The C Terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3′ deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity

Celecoxib concentration predicts decrease in prostaglandin E\u3csub\u3e2\u3c/sub\u3e concentrations in nipple aspirate fluid from high risk women

BACKGROUND: Epidemiologic studies suggest that long term low dose celecoxib use significantly lowers breast cancer risk. We previously demonstrated that 400 mg celecoxib taken twice daily for 2 weeks lowered circulating plasma and breast nipple aspirate fluid (NAF) prostaglandin (PG)E2 concentrations in post- but not premenopausal high risk women. We hypothesized that circulating concentrations of celecoxib influenced PGE2 response, and that plasma levels of the drug are influenced by menopausal status. To address these hypotheses, the aims of the study were to determine: 1) if circulating plasma concentrations of celecoxib correlated with the change in plasma or NAF PGE2 concentrations from baseline to end of treatment, and 2) whether menopausal status influenced circulating levels of celecoxib. METHODS: Matched NAF and plasma were collected from 46 high risk women who were administered celecoxib twice daily for two weeks, 20 subjects receiving 200 mg and 26 subjects 400 mg of the agent. NAF and plasma samples were collected before and 2 weeks after taking celecoxib. RESULTS: In women taking 400 mg bid celecoxib, plasma concentrations of the agent correlated inversely with the change in NAF PGE2 levels from pre- to posttreatment. Nonsignificant trends toward higher celecoxib levels were observed in post- compared to premenopausal women. There was a significant decrease in NAF but not plasma PGE2 concentrations in postmenopausal women who took 400 mg celecoxib (p = 0.03). CONCLUSION: In high risk women taking 400 mg celecoxib twice daily, plasma concentrations of celecoxib correlated with downregulation of PGE2 production by breast tissue. Strategies synergistic with celecoxib to downregulate PGE2 are of interest, in order to minimize the celecoxib dose required to have an effect

Efficacy of turmeric (Curcuma longa) to ameliorate the adverse effects of ochratoxin A in broiler chicks

Abstract only availableA 21-day feeding study was conducted to assess the effectiveness of turmeric (Curcuma longa) powder (TMP), containing a known level of curcumin to offset the adverse effects of ochratoxin A (OA) in broiler chicks. Five pen replicates of 5 chicks each were assigned to each of 6 dietary treatments. Dietary treatments evaluated include: 1) basal diet containing no OA or TMP; 2) basal diet supplemented with 0.67% TMP containing 220 mg/kg total curcuminoids (TCMN); 3) basal diet supplemented with 1 mg/kg OA; 4) basal diet supplemented with 1 mg/kg OA and 220 mg/kg TCMN; 5) basal diet supplemented with 2 mg/kg OA; 6) and basal diet supplemented with 2 mg/kg OA and 220 mg/kg TCMN. The addition of OA to the diet significantly reduced (P < 0.05) feed intake, body weight gain, and caused poor feed conversion . Similarly, there was a significant effect (P < 0.05) of OA on relative liver weight and relative kidney weight.. Results indicated that 220 mg/kg TCMN did not counteract any adverse effects in broiler chicks fed OA at levels of 1 mg/kg and 2 mg/kg. It remains to be seen if OA negatively affected antioxidant status and hepatic gene expression of chicks, and if TCMN will be beneficial in ameliorating any observed adverse effects. Samples are currently being analyzed for antioxidant activity and changes in gene expression.F.B. Miller Undergraduate Research Program in Animal Science

Do Plant Secondary Metabolite‐Containing Forages Influence Soil Processes in Pasture Systems?

Grazed pastures are susceptible to N loss from urine/manure additions, which increases eutrophication, affecting the global N cycle. Plant secondary metabolites (PSM), such as condensed tannins (CT) and terpenes, influence silviculture soil dynamics by generally decreasing N mineralization. We investigated whether cattle‐grazed pastures of non‐traditional grass and legume forage monoculture strips including CT‐containing sainfoin (Onobrychis viciifolia Scop.) and tall fescue (TF) [Schedonorus arundinaceus (Schreb.) Dumort.] influenced soil dynamics compared with traditional grass and legume forage monoculture strips of alfalfa (Medicago sativa L.), without tannins, and TF. Throughout the study, CT in sainfoin averaged 58.9 g kg−1 whereas alfalfa saponins averaged 5.7 g kg−1. We observed greater soil microbial respiration (p = .01) in TF strips than legume strips, indicating greater microbial activity, and between legumes we found greater soil NO3 (p = .01) in alfalfa than in sainfoin, although aboveground biomass and N differences were negligible. We also conducted a laboratory soil‐feces incubation study to determine if feces from cattle foraging diets of legumes with or without CT influenced soil dynamics. Both feces treatments showed lower NO3 (p \u3c .001) than without feces, suggesting microbial inhibition. Dehydrogenase activity (DHEA) was lower (p = .03) in sainfoin than alfalfa feces, suggesting CT from sainfoin inhibit DHEA. To our knowledge this study is the first considering whether CT‐containing sainfoin and saponin‐containing alfalfa influence soil dynamics by assessing general differences in soil parameters. More research is needed to determine whether specific PSM mitigate N loss in pasture systems by slowing N mineralization

Simultaneous Determination of the Predominant Hyperforins and Hypericins in St. John&apos;s Wort (Hypericum perforatum L.) by Liquid Chromatography

Hypericin and hyperforin are believed to be among the active constituents in common St. John&apos;s wort (Hypericum perforatum L.). Presently, dietary supplements are generally standardized to contain specified levels of hypericin and hyperforin, and the related compounds, pseudohypericin and adhyperforin. A rapid method was developed for simultaneous determination of these 4 active constituents by liquid chromatography (LC). A 1 g portion of dried, finely ground leaf/flower sample is extracted with 20 mL methanol for 2 h. A 0.6 mL aliquot of the crude extract is combined with 5.4 mL acetonitrile-methanol (9 + 1) and passed through a mixed solid-phase cleanup column. The eluate is examined by LC for hyperforin, adhyperforin, hypericin, and pseudohypericin on a Hypersil reversed-phase column by using simultaneous ultraviolet (284 nm) and fluorescence detection (excitation, 470 nm; emission, 590 nm). The compounds are easily separated isocratically within 8 min with a mobile phase of acetonitrile-aqueous 0.1M triethylammonium acetate (8 + 2). Average recoveries of hyperforin and adhyperforin were 101.9 and 98.4%, respectively, for 3 sample mixtures containing concentrations ranging from approximately 0.2 to 1.5% combined hyperforins per gram dry weight. Average relative standard deviation (RSD) values for hyperforin and adhyperforin for all 3 mixtures were 18.9 and 18.0%, respectively. Average recoveries of hypericin and pseudohypericin were 88.6 and 93.3% respectively, from 3 sample mixtures containing concentrations ranging from approximately 0.2 to 0.4% combined hypericins per gram dry weight. Average RSD values for hypericin and pseudohypericin for all 3 mixtures were 3.8 and 4.2%, respectively. C ommon St. John&apos;s wort (Hypericum perforatum L.) is a perennial species of the Hypericaceae family, native to Europe. Dietary supplements and other herbal preparations produced from the leaves and flowers of St. John&apos;s wort have gained popularity in the United States in recent years (1, 2). A recent overview of 23 controlled clinical trials concluded that St. John&apos;s wort was more effective than a placebo for the treatment of mild depression (3). Commercial extracts from the leaves and flowers are also being investigated for anticancer and antiviral activities (4). The predominant napthodianthrone derivatives, hypericin and pseudohypericin, and the phloroglucine derivatives, hyperforin and adhyperforin, are among the compounds presently being investigated for their biological activities. Standardized dietary supplements of St. John&apos;s wort currently contain from 0.3 to 0.5% hypericin(s), and/or approximately 3.0% hyperforin(s). In 1998, St. John&apos;s wort herbal products showed exceptional sales growth, increasing nearly 3000% from 1997 to 1998 (2). Several recent papers on the chemical analysis of St. John&apos;s wort have provided the means to measure many of the predominant chemical constituents from diluted, crude extracts (5-12). In general, samples were extracted and filtered, or liquid-liquid extraction was used to remove chlorophylls and other pigments. The use of mixed solid-phase (MSP) cleanup columns has been reported recently in the literature. Wilson and Romer (13) developed a proprietary cleanup column consisting of a mixture of reversed-phase, ion-exclusion, and ion-exchange packing materials used for cleanup of extracts of corn, cottonseed, rice, mixed feeds, and a variety of nuts in the determination of aflatoxins. Similarly, Tacke and Casper (14) developed a C 18 -alumina (1 + 3) MSP cleanup column for wheat extracts in the determination of deoxynivalenol. The following method was developed to provide a rapid, inexpensive, MSP cleanup with simultaneous determination of the 4 compounds of greatest current interest, hypericin, hyperforin, pseudohypericin, and adhyperforin from flower and leaf mixtures of St. John&apos;s wort

A survey of phytoestrogenic activity in Kansas flint hills pastures

The botanical composition and basal cover of three Kansas Flint Hills pastures located in Butler and Chase counties was surveyed to estimate the incidence of plant species that contain appreciable levels of estrogenic activity. Many-flowered scurfpea and Ladino clover were the only plant species classified as high in estrogenic activity. Although significant estrogenic activity existed in specific species, the willingness of livestock to consume those species is unclear

T-2 toxin adsorption by hectorite

The adsorption of T-2 toxin by the natural smectite mineral – hectorite at pH 3.0, 7.0 and 9.0 was investigated. The results of T-2 toxin adsorption on hectorite showed that the T-2 adsorption capacity decreased with increasing concentration of adsorbent in the suspension for all the investigated pH values. From the adsorption isotherms, an increase in T-2 toxin adsorption with increasing initial T-2 toxin concentration was observed for all the investigated pH values. The T-2 toxin adsorption by hectorite followed a non-linear (Langmuir) type of isotherm at pH 3.0, 7.0 and 9.0, with correlation coefficients (r2) of 0.943 at pH 3.0, 0.919 at pH 7.0 and 0.939 at pH 9.0. The estimated maximum T-2 toxin adsorption by hectorite based on the Langmuir fit to the data (9.178 mg/g at pH 3.0, 9.930 mg/g at pH 7.0, and 19.341 mg/g at pH 9.0), indicated that the adsorption of T-2 toxin by hectorite is pH dependent. The obtained data suggest the existence of specific active sites in hectorite onto which the T-2 toxin is adsorbed

Zearalenone and ochratoxin A: adsorption by kaolin modified with surfactant

Octadecyldimethylbenzyl ammonium chloride (OA) was used as a surfactant for the preparation of organokaolin. The natural kaolin (from a plant for production of quartz sand in Rgotina, Serbia) was modified with a surfactant in amount equal to 90% of the kaolin cation exchange capacity (CEC). FTIR spectroscopy was used for characterization of the new product. FTIR spectra confirmed the presence of OA ions at the kaolin surface. Adsorption of mycotoxins – zearalenone (ZEN) and ochratoxin A (OCHRA) was studied by organokaolin at different amounts of adsorbent and pHs. Results showed that the presence of organic cations in the kaolin structure increased adsorption of both ZEN and OCHRA. Adsorption of the mycotoxins by organokaolin increased with increasing amounts of adsorbent and, at the lowest amount of solids in suspension, adsorption of ZEN and OCHRA was slightly higher at pH 7 and 9

Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes.

ArticleBackground: Mean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus. Methods: A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs. Results: Using different statistical methods,MDRI values ranged from 88-94 days at cutoff ODn = 1.0 to 177-183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing formisclassification among long-terminfections indicated that overall PFRs were 0.6%to 2.5%at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C). Conclusions: Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118-142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.This research has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC)

Effects of Aflatoxin B1 and Fumonisin B1 on Blood Biochemical Parameters in Broilers

The individual and combined effects of dietary aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on liver pathology, serum levels of aspartate amino-transferase (AST) and plasma total protein (TP) of broilers were evaluated from 8 to 41 days of age. Dietary treatments included a 3 × 3 factorial arrangement with three levels of AFB1 (0, 50 and 200 μg AFB1/kg), and three levels of FB1 (0, 50 and 200 mg FB1/kg). At 33 days post feeding, with the exception of birds fed 50 mg FB1 only, concentrations of AST were higher (p < 0.05) in all other treatment groups when compared with controls. Plasma TP was lower (p < 0.05) at six days post feeding in groups fed 200 μg AFB1/kg alone or in combination with FB1. At day 33 days post feeding, with the exception of birds fed the highest combination of AFB1 and FB1 which had higher plasma TP than control birds, plasma TP of birds fed other dietary treatments were similar to controls. Broilers receiving the highest levels of AFB1 and FB1 had bile duct proliferation and trabecular disorder in liver samples. AFB1 singly or in combination with FB at the levels studied, caused liver damage and an increase in serum levels of AST