Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Salmonella from gopher tortoises (Gopherus polyphemus) in south Georgia

From 2002 to 2006, gopher tortoises (Gopherus polyphemus) were collected at Moody Air Force Base, Lowndes/Lanier counties, Georgia, USA, and opportunistically surveyed for the presence of Salmonella species. Four of 155 (2.6%) cloacal swabs collected from 80 tortoises were positive for the presence of Salmonella enterica, and the following serovars were identified: Give, Hartford, Javiana, and Luciana. Female tortoises (5%) were infected at a rate similar to male tortoises (5%). All isolates were obtained from adult tortoises (n = 73); subadults (n = 7) were all negative. Each isolated serovar is a potential human pathogen, suggesting appropriate precautions should be emphasized when handling these animals.     

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN⁺], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.     

Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.     

Dissolved and particulate nutrient flux from three adjacent agricultural watersheds: A five-year study

Fluxes of dissolved and particulate nitrogen (N) and phosphorus(P) from three adjacent watersheds were quantified with ahigh-resolution sampling program over a five-year period. The watershedsvary by an order of magnitude in area (12,875, 7968 and 1206 ha), and inall three watersheds intensive agriculture comprises > 90% ofland. Annual fluxes of dissolved N and P per unit watershed area (exportcoefficients) varied 2X among watersheds, and patterns were notdirectly related to watershed size. Over the five-year period, meanannual flux of soluble reactive P (SRP) was 0.583 kg P ·ha^–1 · yr^–1 from the smallestwatershed and 0.295 kg P · ha^–1 ·yr^–1 from the intermediate-sized watershed, which hadthe lowest SRP flux. Mean annual flux of nitrate was 20.53 kg N ·ha^–1 · yr^–1 in the smallestwatershed and 44.77 kg N · ha^–1 ·yr^–1 in the intermediate-sized watershed, which had thehighest nitrate flux. As a consequence, the export ratio of dissolvedinorganic N to SRP varied from 80 (molar) in the smallest watershed to335 in the intermediate-sized watershed. Because most N was exported asnitrate, differences among watersheds in total N flux were similar tothose for nitrate. Hence, the total N:P export ratio was 42(molar) for the smallest watershed and 109 for the intermediate-sizedwatershed. In contrast, there were no clear differences among watershedsin the export coefficients of particulate N, P, or carbon, even though> 50% of total P was exported as particulate P in allwatersheds. All nutrient fractions were exported at higher rates in wetyears than in dry years, but precipitation-driven variability in exportcoefficients was greater for particulate fractions than for dissolvedfractions.Examination of hydrological regimes showed that, for all nutrientfractions, most export occurred during stormflow. However, theproportion of nitrate flux exported as baseflow was much greater thanthe proportion of SRP flux exported as baseflow, for all threewatersheds (25–37% of nitrate exported as baseflow vs.3–13% of SRP exported as baseflow). In addition, baseflowcomprised a greater proportion of total discharge in theintermediate-sized watershed (43.7% of total discharge) than theother two watersheds (29.3 and 30.1%). Thus, higher nitrateexport coefficients in the intermediate-sized watershed may haveresulted from the greater contribution of baseflow in this watershed.Other factors potentially contributing to higher nitrate exportcoefficients in this watershed may be a thicker layer of loess soils anda lower proportion of riparian forest than the other watersheds. Theamong-watershed variability in SRP concentrations and exportcoefficients remains largely unexplained, and might represent theminimum expected variation among similar agriculturalwatersheds.     

Phosphatidylcholine: cholesterol phase diagrams

Two mono-cis-unsaturated phosphatidylcholine (PC) lipid molecules, having very different gel-liquid crystalline phase transition temperatures as a consequence of the relative positions of the double bond, exhibit PC:cholesterol phase diagrams that are very similar to each other and to that obtained previously for a fully saturated PC:cholesterol mixture (Vist, M. R., and J. H. Davis. 1990. Biochemistry 29:451-464). This leads to the conjecture that PC:cholesterol membrane phase diagrams have a universal form which is relatively independent of the precise chemical structure of the PC molecule. One feature of this phase diagram is the observation over a wide temperature range of a fluid but highly conformationally ordered phase at bilayer concentrations of more than ~25 mol% cholesterol. This `liquid ordered' phase is postulated to be the relevant physical state for many biological membranes, such as the plasma membrane of eukaryotic cells, that contain substantial amounts of cholesterol or equivalent sterols.     

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid-liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 x 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/z 854.4--> 286.2 transition of paclitaxel is measured versus that of the m/z 808.5--> 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2-500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (< or = 15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy < or = 15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.