTS: The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). CONCLUSIONS: It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN)2*- and DNA-G*+. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G*+/DNA of E7 = +1.39+/-0.01V.

Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation.

PURPOSE: It has been previously argued that the use of the one-electron oxidants (SCN)2(*-) and Br2(*-) with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl(II)OH(+)) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA containing 10(-3) mol dm(-3) thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10(-7)-10(-4) mol dm(-3)) were irradiated with 137Cs gamma-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. RESULTS: High yields of FPG-sensitive sites produced by the oxidants (SCN)2(*-) and Tl(II)OH(+) were strongly attenuated by the presence of the reducing agents. CONCLUSIONS: From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10(4)-10(7) dm(3) mol(-1) s(-1). Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.

Redox reactivity of guanyl radicals in plasmid DNA.

PURPOSE: It has been previously argued that gamma-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E. coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG), and there that derive from an intermediate guanyl radical species. The wish was to characterize the reactivity of this intermediate with reducing agents. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10(-3) to 10(-1) mol dm(-3)) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10(-7) to 10(-3) mol dm(-3)) were subjected to 137Cs gamma-irradiation (662 keV). After irradiation, the plasmid was incubated with FPG. Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions. RESULTS: The very high yields of FPG-sensitive sites in the presence of SCN- or Br- decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least. CONCLUSIONS: From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5 x 10(5), 2 x 10(5), 10(7) and 10(7) dm3 mol(-1) s(-1) for ferrocyanide, nitrite, promethazine and tryptophan respectively).