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

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

Hypericin and hyperforin are believed to be among the active constituents in common St. John'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'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'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'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's wort currently contain from 0.3 to 0.5% hypericin(s), and/or approximately 3.0% hyperforin(s). In 1998, St. John'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'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's wort

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

Celecoxib concentration predicts decrease in prostaglandin E2 concentrations in nipple aspirate fluid from high risk women

<p>Abstract</p> <p>Background</p> <p>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)E₂concentrations in post- but not premenopausal high risk women. We hypothesized that circulating concentrations of celecoxib influenced PGE₂response, 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 PGE₂concentrations from baseline to end of treatment, and 2) whether menopausal status influenced circulating levels of celecoxib.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>In women taking 400 mg bid celecoxib, plasma concentrations of the agent correlated inversely with the change in NAF PGE₂levels 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 PGE₂concentrations in postmenopausal women who took 400 mg celecoxib (p = 0.03).</p> <p>Conclusion</p> <p>In high risk women taking 400 mg celecoxib twice daily, plasma concentrations of celecoxib correlated with downregulation of PGE₂production by breast tissue. Strategies synergistic with celecoxib to downregulate PGE₂are of interest, in order to minimize the celecoxib dose required to have an effect.</p

In Vivo Electroporation Enhances the Immunogenicity of an HIV-1 DNA Vaccine Candidate in Healthy Volunteers

DNA-based vaccines have been safe but weakly immunogenic in humans to date.We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines.This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate.ClinicalTrials.gov NCT00545987

Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.This research was supported by the National Science Foundation Plant Genome Award DBI0321467 and by research funds provided by the USDA Agricultural Research Service to MDM.Peer reviewe