Measurement of free drug and clinical end-point by high-performance liquid chromatography-mass spectrometry: Applications and implications for pharmacokinetic and pharmacodynamic studies

Free drug measurement and pharmacodymanic markers provide the opportunity for a better understanding of drug efficacy and toxicity. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) is a powerful analytical technique that could facilitate the measurement of free drug and these markers. Currently, there are very few published methods for the determination of free drug concentrations by HPLC-MS. The development of atmospheric pressure ionisation sources, together with on-line microdialysis or on-line equilibrium dialysis and column switching techniques have reduced sample run times and increased assay efficiency. The availability of such methods will aid in drug development and the clinical use of certain drugs, including anti-convulsants, anti-arrhythmics, immunosuppressants, local anaesthetics, anti-fungals and protease inhibitors. The history of free drug measurement and an overview of the current HPLC-MS applications for these drugs are discussed. Immunosuppressant drugs are used as an example for the application of HPLC-MS in the measurement of drug pharmacodynamics. Potential biomarkers of immunosuppression that could be measured by HPLC-MS include purine nucleoside/nucleotides, drug-protein complexes and phosphorylated peptides. At the proteomic level, two-dimensional gel electrophoresis combined with matrix-assisted laser desorption/ionisation time-of-flight (TOF) MS is a powerful tool for identifying proteins involved in the response to inflammatory mediators. (C) 2003 Elsevier Science B.V. All rights reserved.

Feeding ecology of the fish ectoparasite Gnathia sp. (Crustacea: Isopoda) from the Great Barrier Reef, and its implications for fish cleaning behaviour

The feeding rate of a parasitic gnathiid isopod on fish was examined. Individual fish, Hemigymnus melapterus, were exposed to gnathiid larvae and sampled after 5, 10, 30, 60, and 240 min. I recorded whether larvae had an engorged gut, an engorged gut containing red material, or had dropped off the fish after having completed engorgement; variation among sampling times and larval stages was analyzed using generalized linear mixed model analyses. The likelihood that larvae had an engorged gut increased with time and varied with larval stage. First stage (1.45 mm) larvae. After 30 min, however, most (>93%) larvae had an engorged gut regardless of their larval stage. The likelihood of red material in the gut of third stage larvae increased over time (46% after 30 min, 70% after 60 min, and 86% after 240 min) while that of first and second stage larvae remained relatively low (

Responses to neighbours and non-neighbours in the buff-banded rail (Gallirallus philippensis): no dear-enemy relationships

Territorial individuals often respond less aggressively to intrusions by their neighbours than to intrusions by unfamiliar individuals. This commonly observed strategy, the dear-enemy phenomenon, is thought to minimise the costs associated with territory defence. This study determined whether an insular population of territory-holding buff-banded rails (Gallirallus philippensis) exhibits dear-enemy relationships and whether males and females differ in their responses to neighbours and non-neighbours. Playback techniques were used to examine and compare the vocalisations and movements of focal individuals in response to the territorial advertisement calls of their neighbours and of unfamiliar individuals (non-neighbours). Results showed that focal birds did not respond differently to the calls of neighbours and non-neighbours; thus, no evidence for the dear-enemy phenomenon was found in this species. We suggest that this result is due to the significant threats posed by neighbours to territory owners in this territorial system, in addition to a high level of territorial instability in the population. Males and females were shown to differ qualitatively in their general territorial response to intruders but not in their relative responses to neighbours and non-neighbours, suggesting that the relative threats posed by neighbours and non-neighbours do not differ between the sexes.

Reciprocal N ((NH4+)-N-15 or (NO3-)-N-15) transfer between nonN(2)-fixing Eucalyptus maculata and N-2-fixing Casuarina cunninghamiana linked by the ectomycorrhizal fungus Pisolithus sp.

Two-way N transfers mediated by Pisolithus sp. were examined by excluding root contact and supplying (NH4+)-N-15 or (NO3-)-N-15 to 6-month-old Eucalyptus maculata or Casuarina cunninghamiana grown in two-chambered-pots separated by 37 m screens. Mycorrhizal colonization was 35% in Eucalyptus and 66% in Casuarina (c. 29% N-2-fixation). Using an environmental scanning electron microscope, living hyphae were observed to interconnect Eucalyptus and Casuarina. Biomass and N accumulation was greatest in nodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, less in nonnodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, and least in nonnodulated nonmycorrhizal Casuarina/nonmycorrhizal Eucalyptus pairs. In nonnodulated mycorrhizal pairs, N transfers to Eucalyptus or to Casuarina were similar (2.4-4.1 mg per plant in either direction) and were 2.6-4.0 times greater than in nonnodulated nonmycorrhizal pairs. In nodulated mycorrhizal pairs, N transfers were greater to Eucalyptus (5-7 times) and to Casuarina (12-18 times) than in nonnodulated mycorrhizal pairs. Net transfer to Eucalyptus or to Casuarina was low in both nonnodulated nonmycorrhizal (< 0.7 mg per plant) and nonnodulated mycorrhizal pairs (< 1.1 mg per plant). In nodulated mycorrhizal pairs, net transfer to Casuarina was 26.0 mg per plant. The amount and direction of two-way mycorrhiza-mediated N transfer was increased by the presence of Pisolithus sp. and Frankia, resulting in a net N transfer from low-N-demanding Eucalyptus to high-N-demanding Casuarina.

The concurrent validity and test-retest reliability of a visuospatial working memory task

To determine whether the visuospatial n-back working memory task is a reliable and valid measure of cognitive processes believed to underlie intelligence, this study compared the reaction times and accuracy of perforniance of 70 participants, with performance on the Multidimensional Aptitude Battery (MAB). Testing was conducted over two sessions separated by 1 week. Participants completed the MAB during the second test session. Moderate testretest reliability for percentage accuracy scores was found across the four levels of the n-back task, whilst reaction times were highly reliable. Furthermore, participants' performance on the MAB was negatively correlated with accuracy of performance at the easier levels of the n-back task and positively correlated with accuracy of performance at the harder task levels. These findings confirm previous research examining the cognitive basis of intelligence, and suggest that intelligence is the product of faster speed of information processing, as well as superior working memory capacity. (C) 2004 Elsevier Inc. All rights reserved.

Perceived dissimilarity and work group involvement: The moderating effects of group openness to diversity

Perceived dissimilarity and its association with work group involvement were examined in this study. Additionally, perceived group openness to diversity was examined as a moderator of this relationship. A longitudinal study was conducted with nurses in four departments of a public hospital. Results revealed that visible dissimilarity was negatively associated with work group involvement at both times, and informational dissimilarity was negatively associated with work group involvement at Time 1. Openness to diversity interacted with visible and informational dissimilarity in the prediction of work group involvement at both times. This interaction pattern showed that there was a negative relationship between dissimilarity and work group involvement when individuals perceived low group openness to diversity, whereas there was no relationship when individuals perceived high group openness to diversity. Results highlight the importance of managing perceptions of difference and introducing norms that encourage the active involvement of group members.

Design, development, and use of molecular primers and probes for the detection of Gluconacetobacter species in the pink sugarcane mealybug

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of squashed whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.

Understanding the phylogeographic patterns of European hedgehogs, Erinaceus concolor and E-europaeus using the MHC

The genome of the European hedgehog, Erinaceus concolor and E. europaeus, shows a strong signal of cycles of restriction to glacial refugia and postglacial expansion. Patterns of expansion, however, differ for mitochondrial DNA (mtDNA) and preliminary analysis of nuclear markers. In this study, we determine phylogeographic patterns in the hedgehog using two loci of the major histocompatibility complex (MHC), isolated for the first time in hedgehogs. These genes show long persistence times and high polymorphism in many species because of the actions of balancing selection. Among 84 individuals screened for variation, only two DQA alleles were identified in each species, but 10 DQB alleles were found in E. concolor and six in E. europaeus. A strong effect of demography on patterns of DQB variability is observed, with only weak evidence of balancing selection. While data from mtDNA clearly subdivide both species into monophyletic subgroups, the MHC data delineate only E. concolor into distinct subgroups, supporting the preliminary findings of other nuclear markers. Together with differences in variability, this suggests that the refugia history and/or expansion patterns of E. concolor and E. europaeus differ.

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N2O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. NO accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as NO. After mixing SNDPR sludge with other denitrifying sludge, N2O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N2O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source. (C) 2005 Elsevier B.V. All rights reserved.

The effectiveness of heat, cold and 6-dimethylaminopurine shocks for inducing tetraploidy in the Kuruma shrimp, Marsupenaeus japonicus (Bate)

In this study tetraploid Marsupenaeus japonicus (Bate) embryos were produced by preventing the first division in mitosis. The effectiveness of temperature and chemical shocks for producing tetraploid M. japonicus were assessed when applied at different times postspawning and for different durations. Tetraploid M. japonicus embryos (spawned at 27 degrees C) were produced by heat shocks at 35 degrees C and 36 degrees C in three and eight spawning samples respectively, and a cold shock at 5 degrees C in a single spawning sample. All temperature shocks inducing tetraploidy were applied 18-23 min postspawning for a 5-10 min duration. The percentage of spawnings successfully inducing tetraploid embryos (i.e., frequency of induction) ranged from 33.33% to 66.67% for the 21, 22 and 23 min postspawning heat shock treatment regimes. The percentage of tetraploid embryos within an induction (i.e., induction rate), as determined by flow cytometry, ranged from 8.82% to 98.12% (ave. S.E.) (34.4 +/- 21.4%) for the 35 degrees C shock treatments, from 13.12% to 61.02% (35.0 +/- 5.0%) for the 36 degrees C shock treatments and was 15% for the 5 degrees C cold shock treatment. No tetraploids were produced for spawnings that received heat shocks above 36 degrees C or below 35 degrees C, or for cold shocks above 5 degrees C for any of the tested postspawning treatment and duration times. Chemical shock with 150 mu M 6-dimethylaminopurine did not result in tetraploid M. japonicus embryos at any of the tested postspawning treatment times and durations. Tetraploid M. japonicus embryos were nonviable, with no tetraploid larvae being detected by flow cytometry. Based on our results heat shocking of M. japonicus embryos at 36 degrees C, 23 min postspawning for a 5-10 min duration is the most effective means to produce tetraploids through inhibition of the first mitotic division (taking into consideration the importance of frequency and induction rate equally).

A comparison of the effect of high- and low-dose fentanyl on the incidence of postoperative cognitive dysfunction after coronary artery bypass surgery in the elderly

Background. Postoperative cognitive dysfunction (POCD) after coronary artery bypass graft surgery is a common complication for which, despite many clinical investigations, no definitive etiology has been found. The current use of both high and low-dose fentanyl as anesthetic techniques allowed us to investigate the effect of fentanyl on the incidence of POCD. Methods. Three hundred fifty patients scheduled to undergo elective coronary artery bypass graft surgery were randomized to receive either high-dose fentanyl (50 mu g/kg) or low-dose fentanyl (10 mu g/kg) as the basis of the anesthetic. All patients underwent neuropsychological testing before surgery and at 1 week, 3 months, and 12 months after surgery. Results. One hundred sixty-eight patients in the low-dose group and 158 patients in the high-dose group were included in the final analysis. Neuropsychological testing was performed on 88%, 93%, and 92% of patients at 1 week, 3 months, and 12 months, respectively. There was no difference between group mean scores at any of the three testing times. Analysis of individual patients by the 20% rule did not detect any differences between groups. The one SD rule, which has fewer false-positive results, detected significantly more patients with POCD in the low-dose group than in the high-dose group at 1 week (23.6% vs. 13.7%; P = 0.03) but not at the other testing times. Patients with POCD spent an average of 1.2 days longer in the hospital than those without POCD (P = 0.021). Conclusions: High-dose fentanyl is not associated with a difference in the incidence of POCD at 3 or 12 months after surgery. Low-dose fentanyl leads to shorter postoperative ventilation times and may be associated with a greater incidence of POCD 1 week after surgery. Early POCD is associated with an increased duration of stay in the hospital.