A pleiotropy scan to discover new susceptibility loci for pancreatic ductal adenocarcinoma.

Pleiotropic variants (i.e., genetic polymorphisms influencing more than one phenotype) are often associated with cancer risk. A scan of pleiotropic variants was successfully conducted ten years ago in relation to pancreatic ductal adenocarcinoma susceptibility. However, in the last decade, genetic association studies performed on several human traits have greatly increased the number of known pleiotropic variants. Based on the hypothesis that variants already associated with a least one trait have a higher probability of association with other traits, 61,052 variants reported to be associated by at least one genome wide association study (GWAS) with at least one human trait were tested in the present study consisting of two phases (discovery and validation), comprising a total of 16,055 pancreatic ductal adenocarcinoma (PDAC) cases and 212,149 controls. The meta-analysis of the two phases showed two loci (10q21.1-rs4948550 (P=6.52×10-5) and 7q36.3-rs288762 (P=3.03×10-5) potentially associated with PDAC risk. 10q21.1-rs4948550 shows a high degree of pleiotropy and it is also associated with colorectal cancer risk while 7q36.3-rs288762 is situated 28,558 base pairs upstream of the Sonic Hedgehog (SHH) gene, which is involved in the cell differentiation process and PDAC etiopathogenesis. In conclusion, none of the single nucleotide polymorphisms (SNPs) showed a formally statistically significant association after correction for multiple testing. However, given their pleiotropic nature and association with various human traits including colorectal cancer, the two SNPs showing the best associations with PDAC risk merit further investigation through fine mapping and ad hoc functional studies.

Germline BRCA2 K3326X and CHEK2 I157T mutations increase risk for sporadic pancreatic ductal adenocarcinoma.

Rare truncating BRCA2 K3326X (rs11571833) and pathogenic CHEK2 I157T (rs17879961) variants have previously been implicated in familial pancreatic ductal adenocarcinoma (PDAC), but not in sporadic cases. The effect of both mutations in important DNA repair genes on sporadic PDAC risk may shed light on the genetic architecture of this disease. Both mutations were genotyped in germline DNA from 2,935 sporadic PDAC cases and 5,626 control subjects within the PANcreatic Disease ReseArch (PANDoRA) consortium. Risk estimates were evaluated using multivariate unconditional logistic regression with adjustment for possible confounders such as sex, age and country of origin. Statistical analyses were two-sided with p values <0.05 considered significant. K3326X and I157T were associated with increased risk of developing sporadic PDAC (odds ratio (ORdom ) = 1.78, 95% confidence interval (CI) = 1.26-2.52, p = 1.19 × 10-3 and ORdom = 1.74, 95% CI = 1.15-2.63, p = 8.57 × 10-3 , respectively). Neither mutation was significantly associated with risk of developing early-onset PDAC. This retrospective study demonstrates novel risk estimates of K3326X and I157T in sporadic PDAC which suggest that upon validation and in combination with other established genetic and non-genetic risk factors, these mutations may be used to improve pancreatic cancer risk assessment in European populations. Identification of carriers of these risk alleles as high-risk groups may also facilitate screening or prevention strategies for such individuals, regardless of family history.

Determination of circular dichroism and ultraviolet spectral parameters of norgestimate- and other Delta(4)-3-ketosteroid oxime isomers via normal phase HPLC method.

The oxime formation reaction of therapeutical progestogen (levonorgestrel, levonorgestrel acetate, norethisterone), androgen (methyltestosterone, testosterone phenylpropionate) and anabolic (nortestosterone phenylpropionate) Delta(4)-3-ketosteroids has been investigated. The ketosteroid-hydroxylamine reaction was monitored by reversed phase HPLC system. It was established, that under the experimental conditions applied the oxime formation was complete within 2 h. The reaction leads to the formation of Z and E oxime isomers. The isomers of norgestimate (levonorgestrel 17-acetate oxime) and other Delta(4)-3-ketosteroid oximes have been separated by a new normal phase HPLC method. The identification (elution order assignation) and determination of the formation ratio of the isomers have been performed by 1H NMR spectroscopy on the basis of the chemical shift differences of 4-H signals. The on-line CD and UV spectra of the pure oxime isomers were recorded and then molar ellipticities and absorbances of the isomers were calculated in the wavelength range of 200-300 nm via parameter estimation method.

6-oxo-morphinane oximes: pharmacology, chemistry and analytical application.

The permanent therapeutic importance of morphine derivatives in pain treatment has inspired continual synthetic efforts to modify the rigid pentacyclic systems in search for new selective analgesic agents. As a result, several morphinane oximes have been synthesized recently, which have the additional advantage of possessing an oxime group that can provide a method for selective determination of opiate alkaloids in biological matrices. The oximes of hydrocodone and oxycodone have stronger analgesic effect than the parent ketones and they also proved to be effective in preventing the respiratory depressant and hypotensive actions of fentanyl. In this work a review is given on the present status of oxime pharmacology, chemistry and analysis and also the oxime and O-methyl oxime formation of 6-oxo-morphinanes with therapeutic interest (codeinone, oxycodone, hydrocodone and 14-OH-codeinone). The oxime formation was monitored by reversed-phase HPLC and the chromatographic properties of oxime isomers have been characterized. The assignation of the individual isomers isolated by preparative HPLC was performed by (1)H NMR spectroscopy based on the chemical shift differences of the 5-H signals. In this way the isomeric ratio in the oxime products could also be determined. It was found that in the case of Delta(7)-6-oxo-morphinanes, depending on the substituents, the formation of the Z-isomer highly dominates (73-96%) over that of the E-isomer. However, for the saturated 7,8-(dihydro) derivatives the E-isomer is definitely preferred (>98%). In conclusion of a survey on the theoretical background of oxime isomerism, the conformational differences between the saturated and unsaturated morphinane systems were found responsible for the different E/Z ratios. On the basis of the isomeric ratio and the on-line CD and UV spectra of the pure isomers, the molar ellipticities and absorbancies of the isomers were calculated by a parameter estimation method.

Evaluation of CD detection in an HPLC system for analysis of DHEA and related steroids.

The biological importance of dehydroepiandrosterone (DHEA) is reflected by the fact that DHEA is a crucial precursor of the biosynthesis of the steroidal sex hormones. Simultaneous separation of DHEA, dehydroepiandrosterone sulfate (DHEA-S), pregnenolone, androstenedione and testosterone has been accomplished by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) based on isocratic elution applying circular dichroism (CD) detection at 295 nm. Addition of tetrabutylammonium hydrogensulfate to the mobile phase increases the retention of DHEA-S on the C8-silica column by an apparent ion-pairing mechanism without affecting the retention of the other (non-ionic) steroids. CD spectroscopy provides highly selective detection of compounds possessing optically active absorption bands and the separation is even more selective in the higher wavelength range applied. The linearity of the steroid concentration (c, mg mL(-1)) versus peak area was tested in the concentration range of 0.5-2 mg mL(-1) (injected quantities were 10-40 microg). The relative standard deviation (RSD) values for DHEA and DHEA-S indicated a good intra-assay and inter-assay precision of the method.

Preference for magnesium sulfate-treated leguminous seeds in egg-laying bean weevil (Acanthoscelides obtectus say, col., bruchidae).

Unlike many secondary plant substances, a wide range of concentrations (4-1000 mM) of magnesium sulfate, applied to dry beans, significantly increased egg-laying by the dry bean weevil in binary choice tests, in favor of treated seeds. No other magnesium-containing compounds studied exerted such an effect, nor was a similar response noted on treated beans in no-choice situations. The total number of eggs laid per female was in the same range in both types of test. Variably enhanced or suppressed oviposition responses were shown on magnesium sulfate-treated secondary hosts and on nonhosts or on indifferent substrates. No specific behavior by egg-laying bean weevil females on Mg-treated seeds could be detected. The results are explained by assuming the functioning of magnesium as a supernormal stimulus for egg-laying. However, a physiological effect on neuromuscular synaptic transmission, as a consequence of probable Mg uptake resulting in a decreased propensity to move, is also hypothesized.

Fate of quinolizidine alkaloids through three trophic levels:Laburnum anagyroides (Leguminosae) and associated organisms.

The quinolizidine alkaloids (QA) of golden rain,Laburnum anagyroides, and those of phytophagous insects associated with the plant, as well as of parasitoids of the latter, were analyzed by capillary GLC and GLC-MS. The alkaloid content in samples of vegetative plant parts was high at the beginning of the season, then decreased, while that of reproductive organs was high throughout flowering, pod formation, and maturation. The analyses showed that the QA of the plant passed through two higher trophic levels (herbivorous insects and their parasitoids) and that the alkaloid pattern changed little during the passage. The alkaloids were present in two phytophagous insect species associated with golden rain: the predispersal seed predator,Bruchidius villosus [5-13µg/g fresh weight (fw)], andAphis cytisorum (182-1012µg/g fw), an aphid that feeds on shoots, leaves, and inflorescences. Braconid and chalcidoid parasitoids emerging from the bruchid host also contained alkaloids (1.3-3µg/g fw), as did three foraging ant species,Lasius niger, Formica rufibarbis, andF. cunicularia (45µg/g fw), that visited the aphid colonies or honeydew-covered leaves of aphid-infested plants. The hypothesis that developing bruchid larvae and/or the plant "manipulate" QA supply to infested seeds was not supported, because QA content of leftover endosperm in seeds after bruchid development was similar to that of uninfested seeds. The frass of developing bruchid larvae was rich in QA (31µg/ g dry weight). While aphids sequestered, the bruchid larvae took up and eliminated QA with the frass without chemical transformation.

Determination of delta4-3-ketosteroids based on oxime formation by difference circular dichroism spectroscopy.

delta4-3-Ketosteroids exhibit an intensive negative Cotton effect on the circular dichroism (CD) spectra in the wavelength range for the n-pi* electronic transition (270-350 nm). With hydroxylamine hydrochloride, delta4-3-ketosteroid compounds can be transformed into oxime derivatives. Following oxime formation, positive ellipticity with low intensity can be registered in this wavelength range. The quantitative determination of delta4-3-ketosteroids is based on the considerable difference between the ellipticities before and after oxime formation. The difference ellipticity for the six ketosteroids examined (norethisterone, levonorgestrel, levonorgestrel acetate, methyltestosterone, testosterone phenylpropionate, nortestosterone phenylpropionate) varies linearly with the concentration in the interval 6 x 10(-6)-3 x 10(-3) mol/L. The method can be well applied to determination of delta4-3-ketosteroid contamination of norgestimate [(+)-13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn4-en-20-yn-3-one oxime acetate]; 0.02-10% impurity can be measured.

Determination of 6-oxo-morphinans, as the oximes, by difference circular dichroism spectroscopy.

A negative Cotton effect is observed in the circular dichroism (CD) spectra of 6-oxo-morphinans in the wavelength range of n-pi* electron transitions. 6-oxo-Morphinans can be transformed into oxime derivatives with hydroxylamine and after oxime formation the CD spectra are significantly different. Oxime formation was monitored by CD and by HPLC. It was established that under the experimental conditions used oxime formation was complete within 90 min. The method suggested for the determination of 6-oxo-morphinans is based on the considerable differences between the ellipticities before and after the oxime formation. The ellipticity difference varies linearly with concentration in the range 2 x 10(-5)-5 x 10(-4) mol L(-1) for the three 6-oxo-morphinans examined (oxycodone, hydrocodone, and 14-hydroxycodeinone). For hydrocodone the dependence is also linear in a lower concentration range (5 x 10(-6)-10(-4) mol L(-1)). The new difference CD spectroscopic method can be applied to the selective determination of 6-oxo-morphinans in bulk and dosage forms.