Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.     

Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.     

Ascorbate inhibits edema in brain slices

Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol. Slices of mammalian brain rapidly lose ascorbate, however, when incubated in ascorbate-free media; brain slices also take up water and swell. Here we investigated water gain in coronal slices of rat forebrain incubated with and without ascorbate for 1-3 h at 34 degrees C. Slices progressively gained water in ascorbate-free media, with a significant 12% water increase after 3 h at 34 degrees C, compared with the water content of slices after a 1-h recovery period at 24 degrees C, immediately following slice preparation. Inclusion of 400 micro M ascorbate in the medium led to an increase in tissue ascorbate content and prevented water gain at 34 degrees C. By contrast, water gain was not inhibited by isoascorbate or thiourea, which are antioxidants that are not accumulated in brain cells. The oxidant H2O2 enhanced water gain, whereas a cocktail of NMDA and non-NMDA receptor blockers inhibited edema formation to the same extent as ascorbate. These data demonstrate that brain edema, linked to glutamate-receptor activation, can result from intracellular oxidative stress and that this can be prevented by ascorbate.     

The Caspase-3 Precursor Has a Cytosolic and Mitochondrial Distribution: Implications for Apoptotic Signaling

Caspase-3–mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.     

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABA_A receptors (GABA_ARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABA_ARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABA_ARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.     

NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat

The metabolic profiles of three wild mammals that vary in their trophic strategies, the herbivorous bank vole (Clethrionomys glareolus), the granivorous wood mouse (Apodemus sylvaticus), and the insectivorous white-toothed shrew (Crocidura suaveolens), were compared with that of a widely used strain of laboratory rat (Sprague Dawley). In conjunction with NMR spectroscopic investigations into the urine and blood plasma composition for these mammals, high resolution magic angle spinning (HRMAS) 1H-nuclear magnetic resonance (NMR) spectroscopy was applied to investigate the composition of intact kidney samples. Adaptation to natural diet affects both renal metabolism and urinary profiles, and while these techniques have been used to study the metabolism of the laboratory rat little is known about wild small mammals. The species were readily separated by their urinary profiles using either crude metabolite ratios or statistical pattern recognition. Bank vole urine contained higher concentrations of aromatic amino acids compared with the other small mammals, while the laboratory rats produced relatively more hippurate. HRMAS 1H-NMR demonstrated striking differences in both lipid concentration and composition between the wild mammals and Sprague Dawley rats. Bank voles contained high concentrations of the aromatic amino acids phenylalanine, tyrosine and tryptophan in all tissue and biofluids studied. This study demonstrates the analytical power of combined NMR techniques for the study of inter-species metabolism and further demonstrates that metabolic data acquired on laboratory animals cannot be extended to wild species.     

Transgenomic metabolic interactions in a mouse disease model: interactions of Trichinella spiralis infection with dietary Lactobacillus paracasei supplementation

Irritable Bowel Syndrome (IBS) is a common multifactorial intestinal disorder for which the aetiology remains largely undefined. Here, we have used a Trichinella spiralis (T. spiralis)-induced model of post-infective IBS, and the effects of probiotic bacteria on gut dysfunction have been investigated using a metabonomic strategy. A total of 44 mice were divided into four groups: an uninfected control group and three T. spiralis-infected groups, one as infected control and the two other groups subsequently treated with either Lactobacillus paracasei (L. paracasei) NCC2461 in spent culture medium (SCM) or with L. paracasei-free SCM. Plasma, jejunal wall and longitudinal myenteric muscle samples were collected at day 21 post-infection. An NMR-based metabonomic approach characterized that the plasma metabolic profile of T. spiralis-infected mice showed an increased energy metabolism (lactate, citrate, alanine), fat mobilization (acetoacetate, 3-D-hydroxybutyrate, lipoproteins) and a disruption of amino acid metabolism due to increased protein breakdown, which were related to the intestinal hypercontractility. Increased levels of taurine, creatine and glycerophosphorylcholine in the jejunal muscles were associated with the muscular hypertrophy and disrupted jejunal functions. L. paracasei treatment normalized the muscular activity and the disturbed energy metabolism as evidenced by decreased glycogenesis and elevated lipid breakdown in comparison with untreated T. spiralis-infected mice. Changes in the levels of plasma metabolites (glutamine, lysine, methionine) that might relate to a modulation of immunological responses were also observed in the presence of the probiotic treatment. The work presented here suggests that probiotics may be beneficial in patients with IBS.     

Evolution of population structure in a highly social top predator, the killer whale

Intraspecific resource partitioning and social affiliations both have the potential to structure populations, though it is rarely possible to directly assess the impact of these mechanisms on genetic diversity and population divergence. Here, we address this for killer whales (Orcinus orca), which specialize on prey species and hunting strategy and have long-term social affiliations involving both males and females. We used genetic markers to assess the structure and demographic history of regional populations and test the hypothesis that known foraging specializations and matrifocal sociality contributed significantly to the evolution of population structure. We find genetic structure in sympatry between populations of foraging specialists (ecotypes) and evidence for isolation by distance within an ecotype. Fitting of an isolation with migration model suggested ongoing, low-level migration between regional populations (within and between ecotypes) and small effective sizes for extant local populations. The founding of local populations by matrifocal social groups was indicated by the pattern of fixed mtDNA haplotypes in regional populations. Simulations indicate that this occurred within the last 20,000 years (after the last glacial maximum). Our data indicate a key role for social and foraging behavior in the evolution of genetic structure among conspecific populations of the killer whale.     

Diagnostic sensitivity and specificity of pulmonary cytology: comparison of techniques used in conjunction with flexible fiber optic bronchoscopy

We examined the accuracy of pulmonary cytology in 224 consecutive patients being evaluated for lung cancer. The diagnostic yeild of specimens obtained by various methods, including flexible fiber optic bronchoscopy (FFB), was compared. Among 69 patients with lung cancer, a cytologic diagnosis was made in 87%, including 73% with peripheral tumors. Prebronchoscopy sputa were positive in 50%, bronchial washings in 63%, postbronchoscopy sputa in 82% and bronchial brushings in 59% of the patients. In only one patient was the bronchial brush specimen the only positive cytologic specimen. Normal FFB and small cell undifferentiated cancer were found with increased frequency (P less than 0.05) among the nine patients (13%) with false-negative cytology. Among 155 patients with nonmalignant lung disease, 16 (10%) had false-positive specimens; this finding was significantly related (P less than 0.05) to necrotizing pneumonia in 13 of the 16 patients (81%). The overall diagnostic accuracy of cytology showed 87% sensitivity and 90% specificity, and the predictive value of a positive specimen was 79%. In the absence of necrotizing pneumonia these values exceeded 95%.     

Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype

The postnatal environment, including factors such as weaning and acquisition of the gut microbiota, has been causally linked to the development of later immunological diseases such as allergy and autoimmunity, and has also been associated with a predisposition to metabolic disorders. We show that the very early-life environment influences the development of both the gut microbiota and host metabolic phenotype in a porcine model of human infants. Farm piglets were nursed by their mothers for 1 day, before removal to highly controlled, individual isolators where they received formula milk until weaning at 21 days. The experiment was repeated, to create two batches, which differed only in minor environmental fluctuations during the first day. At day 1 after birth, metabolic profiling of serum by ¹H nuclear magnetic resonance spectroscopy demonstrated significant, systemic, inter-batch variation which persisted until weaning. However, the urinary metabolic profiles demonstrated that significant inter-batch effects on 3-hydroxyisovalerate, trimethylamine-N-oxide and mannitol persisted beyond weaning to at least 35 days. Batch effects were linked to significant differences in the composition of colonic microbiota at 35 days, determined by 16 S pyrosequencing. Different weaning diets modulated both the microbiota and metabolic phenotype independently of the persistent batch effects. We demonstrate that the environment during the first day of life influences development of the microbiota and metabolic phenotype and thus should be taken into account when interrogating experimental outcomes. In addition, we suggest that intervention at this early time could provide ‘metabolic rescue'' for at-risk infants who have undergone aberrant patterns of initial intestinal colonisation.