Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolite (EDDP) in human plasma by liquid chromatography/mass spectrometry

A previously reported enantioselective LC-MS assay for the determination of (R)- and (S)-methadone [Met] and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine [EDDP] (the primary metabolite of Met) has been adapted for use in the simultaneous determination of the plasma concentrations of Met, EDDP, buprenorphine (Bu) and norbuprenorphine (norBu). All of the target compounds were separated within 15 min using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile: ammonium acetate buffer [10 mM, pH 7.0] in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min at 25 degrees C. Deuterium labeled compounds were used as internal standards [d4-Bu, d3-norBu, (R,S)-d3-Met and (R,S)-d3-EDDP] and linear relationships between peak height ratios and drug concentrations were obtained for Bu and norBu in the range 0.2-12 ng/ml with correlation coefficients greater than 0.999. The relative standard deviations (%R.S.D.) for the intra- and inter-day precision of the method were <4.5% and for accuracy was <4.0%. The method was validated and used to analyze plasma samples obtained from opioid dependent methadone-maintained adults enrolled in a research study.     

Molecular mimicry in Campylobacter jejuni: role of the lipo-oligosaccharide core oligosaccharide in inducing anti-ganglioside antibodies

Campylobacter jejuni is recognized as the most common identifiable pathogen associated with the development of Guillain-Barré syndrome (GBS), an acute autoimmune-mediated disease affecting the peripheral nervous system. The immune response to ganglioside-like structures in lipo-oligosaccharides (LOSs) of certain C. jejuni strains is thought to cross-react with human nerve gangliosides and induce GBS. To study the involvement of LOSs in the pathogenesis of Campylobacter-induced GBS, we created truncated LOS molecules by inactivating the waaF gene in a GBS-associated isolate of C. jejuni. Gas Chromatography-MS analysis of the waaF mutant LOSs revealed a marked reduction in sugar content, including sialic acid and galactose. GM1 and GD1a-like mimicry was not detected in the waaF mutant by Western blot analysis with cholera toxin B and anti-GD1a antibodies. Mice immunized with the waaF mutant failed to develop anti-GM1 or anti-GD1a antibodies. The waaF mutant also showed reduced adherence to and invasion of INT-407 cells. The results indicate that the LOS of C. jejuni HB93-13 is essential for adherence and invasion as well as for anti-ganglioside antibody induction.     

Sex differences in the onset of seasonal reproductive quiescence in hamsters

Day length is the primary cue used by many mammals to restrict reproduction to favourable spring and summer months, but it is unknown for any mammal whether the seasonal loss of fertility begins at the same time and occurs at the same rate in females and males; nor it established whether the termination of mating behaviour in males and females coincides with the loss of fertility. We speculated that females, owing to their greater energetic investment in reproduction, are the limiting sex in terminating offspring production in short days (SDs). Oestrous cycles and production of young were monitored in Syrian hamsters (Mesocricetus auratus) transferred from long days (LDs) to SDs. Females were mated to LD males after three to eight weeks of SD treatment; in a parallel experiment, males housed in SDs were mated to LD females. After five and eight weeks in SDs, at least twice as many males as females were fertile. Both males and females continued to copulate for several weeks after becoming infertile. The onset of seasonal infertility occurs earlier in females than males and the decline in fertility precedes the seasonal loss of mating behaviour in both sexes.     

Xanthatin and xanthinosin from the burs of Xanthium strumarium L. as potential anticancer agents

Xanthatin and xanthinosin, 2 sesquiterpene lactones isolated from the burs of Xanthiun strumarium L. (cocklebur), showed moderate to high in vitro cytotoxic activity in the human cancer cell lines WiDr ATCC (colon), MDA-MB-231 ATCC (breast), and NCI-417 (lung). Xanthatin and xanthinosin were purified as the result of a multi-screening bioassay-guided study of wild plant species of the family Asteraceae, collected from various sites in Saskatchewan, Canada. Seventy-five extracts at a single concentration of 100 microg/mL were evaluated for in vitro cytotoxicity to the human cancer cell lines used. The chloroform extract of Carduus nutans L. (nodding thistle) aerial parts (IC50, 9.3 microg/mL) and the hexane extract of Echinacea angustifolia DC. (narrow-leaved purple coneflower) root (IC50, 4.0 microg/mL) were moderately to highly cytotoxic to the lung cancer cell line. The chloroform extracts of X. strumarium L. burs and Tanacetum vulgare L. (tansy) aerial parts exhibited the highest cytotoxicity for all cell lines tested; their IC50 values, obtained from multidose testing, ranged from 0.1 to 6.2 microg/mL (X. strumarium) and from 2.4 to 9.1 microg/mL (T. vulgare). Further purification of the chloroform fraction of X. strumarium yielded xanthatin and xanthinosin in high yields. This is the first time that these compounds have been reported in the burs of X. strumarium. Their IC50 values are also reported herein.     

Short duration testosterone infusions maintain male sex behavior in Syrian hamsters

In most mammalian species, reduced androgen availability is associated with marked reductions in male sexuality; conversely, androgen replacement in castrated males restores sex behavior within a few weeks. Testosterone (T) pulse duration, amplitude, frequency, and inter-pulse interval may be as important as total amount of hormone in determining target tissue responsiveness. We remain ignorant of the number and duration of daily T pulses necessary and sufficient to sustain male mating behavior. An in-dwelling infusion system was employed to vary T-pulse frequencies and durations. Daily 4 h infusions of aqueous T (100 microg/0.064 ml) and twice daily 4 h pulses of T (each 50 microg/0.064 ml) were sufficient to maintain ejaculatory behavior of sexually experienced castrated hamsters for 11 weeks post-castration; castrated hamsters infused with vehicle ceased to display the ejaculatory pattern 3 weeks after gonadectomy. Circulating T concentrations of hormone-infused hamsters declined markedly 7 h after the termination of each infusion. These results establish that male sex behavior can be sustained with infusions of relatively low T concentrations for 4 h/day and suggests that the basal concentrations of T sustained by the gonad during inter-pulse intervals may not be necessary for maintenance of sex behavior. 4 h T infusions were sufficient to maintain penile and seminal vesicles weights, but not ventral prostate weights or flank gland dimensions; the threshold for maintaining male sex behavior is lower than that for some androgen-dependent peripheral structures. Development of effective androgen replacement regimens that sustain sex behavior in castrated animals may be useful in the design of androgen replacement therapy for hypogonadal men.     

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.     

Abundance and nightly activity behavior of a sylvan population of Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) from the Yucatan,México

Triatoma dimidiata is the vector of Trypanosoma cruzi in the Yucatan Peninsula (YP). Earlier studies have shown that domestic and peri‐domestic populations of the vector originated from the sylvan stock and that effectiveness of insecticide‐spraying was affected by re‐infestations of houses from the sylvan T. dimidiata population. In addition, in the YP most previously published reports have focused on domestic and peri‐domestic populations and very little is known about the nocturnal behavior of the sylvan populations. The main aim of our study was to determine the nightly activity patterns of adult T. dimidiata in a selected location in the YP. Secondly, we sought to document the reproductive status and infection rate of active females. During eight sampling nights spaced from late March to late July, 2007, we collected 544 adult T. dimidiata. We found that square‐cloth illuminated white traps were effective to attract the sylvan individuals and that T. dimidiata adults exhibited a unimodal activity pattern throughout the night. The accumulated mean of captured bugs also showed a non‐linear distribution for females and males. Furthermore, we found that male and female catches were significantly correlated with the means of temperature and humidity recorded during the sampling period. Out of 46 dissected females, we observed that 43.5% of females had fully‐formed eggs in their abdomens, and only two females (4.4%) had sperm within the spermatheca. The infection rate of T. dimidiata harboring T. cruzi was found to be 3.7%. The implications of the light attraction to bugs and potential dispersal capabilities are discussed in the paper in the context of infestation/re‐infestation of rural houses by sylvan T. dimidiata flying adults.     

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P₂ substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P₂ position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P₂ Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.     

Infrequent low dose testosterone treatment maintains male sexual behavior in Syrian hamsters

Testosterone (T) secreted in short pulses several times each day is essential for the maintenance of male sex behavior (MSB) in mammals. Blood T concentrations are relatively low during inter-pulse intervals. Assessment of androgenic influences on MSB of rodents has, with very few exceptions, involved either injections of pure or esterified hormones dissolved in oil or implantation of constant release capsules that generate supraphysiological and/or constantly elevated T concentrations. The minimum daily concentration of T necessary to maintain and restore MSB when T is delivered as a discrete short pulse remains unspecified; nor is it known whether infrequent T pulses in the physiological range sustain MSB. To address these questions, we varied T injection concentrations and frequencies in castrated, sexually-experienced Syrian hamsters. All males injected daily with an aqueous vehicle failed to display the ejaculatory reflex 5 weeks after castration. Once daily 15 μg subcutaneous T injections both maintained and restored MSB, whereas once daily 5 μg T injections resulted in fewer males ejaculating and longer ejaculation latencies. Substantially higher T doses were required to restore MSB in previous studies when T was administered in an oil vehicle. 50 μg T maintained MSB in most hamsters injected once every 4 or 7 days, despite long intervals between injections during which circulating T was undetectable or well below physiological concentrations. Some T regimens that maintained MSB were associated with subnormal seminal vesicle and ventral prostate weights. The demonstration that relatively brief, infrequent elevations of T are sufficient to support MSB provides a useful model to assess the neuroendocrine basis of MSB and raises the possibility that infrequent low dose androgen replacement protocols may restore sex behavior to hypogonadal men without inducing some of the negative side-effects associated with more frequent, higher dose treatments.