Interaction of bracken-fern extract with vitamin C in human submandibular gland and oral epithelium cell lines

The consumption of bracken-fern (Pteridium aquilinum) as food is associated with a high incidence of cancer in humans and animals. Thus far, the carcinogenic effects of bracken-fern consumption could be related to chromosome aberrations verified in animal and in human peripheral lymphocytes. We tested the in vitro effects of vitamin C (10 and 100 microg/ml) on the reversibility of DNA damage caused by bracken-fern on human submandibular gland (HSG) cells and on oral epithelium cells (OSCC-3) previously exposed to bracken-fern extract. DNA damage (i.e. nuclei with increased levels of DNA migration) was determined by comet assay, cell morphology was evaluated by light microscopy and cellular degeneration was assessed by the acridine orange/ethidium bromide fluorescent-dyeing test. Results showed that vitamin C alone did not reduce DNA damage caused by bracken-fern in HSG and OSSC-3 cells. However, at a higher concentration (100 microg/ml), vitamin C induced DNA damage in both cell lines. Moreover, vitamin C (10 and 100 microg/ml) together with bracken-fern extract showed synergistic effects on the frequency of DNA damage in HSG cells. In addition, cells treated with bracken-fern extract or vitamin C alone, or with their association, showed apoptosis morphological features, such as chromatin condensation, cytoplasmic volume loss, changes in membrane symmetry and the appearance of vacuoles; these alterations were observed in both cell lines. These results demonstrate that bracken-fern extract was cytotoxic to HSG and OSCC-3 cells, causing cell death by apoptosis, and that vitamin was not able to revert these effects.     

Gating deficits in isolation-reared rats are correlated with alterations in protein expression in nucleus accumbens

The isolation-rearing (IR) paradigm, consisting of the social deprivation for 6–9 weeks after weaning, induces a spectrum of aberrant behaviors in adult rats. Some of these alterations such as sensorimotor gating deficits are reminiscent of the dysfunctions observed in schizophrenia patients. Although gating impairments in IR rats have been linked to impairments in the cortico-mesolimbic system, the specific molecular mechanisms underlying this relation are unclear. To elucidate the neurochemical modifications underlying the gating disturbances exhibited by IR rats, we compared their pre-pulse inhibition (PPI) of the acoustic startle reflex with that of socially reared (SR) controls, and correlated this index to the results of proteomic analyses in prefrontal cortex and nucleus accumbens from both groups. As expected, IR rats exhibited significantly lower startle amplitude and PPI than their SR counterparts. Following behavioral testing, IR and SR rats were killed and protein expression profiles of their brain regions were examined using two-dimensional electrophoresis based proteomics. Image analysis in the Coomassie blue-stained gel revealed that three protein spots were differentially expressed in the nucleus accumbens of IR and SR rats. Mass spectrometry (matrix-assisted laser desorption ionization-time of flight and MS/MS) identified these spots as heat shock protein 60 (HSP60), α-synuclein (α-syn), and 14-3-3 protein ζ/δ. While accumbal levels of HSP60 was decreased in IR rats, α-syn and 14-3-3 proteins were significantly increased in IR in comparison with SR controls. Notably, these two last alterations were significantly correlated with different loudness intensity-specific PPI deficits in IR rats. In view of the role of these proteins in synaptic trafficking and dopaminergic regulation, these findings might provide a neurochemical foundation for the gating alterations and psychotic-like behaviors in IR rats.     

Identification of a regulatory loop for the synthesis of neurosteroids: a steroidogenic acute regulatory protein-dependent mechanism involving hypothalamic-pituitary-gonadal axis receptors

Brain sex steroids are derived from both peripheral (primarily gonadal) and local (neurosteroids) sources and are crucial for neurogenesis, neural differentiation and neural function. The mechanism(s) regulating the production of neurosteroids is not understood. To determine whether hypothalamic‐pituitary‐gonadal axis components previously detected in the extra‐hypothalamic brain comprise a feedback loop to regulate neuro‐sex steroid (NSS) production, we assessed dynamic changes in expression patterns of steroidogenic acute regulatory (StAR) protein, a key regulator of steroidogenesis, and key hypothalamic‐pituitary‐gonadal endocrine receptors, by modulating peripheral sex hormone levels in female mice. Ovariectomy (OVX; high serum gonadotropins, low serum sex steroids) had a differential effect on StAR protein levels in the extrahypothalamic brain; increasing the 30‐ and 32‐kDa variants but decreasing the 37‐kDa variant and is indicative of cholesterol transport into mitochondria for steroidogenesis. Treatment of OVX animals with E₂, P₄, or E₂ + P₄ for 3 days, which decreases OVX‐induced increases in GnRH/gonadotropin production, reversed this pattern. Suppression of gonadotropin levels in OVX mice using the GnRH agonist leuprolide acetate inhibited the processing of the 37‐kDa StAR protein into the 30‐kDa StAR protein, confirming that the differential processing of brain StAR protein is regulated by gonadotropins. OVX dramatically suppressed extra‐hypothalamic brain gonadotropin‐releasing hormone 1 receptor expression, and was further suppressed in E₂‐ or P₄‐treated OVX mice. Together, these data indicate the existence of endocrine and autocrine/paracrine feedback loops that regulate NSS synthesis. Further delineation of these feedback loops that regulate NSS production will aid in developing therapies to maintain brain sex steroid levels and cognition.     

Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis

Biofilm production is thought to be an important step in many enterococcal infections. In several Gram-positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non-polar deletion mutant of a putative glucosyltransferase designated biofilm-associated glycolipid synthesis A ( bgsA ) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030Δ bgsA was devoid of diglucosyl–diacylglycerol (DGlcDAG), while monoglucosyl–diacylglycerol was overrepresented. The cell walls of 12030Δ bgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild-type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA , on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA -deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalis Δ bgsA to Caco-2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030Δ bgsA was cleared more rapidly from the bloodstream than the wild-type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo .     

Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

Aim The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post‐glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. Methods As our model system we used Pulsatilla vernalis, a widely distributed European herbaceous plant occurring both in the high‐mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution‐wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment‐length polymorphisms (PCR–RFLPs) (trnH–trnK, trnK–trnK, trnC–trnD, psbC–trnS, psaA–trnS, trnL–trnF) and further sequencing of trnL–trnF and trnH–psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus‐scale cpDNA variation. Results Eleven PCR–RFLP polymorphisms were detected in P. vernalis, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section Pulsatilla was low, but P. vernalis did not share haplotypes with other species. Main conclusions The genetic distinctiveness of P. vernalis populations from the south‐western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high‐mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post‐glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post‐glacial colonization of these two areas, with possible founder effects.     

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µ M sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N -[³H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µ M . Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [³H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.     

Wing shape versus asymmetry as an indicator of changing environmental conditions in insects

Abstract In insects, the fluctuating asymmetry of bilaterally symmetrical traits has been suggested as an indicator of environmental stress because asymmetry is expected to increase when stressful conditions disturb the normal development of organisms. However, the extensive literature on asymmetry–stress associations is indeterminate. Here we contrast changes in asymmetry with changes in an alternate stress indicator, the shape of insect wings. The development of wing shape involves numerous genes that act throughout egg-to-adult development, so stresses that act at a specific time could alter shape in specific ways. Shape changes, as measured by the Procrustes technique, were considered in five data sets: exposure of Drosophila melanogaster (Meigen) to multiple stresses involving ethanol, low nutrition and cold shocks; exposure of a chironomid ( Chironomus tepperi (Skuse)), a blowfly ( Lucilia cuprina (Wiedemann)), and lightbrown apple moth ( Epiphyas postvittana (Walker)) to pesticides; and development of C. tepperi under saline conditions. All these conditions influenced viability and development time. In none of these cases was a change in symmetry of wing size or wing shape detected. In contrast, in four of the five data sets there was a change in wing shape. These results suggest that wing shape may be altered more commonly by stress than trait asymmetry. Wing-shape monitoring may be useful in detecting stressful environmental conditions during development, at least under controlled conditions.     

Calcium Binding Protein in Squid Brain: Biochemical Similarity to the 28,000-Mr Vitamin D-Dependent Calcium Binding Protein (Calbindin-D28k)

A calcium binding protein that is biochemically similar to vertebrate 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60 degrees C, and showed increased electrophoretic mobility in the presence of chelator. Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin-D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfate-gel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocytochemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D-dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.