Extrapolation of Rodent Studies on Amniotic Fluid Contaminatnts to Human Populations

If endocrine active chemicals (EACs) adversely affect human development, then there must be evidence of effects in animal models at properly scaled levels of exposure during pertinent sensitive periods as derived from quantified exposures of the human fetus. Our recent studies attempt to address both effects and exposures. First Study: Dams were gavaged from Gestation Day (GD) 14 through weaning on Post-Natal Day (PND) 21 with either corn oil alone (unexposed controls) or Low DES (0.5?mg/kg BW); High DES (5.0?mg/kg BW); GEN (15?mg/kg BW); GEN + DES (GEN at 15?mg/kg BW and DES at 0.5?mg/kg BW). No treatments affected duration of gestation, litter size or birth anogenital distance / birth body weights ((bAGD/bBW) or ratios of bAGD/cube root of bBW of pups of either sex. The ratio of weaning AGD to weaning body weight (wAGD/wBW) differed significantly between the control group and each of the estrogenic treatments in both sexes with larger wAGD/wBW values associated with each of the estrogenic treatments. Males exposed to High DES and GEN alone exhibited earlier onset of puberty. Only females in the low DES group showed an earlier onset of puberty. At 50 to 70 days of age, the ratios of male reproductive organ weights/body weight were unaffected by estrogen treatment in all groups except high DES which increased testicle weight and decreased epididymis, seminal vesicle, and prostate weights. Initial vaginal cycle lengths were affected only in the high DES group. Thus low doses of DES and GEN at levels comparable to the upper range of human exposure affect some but not all markers of sexual development. Second Study: Amniotic fluid samples obtained at routine amniocentesis between 15 and 23 weeks of gestation were assayed by gas chromatographic/mass spectrometric (GC/MS) analysis. The first group of amniotic fluid samples (n = 53) from 51 women were analysed for several xenobiotic EACs. Alpha-hexachlorocyclohexane, with a mean (± SD) concentration of 0.15 ± 0.06 (ng/ml), and p,p′-DDE, with a mean (± SD) concentration of 0.21 ± 0.18?ng/ ml, were detected in several specimens. Overall one in three amniotic fluid samples tested positive for at least one xenobiotic EAC. Another group of amniotic fluid samples (n = 62) from 56 women were analysed for phytoestrogenic EACs. The mean (± SD) concentration of daidzein and genistein in amniotic fluid were 1.14 ± 1.04 and 1.37 ± 1.00?ng/ml with maximum levels of 5.52 and 4.86?ng/ml, respectively. Overall, 26 and 34 of the samples had quantifiable levels of daidzein and genistein, respectively. Conclusions: One in three human fetuses were exposed to xenobiotic EACs and two of three human fetuses were exposed to phytoestrogenic EACs in utero. Our demonstrations that EACs have developmental effects in an animal model at levels of exposure that mimic those found in humans in North America during sensitive time-frames sustains concerns about potential adverse health effects of developmental exposures to EACs for the human fetus/neonate.     

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Most recent common ancestor probability distributions in gene genealogies under selection

A computational study is made of the conditional probability distribution for the allelic type of the most recent common ancestor in genealogies of samples of n genes drawn from a population under selection, given the initial sample configuration. Comparisons with the corresponding unconditional cases are presented. Such unconditional distributions differ from samples drawn from the unique stationary distribution of population allelic frequencies, known as Wright's formula, and are quantified. Biallelic haploid and diploid models are considered. A simplified structure for the ancestral selection graph of S. M. Krone and C. Neuhauser (1997, Theor. Popul. Biol. 51, 210-237) is enhanced further, reducing the effective branching rate in the graph. This improves efficiency of such a nonneutral analogue of the coalescent for use with computational likelihood-inference techniques.     

Simulation of selected genealogies

Algorithms for generating genealogies with selection conditional on the sample configuration of n genes in one-locus, two-allele haploid and diploid models are presented. Enhanced integro-recursions using the ancestral selection graph, introduced by S. M. Krone and C. Neuhauser (1997, Theor. Popul. Biol. 51, 210-237), which is the non-neutral analogue of the coalescent, enables accessible simulation of the embedded genealogy. A Monte Carlo simulation scheme based on that of R. C. Griffiths and S. Tavaré (1996, Math. Comput. Modelling 23, 141-158), is adopted to consider the estimation of ancestral times under selection. Simulations show that selection alters the expected depth of the conditional ancestral trees, depending on a mutation-selection balance. As a consequence, branch lengths are shown to be an ineffective criterion for detecting the presence of selection. Several examples are given which quantify the effects of selection on the conditional expected time to the most recent common ancestor.     

Phylogeography of Bufo marinus from its natural and introduced ranges.

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

Microbial metabolism of cannflavin A and B isolated from Cannabis sativa

Microbial metabolism of cannflavin A (1) and B (2), two biologically active flavonoids isolated from Cannabis sativa L., produced five metabolites (3–7). Incubation of 1 and 2 with Mucor ramannianus (ATCC 9628) and Beauveria bassiana (ATCC 13144), respectively, yielded 6″S,7″-dihydroxycannflavin A (3), 6″S,7″-dihydroxycannflavin A 7-sulfate (4) and 6″S,7″-dihydroxycannflavin A 4′-O-α-l-rhamnopyranoside (5), and cannflavin B 7-O-β-d-4?-O-methylglucopyranoside (6) and cannflavin B 7-sulfate (7), respectively. All compounds were evaluated for antimicrobial and antiprotozoal activity.     

Oestrous state dynamics in chemical communication by captive female Asian elephants

In many mammals, reproductive status is revealed through chemical cues in urine. The reproductive status of receivers may influence their interest in such signals. For social mammals that live in matrilineal groups, females may benefit by detecting the reproductive condition of herdmates. Responses to urine during oestrous cycles of senders and receivers are potential indicators of signal functions. We examined the chemosensory responses, first by four captive female Asian elephants,Elephas maximus , over their oestrous cycles to familiar follicular and luteal phase urine and second by 14 different female Asian elephants to unfamiliar conspecific follicular and luteal phase urine. We asked whether females could distinguish the reproductive state of another female as measured by their differential response to luteal- and follicular-phase urine. We further examined the influence of the receiver's reproductive status on response levels. Females responded more with specific tactolfactory trunk behaviours to follicular- than to luteal-phase urine, but only when the receiving female was in her follicular phase. Like their male conspecifics, Asian elephant females can detect changes in the reproductive state of conspecifics. The functional significance of this ability has yet to be determined but may be related more to the resource holding power of females in follicular phase than to a means for females to synchronize oestrous cycles. Such female-female communication may have important effects on social group dynamics. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Tropical dung beetle morphological traits predict functional traits and show intraspecific differences across land uses

Functional traits and functional diversity measures are increasingly being used to examine land use effects on biodiversity and community assembly rules. Morphological traits are often used directly as functional traits. However, behavioral characteristics are more difficult to measure. Establishing methods to derive behavioral traits from morphological measurements is necessary to facilitate their inclusion in functional diversity analyses. We collected morphometric data from over 1,700 individuals of 12 species of dung beetle to establish whether morphological measurements can be used as predictors of behavioral traits. We also compared morphology among individuals collected from different land uses (primary forest, logged forest, and oil palm plantation) to identify whether intraspecific differences in morphology vary among land use types. We show that leg and eye measurements can be used to predict dung beetle nesting behavior and period of activity and we used this information to confirm the previously unresolved nesting behavior for Synapsis ritsemae. We found intraspecific differences in morphological traits across different land use types. Phenotypic plasticity was found for traits associated with dispersal (wing aspect ratio and wing loading) and reproductive capacity (abdomen size). The ability to predict behavioral functional traits from morphology is useful where the behavior of individuals cannot be directly observed, especially in tropical environments where the ecology of many species is poorly understood. In addition, we provide evidence that land use change can cause phenotypic plasticity in tropical dung beetle species. Our results reinforce recent calls for intraspecific variation in traits to receive more attention within community ecology.     

Sources of Variability in the Measurement of Fungal Spore Yields

Variability in the production of fungal spores and in the measurement of spore yields was investigated in four species of fungi: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean ± 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.