Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice.

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Detection of α-, ß-, and γ-amanitin in urine by LC-MS/MS using 15N10-α-amanitin as the internal standard.

The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect α-, ß-, and γ-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N10-α-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N10-α-amanitin internal standard, precision and accuracy of α-amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1-200 ng/mL. ß- and γ-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0-200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled α-amanitin with the ability to detect and confirm human exposures to α-, ß-, and γ-amanitin.

Quantification of Microcystin-LR in Human Urine by Immunocapture Liquid Chromatography Tandem Mass Spectrometry.

Microcystins are toxins produced by many cyanobacteria species, which are often released into waterways during blue-green algal blooms in freshwater and marine habitats. The consumption of microcystin-contaminated water is a public health concern as these toxins are recognized tumor promoters and are hepatotoxic to humans and animals. A method to confirm human exposures to microcystins is needed; therefore, our laboratory has developed an immunocapture liquid chromatography tandem mass spectrometry (LC-MS/MS) method targeting the conserved adda portion of microcystins for the quantitation of a prevalent and highly toxic congener of microcystin, microcystin-LR (MC-LR). An acute exposure method was initially evaluated for accuracy and precision by analyzing calibrators and quality control (QC) samples ranging from 0.500 to 75.0 ng/mL in urine. All calibrators and QC samples characterized were within 15% of theoretical concentrations. An analysis of acutely exposed mouse urine samples using this method identified MC-LR levels from 10.7 to 33.9 ng/mL. Since human exposures are anticipated to result from low-dose or chronic exposures, a high-sensitivity method was validated with 20 calibration curves and QC samples ranging from 0.0100 to 7.50 ng/mL. Relative standard deviations (RSDs) and inaccuracies of these samples were within 15%, meeting United States Food and Drug Administration (FDA) guidelines for analytical methods, and the limit of detection was 0.00455 ng/mL. In conclusion, we have developed a method which can be used to address public health concerns by precisely and accurately measuring MC-LR in urine samples.

Quantification of saxitoxin in human blood by ELISA.

Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half-life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost-effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost-efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross-reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross-reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross-reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements.

Pheromone production in bark beetles.

The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids. The structural similarity between the major pheromone components of bark beetles and the monoterpenes found in the host trees, along with the association of monoterpenoid production with plant tissue, led to the paradigm that most if not all bark beetle pheromone components were derived from host tree precursors, often with a simple hydroxylation producing the pheromone. In the 1990 s there was a paradigm shift as evidence for de novo biosynthesis of pheromone components began to accumulate, and it is now recognized that most bark beetle monoterpenoid aggregation pheromone components are biosynthesized de novo. The bark beetle aggregation pheromones are released from the frass, which is consistent with the isoprenoid aggregation pheromones, including ipsdienol, ipsenol and frontalin, being produced in midgut tissue. It appears that exo-brevocomin is produced de novo in fat body tissue, and that verbenol, verbenone and verbenene are produced from dietary α-pinene in fat body tissue. Combined biochemical, molecular and functional genomics studies in Ips pini yielded the discovery and characterization of the enzymes that convert mevalonate pathway intermediates to pheromone components, including a novel bifunctional geranyl diphosphate synthase/myrcene synthase, a cytochrome P450 that hydroxylates myrcene to ipsdienol, and an oxidoreductase that interconverts ipsdienol and ipsdienone to achieve the appropriate stereochemistry of ipsdienol for pheromonal activity. Furthermore, the regulation of these genes and their corresponding enzymes proved complex and diverse in different species. Mevalonate pathway genes in pheromone producing male I. pini have much higher basal levels than in females, and feeding induces their expression. In I. duplicatus and I. pini, juvenile hormone III (JH III) induces pheromone production in the absence of feeding, whereas in I. paraconfusus and I. confusus, topically applied JH III does not induce pheromone production. In all four species, feeding induces pheromone production. While many of the details of pheromone production, including the site of synthesis, pathways and knowledge of the enzymes involved are known for Ips, less is known about pheromone production in Dendroctonus. Functional genomics studies are under way in D. ponderosae, which should rapidly increase our understanding of pheromone production in this genus. This chapter presents a historical development of what is known about pheromone production in bark beetles, emphasizes the genomic and post-genomic work in I. pini and points out areas where research is needed to obtain a more complete understanding of pheromone production.