Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

Recovery of aSasa tsuboiana population after mass flowering and death

The recovery process of aSasa tsuboiana population after a mass flowering and death in 1977 was investigated by 15 years of observation in the Hira Mountains, Kinki district, western Japan. Seed production was high (6600–13 800 seeds m⁻² inSasa plots and 3900 seeds m⁻² in a forest plot) but emergent seedling density was low (14–21 seedlings m⁻²), probably because of seed predation byMicrotus montebelli occurring between seed shedding and the next spring. The seedling density had decreased further by the next year and theS. tsuboiana population recovered from only a limited number of seedlings. In spite of such a low initial density, theS. tsuboiana population was able to regenerate successfully and attained the previous full stand height in 7–16 years.Miscantbus sinensis invaded and delayed the recovery ofS. tsuboiana in one plot, butS. tsuboiana became dominant as it caught up with the height ofM. sinensis. Seedling growth patterns, such as frequent tillering, the onset of rhizome extension in the early stage of seedling growth and frequent culm production from rhizomes, played important roles in the successful regeneration ofS. tsuboiana.     

ECOLOGICAL CORRELATES OF REPRODUCTIVE TRAITS OF MEXICAN RAIN FOREST TREES

Sexual systems of 139 tree species from a tropical rain forest at Los Tuxtlas, Mexico were investigated to: 1) estimate the relative proportions of hermaphroditic, monoecious, and dioecious species; 2) describe flowers, fruits, and seeds in terms of size and weight; 3) describe flowering and fruiting phenology; and 4) correlate sexuality to pollination and dispersal syndromes, and the successional status occupied in the forest. Hermaphroditism occurred in 63% of the species, monoecism in 9%, and dioecy in 27%. Nondioecious species had larger flowers, but dioecious species had more seeds per fruit. The dioecious condition was associated with small flowers pollinated with unspecialized insects and fleshy fruits dispersed by animals at both species and generic levels. Reproductive traits were more correlated among nondioecious species than dioecious species. Pioneer species had more seeds per fruit, and longer flowering and fruiting periods, but persistent species produced heavier seeds and fruits. Flower and fruit morphological traits, sexual systems, and tree guilds are related in a comprehensive way, and a flow model based on data from this study is presented.     

THE EVOLUTIONARY ECOLOGY OF AN ANTIPREDATOR REACTION NORM: DAPHNIA PULEX AND CHAOBORUS AMERICANUS

Ponds containing the parthenogenetic zooplankter Daphnia pulex with and without chaoborid predators were sampled over the course of a season. A significant (P < 0.05) Spearman rank correlation was found between predator density and the expression of an antipredator defense (neckteeth) by the Daphnia. The reaction norms (percent induction of a single genotype versus predator density) of clones isolated from predator-free and predator-rich habitats were determined in a laboratory setting. There was a statistically significant different response among the six clones tested (P < 0.05). Clones isolated from chaoborid ponds showed significantly greater sensitivity to the presence of predator than clones from predator-free ponds (P < 0.05). In the laboratory, food levels under which prey were cultured affected induction of the antipredator response. Highest induction was found at the lowest food level used.     

THE DISTRIBUTION OF LIFE-HISTORY VARIATION IN THE DAPHNIA PULEX COMPLEX

In the midwestern United States the Daphnia pulex complex consists of a mosaic of sexual and asexual populations, providing a useful model system for studying the evolutionary forces underlying the maintenance of sex. One asexual and two sexual populations were surveyed for genetic variation for isozymes, mitochondrial DNA, and life-history characters. While the sexual populations exhibited substantial levels of genetic variance for fitness characters, no variation was detected in the asexual population at any level. However, a parallel survey among asexual clones derived from other ponds revealed large amounts of quantitative variation among clones, even among those with the same molecular profile. As a group, the asexuals are more variable for life histories than are the sexual populations. The molecular data indicate a relatively recent origin for the extant asexual D. pulex. The polyphyletic origin of these clones, combined with their microevolutionary potential, provides an explanation for their broad geographic distribution. The distribution of sex in the complex cannot be explained with the standard models that assume an invariant asexual population in reproductive isolation from the parental species. Although the frequency of asexuality may be driven by the spread of a sex-limited meiosis suppressor through sexual populations, the complete displacement of sexuality may be prevented by ecological distinctions between the two classes of individuals. On average, the asexuals are larger but produce smaller clutches than the sexuals.     

The braincase and palate of the tetrapodomorph sarcopterygian mandageria fairfaxi: morphological variability near the fish–tetrapod transition

The braincase of the Late Devonian tristichopterid sarcopterygian Mandageria fairfaxi , from Canowindra, NSW, Australia, differs radically from the conservative pattern present in other 'osteolepiforms' (stem–group tetrapodomorph fishes) and non–dipnoan sarcopterygian fishes in general. The basioccipital region is short, displaced anteriorly, and either unossified or loosely articulated to the exoccipital, leaving most or all of the notochordal tunnel open ventrally. The exoccipital complex, which is developed into a large saddle that would have rested on top of the notochord, carries large, triangular articular facets on its posterior face and appears to have formed part of a functional neck joint, a synovial articulation between the skull and vertebral column that allows the former to rotate against the latter. Such a joint is characteristic of post–Devonian tetrapods, but unknown in other sarcopterygians. We infer that the ventrally open notochordal tunnel allowed gentle flexion of the cranial notochord during (predominantly vertical) rotational movement at the occiput; this is a mechanically unique solution to the problem of creating a mobile neck. Other unusual features of Mandageria include a posteriorly located lateral commissure, and structures on the entopterygoid and lateral commissure that may have been associated with an elaborate spiracular tract.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.