Carcinogen-induced alteration of DNA structure

We have prepared covalent complexes between defined length DNA fragments and a diol epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene. We have studied the structure of these complexes, using transient electric dichroism, circular dichroism, fluorescence quenching, thermal denaturation, polyacrylamide gel electrophoresis, and nuclease digestion. Our observations suggest that th covalently bound carcinogen is intercalated within the helix, forming a wedge-shaped complex. Binding of the carcinogen distorts the structure of the DNA over a region extending beyond the immediate binding site. The most striking aspect of this distortion is that it produces a bend in the helix.     

Quantitative Analysis of Cell Migration Using Optical Flow

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Background selection and FST: Consequences for detecting local adaptation

Background selection is a process whereby recurrent deleterious mutations cause a decrease in the effective population size and genetic diversity at linked loci. Several authors have suggested that variation in the intensity of background selection could cause variation in F_ST across the genome, which could confound signals of local adaptation in genome scans. We performed realistic simulations of DNA sequences, using recombination maps from humans and sticklebacks, to investigate how variation in the intensity of background selection affects F_ST and other statistics of population differentiation in sexual, outcrossing species. We show that, in populations connected by gene flow, Weir and Cockerham's (1984; Evolution, 38 , 1358) estimator of F_ST is largely insensitive to locus‐to‐locus variation in the intensity of background selection. Unlike F_ST, however, d_XY is negatively correlated with background selection. Moreover, background selection does not greatly affect the false‐positive rate in F_ST outlier studies in populations connected by gene flow. Overall, our study indicates that background selection will not greatly interfere with finding the variants responsible for local adaptation.     

Dioxin-inducible transactivation in a chromosomal setting. Analysis of the acidic domain of the Ah receptor

We analyzed the transactivation function of the acidic segment of the Ah receptor (amino acids 515-583) by reconstituting AhR-defective mouse hepatoma cells with mutants. Our data reveal that both hydrophobic and acidic residues are important for transactivation and that these residues are clustered in two regions of the acidic segment of AhR. Both regions are crucial for function, because disruption of either one substantially impairs transactivation of the chromosomal CYP1A1 target gene. Neither region contains an amino acid motif that resembles those reported for other acidic activation domains. Furthermore, proline substitutions in both regions do not impair transactivation in vivo, a finding that implies that alpha-helix formation is not required for function.     

Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.     

Phylogeny of the core Malvales: evidence from ndhF sequence data

The monophyly of the group comprising the core malvalean families, Bombacaceae, Malvaceae, Sterculiaceae, and Tiliaceae, was recently confirmed by molecular studies, but the internal structure of this clade is poorly understood. In this study, we examined sequences of the chloroplast ndhF gene (aligned length 2226 bp) from 70 exemplars representing 35 of the 39 putative tribes of core Malvales. The monophyly of one traditional family, the Malvaceae, was supported in the trees resulting from these data, but the other three families, as traditionally circumscribed, are nonmonophyletic. In addition, the following relationships were well supported: (1) a clade, /Malvatheca, consisting of traditional Malvaceae and Bombacaceae (except some members of tribe Durioneae), plus Fremontodendron and Chiranthodendron, which are usually treated as Sterculiaceae; (2) a clade, /Malvadendrina, supported by a unique 21-bp (base pair) deletion and consisting of /Malvatheca, plus five additional subclades, including representatives of Sterculiaceae and Tiliaceae, and Durionieae; (3) a clade, /Byttneriina, with genera traditionally assigned to several tribes of Tiliaceae, plus exemplars of tribes Byttnerieae, Hermannieae, and Lasiopetaleae of Sterculiaceae. The most striking departures from traditional classifications are the following: Durio and relatives appear to be more closely related to Helicteres and Reevesia (Sterculiaceae) than to Bombacaceae; several genera traditionally considered as Bombacaceae (Camptostemon, Matisia, Phragmotheca, and Quararibea) or Sterculiaceae (Chiranthodendron and Fremontodendron) appear as sister lineages to the traditional Malvaceae; the traditional tribe Helictereae (Sterculiaceae) is polyphyletic; and Sterculiaceae and Tiliaceae, as traditionally circumscribed, represent polyphyletic groups that cannot sensibly be maintained with their traditional limits for purposes of classification. We discuss morphological characters and conclude that there has been extensive homoplasy in characters previously used to delineate major taxonomic groups in core Malvales. The topologies here also suggest that /Malvatheca do not have as a synapormophy monothecate anthers, as has been previously supposed but, instead, may be united by dithecate, transversely septate (polysporangiate) anthers, as found in basal members of both /Bombacoideae and /Malvoideae. Thus, “monothecate” anthers may have been derived at least twice, independently, within the /Bombacoideae (core Bombacaceae) and /Malvoideae (traditional Malvaceae).     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.     

Fixation probability and time in subdivided populations

New alleles arising in a population by mutation ultimately are either fixed or lost. Either is possible, for both beneficial and deleterious alleles, because of stochastic changes in allele frequency due to genetic drift. Spatially structured populations differ from unstructured populations in the probability of fixation and the time that this fixation takes. Previous results have generally made many assumptions: that all demes contribute to the next generation in exact proportion to their current sizes, that new mutations are beneficial, and that new alleles have additive effects. In this article these assumptions are relaxed, allowing for an arbitrary distribution among demes of reproductive success, both beneficial and deleterious effects, and arbitrary dominance. The effects of population structure can be expressed with two summary statistics: the effective population size and a variant of Wright's F(ST). In general, the probability of fixation is strongly affected by population structure, as is the expected time to fixation or loss. Population structure changes the effective size of the species, often strongly downward; smaller effective size increases the probability of fixing deleterious alleles and decreases the probability of fixing beneficial alleles. On the other hand, population structure causes an increase in the homozygosity of alleles, which increases the probability of fixing beneficial alleles but somewhat decreases the probability of fixing deleterious alleles. The probability of fixing new beneficial alleles can be simply described by 2hs(1 - F(ST))N(e)/N(tot), where hs is the change in fitness of heterozygotes relative to the ancestral homozygote, F(ST) is a weighted version of Wright's measure of population subdivision, and N(e) and N(tot) are the effective and census sizes, respectively. These results are verified by simulation for a broad range of population structures, including the island model, the stepping-stone model, and a model with extinction and recolonization.