Prior exposure to indole-3-carbinol decreases the incidence of specific cyclophosphamide-induced developmental defects in mice

BACKGROUND: Indole-3-carbinol (I3C) is a product of the hydrolysis of glucobrassicin that is found in cruciferous vegetables. I3C can intervene in toxic processes that are mediated by oxidative mechanisms because it possesses the chemical and pharmacokinetic properties necessary to provide a free radical trap. Cyclophosphamide (CP) is a bifunctional alkylating agent known to produce DNA damage and to cause developmental toxicity, including malformations, in laboratory animals. METHODS: Pregnant CD-1 mice were given a 100 mg/kg dose of I3C 24 or 48 hr before administration of 20 mg/kg CP on gestation day 10 (GD 10). Controls were given the vehicle (DMSO), I3C, or CP. This regimen was carried out to determine if I3C could protect against the developmental toxicity of alkylating agents, such as CP. Dams were sacrificed on GD 17 and their litters were examined for adverse effects. RESULTS: Treatment with I3C 48 hr before CP administration was associated with decreased fetal limb and tail malformations. Limb malformation incidences were reduced from 42% litters affected in the CP control to 16% in the I3C/CP 48-hr treatment group, and tail malformations were reduced from 45% in the CP control to 16% in the I3C/CP 48-hr treatment group, indicating a protective effect of prior exposure to I3C. I3C given 24 hr before CP had no significant protective effect, while having an apparently adverse consequence with regard to the incidence of talipes. CONCLUSIONS: Exposure of a developing mammal to indole-3-carbinol before exposure to cyclophosphamide during organogenesis can influence the teratogenicity of cyclophosphamide.     

Incorporation of galactomannans in the diet of newly weaned piglets: effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11-12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log10 CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.     

Insights into recombination from patterns of linkage disequilibrium in humans

An ability to predict levels of linkage disequilibrium (LD) between linked markers would facilitate the design of association studies and help to distinguish between evolutionary models. Unfortunately, levels of LD depend crucially on the rate of recombination, a parameter that is difficult to measure. In humans, rates of genetic exchange between markers megabases apart can be estimated from a comparison of genetic and physical maps; these large-scale estimates can then be interpolated to predict LD at smaller ("local") scales. However, if there is extensive small-scale heterogeneity, as has been recently proposed, local rates of recombination could differ substantially from those averaged over much larger distances. We test this hypothesis by estimating local recombination rates indirectly from patterns of LD in 84 genomic regions surveyed by the SeattleSNPs project in a sample of individuals of European descent and of African-Americans. We find that LD-based estimates are significantly positively correlated with map-based estimates. This implies that large-scale, average rates are informative about local rates of recombination. Conversely, although LD-based estimates are based on a number of simplifying assumptions, it appears that they capture considerable information about the underlying recombination rate or at least about the ordering of regions by recombination rate. Using LD-based estimators, we also find evidence for homologous gene conversion in patterns of polymorphism. However, as we demonstrate by simulation, inferences about gene conversion are unreliable, even with extensive data from homogeneous regions of the genome, and are confounded by genotyping error.     

Crystal structure of quinone reductase 2 in complex with resveratrol

Resveratrol has been shown to have chemopreventive, cardioprotective, and antiaging properties. Here, we report that resveratrol is a potent inhibitor of quinone reductase 2 (QR2) activity in vitro with a dissociation constant of 35 nM and show that it specifically binds to the deep active-site cleft of QR2 using high-resolution structural analysis. All three resveratrol hydroxyl groups form hydrogen bonds with amino acids from QR2, anchoring a flat resveratrol molecule in parallel with the isoalloxazine ring of FAD. The unique active-site pocket in QR2 could potentially bind other natural polyphenols such as flavonoids, as proven by the high affinity exhibited by quercetin toward QR2. K562 cells with QR2 expression suppressed by RNAi showed similar properties as resveratrol-treated cells in their resistance to quinone toxicity. Furthermore, the QR2 knockdown K562 cells exhibit increased antioxidant and detoxification enzyme expression and reduced proliferation rates. These observations could imply that the chemopreventive and cardioprotective properties of resveratrol are possibly the results of QR2 activity inhibition, which in turn, up-regulates the expression of cellular antioxidant enzymes and cellular resistance to oxidative stress.     

A guide through present computational approaches for the identification of mammalian microRNA targets

Computational microRNA (miRNA) target prediction is a field in flux. Here we present a guide through five widely used mammalian target prediction programs. We include an analysis of the performance of these individual programs and of various combinations of these programs. For this analysis we compiled several benchmark data sets of experimentally supported miRNA-target gene interactions. Based on the results, we provide a discussion on the status of target prediction and also suggest a stepwise approach toward predicting and selecting miRNA targets for experimental testing.     

GEOGRAPHIC VARIATION IN THE SONGS OF NEOTROPICAL SINGING MICE: TESTING THE RELATIVE IMPORTANCE OF DRIFT AND LOCAL ADAPTATION

Patterns of geographic variation in communication systems can provide insight into the processes that drive phenotypic evolution. Although work in birds, anurans, and insects demonstrates that acoustic signals are sensitive to diverse selective and stochastic forces, processes that shape variation in mammalian vocalizations are poorly understood. We quantified geographic variation in the advertisement songs of sister species of singing mice, montane rodents with a unique mode of vocal communication. We tested three hypotheses to explain spatial variation in the song of the lower altitude species, Scotinomys teguina: selection for species recognition in sympatry with congener, S. xerampelinus, acoustic adaptation to different environments, and stochastic divergence. Mice were sampled at seven sites in Costa Rica and Panamá; genetic distances were estimated from mitochondrial control region sequences, between‐site differences in acoustic environment were estimated from climatic data. Acoustic, genetic and geographic distances were all highly correlated in S. teguina, suggesting that population differentiation in song is largely shaped by genetic drift. Contrasts between interspecific genetic‐acoustic distances were significantly greater than expectations derived from intraspecific contrasts, indicating accelerated evolution of species‐specific song. We propose that, although much intraspecific acoustic variation is effectively neutral, selection has been important in shaping species differences in song.     

In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.