Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with N^w-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals. In brain, melatonin and L-NAME increased both tGSH levels and the activity of GSH-PX in LPS-treated animals. The results suggest that melatonin protects against LPS-induced oxidative toxicity in PB-treated animals in both liver and brain, and the findings are consistent with previously published observations related to the antioxidant activity of the pineal hormone.     

Fluorescence in situ hybridisation studies provide evidence for somatic mosaicism in de novo dystrophin gene deletions

The fluorescence in situ hybridisation (FISH) technique was tested for its ability to detect somatic mosaicism in mothers of isolated deletion cases of Duchenne/ Becker muscular dystrophy. A control female with known germline and somatic mosaicism was examined, and both the normal cell line and the carrier cell line were detected. Subsequent FISH analysis of three other mothers of boys with apparent de novo dystrophin gene deletions revealed a second patient with a high level of somatic mosaicism, suggesting that a proportion of de novo dystrophin gene deletions occur as mitotic errors early in development rather than as meiotic errors during gametogenesis.     

Reproductive ecology of five pipefish species in one eelgrass meadow

Synopsis Synopsis Pipefishes have rarely been watched in the wild and have never before been followed in their common seagrass habitats. This study explores the reproductive ecology of five species of pipefishes living in a Swedish eelgrass meadow during parts of four breeding seasons, tagging four of the species. Pipefish are remarkable for their specialised paternal care: only males aerate, osmoregulate and nourish the developing embryos. Two of the species (Entelurus aequoreus andNerophis ophidion) have simple ventral gluing of eggs on the trunk while three species (Syngnathus acus, S. rostellatus andS. typhle) have fully enclosed brood pouches on their tails. Males of the former species receive eggs from one female while males of the genusSyngnathus receive partial clutches from several females. Sex ratios of adults on the site differed from equal to male-biased to female-biased, according to species.S. typhle were most numerous and were resighted most often. They were present throughout the breeding season whereas there were temporal shifts in the presence of the other species on the meadow and in some sex ratios. Most species occurred in the deeper, denser part of the meadow but there was some habitat separation by species and sex. All species tended to stay low in the eelgrass, primarily coming up above the eelgrass to display and mate. No species showed site fidelity either to a home range or to the meadow, withE. aequoreus adults spending least time on the meadow. Sexual size dimorphism differed: males were larger inS. rostellatus, the same size inS acus and smaller in the other species. Although the species overlap in habitat requirements and breeding season, the only observed interspecific interactions were abortive courtships betweenSyngnathus species.     

The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Summary Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by in vivo administration or by in vitro incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G₀ and early G₁, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.     

Chemotactic Responses of Marine Vibrio sp. Strain S14 (CCUG 15956) to Low-Molecular-Weight Substances under Starvation and Recovery Conditions

The chemotactic responses by starved cells of marine Vibrio sp. strain S14 differed from those elicited by cells that were not nutrient limited. The rate of chemotaxis at different concentrations of several attractants varied for starved and growing cells. Vibrio sp. strain S14 showed positive chemotaxis to leucine, valine, arginine, and glucose at the onset of energy and nutrient deprivation. A continued, though decreased, positive response was demonstrated fro leucine, arginine, and glucose at 10 h of starvation. Cells starved for 3 h displayed a stronger response to glucose than those starved for shorter or longer times. However, cells starved for 5 and 10 h responded more strongly to a lower concentration of glucose than did cells starved for 0 and 3 h. Starvation for 24 h elicited no measurable chemotaxis to leucine, arginine, or glucose. The motility decreased by over 95% in the cell population after 24 h of starvation, which resulted in a low sensitivity in the chemotaxis assay. A switch in the response to valine was observed by 3 h of starvation. The addition of nutrients of 22-h-starved cells elicited a temporary positive chemotactic response to leucine by 2 and 4 h of nutrient recovery, while cells at 1 and 6 h of recovery showed no response. At 2 h of recovery, the greatest response was recorded to 10⁻⁴ M leucine, whereas at 4 h it was to 10⁻² M leucine. Ten to fifty percent of the 22-h-starved cell population regained their motility after 4 h of nutrient-aided recovery. It is possible that two types of chemosensory systems exist in marine bacteria. Starved and growing cells responded to different concentrations of the attractant, and growing cells displayed a saturated chemotactic system with leucine as the attractant, unlike the response during starvation.     

Chromosome polymorphism in populations of the grasshopper Trimerotropis pallidipennis from southern Argentina

Three populations of the grasshopper Trimerotropis pallidipennis from southern Argentina have been studied cytologically. A very characteristic B-chromosome was found in all three. They also showed geographical variability in respect of the presence of pericentric inversions, and the inversion system was found to influence chiasma frequency. The Laguna Blanca population, which is on the hypothetical pathway the species is believed to have followed during its migration from northern to southern latitudes, has the same karyotype composition as the N. American form, with fixed inversions in the 3 largest autosomes and the X-chromosome. Its members have a high total chiasma frequency and a great number of interstitial chiasmata. The Sierra de la Ventana population, situated at the absolute eastern border of the species distribution is highly polymorphic with respect to the presence of inversions in the medium chromosomes. Its members have the lowest total chiasma frequency and a greatly reduced number of interstitial chiasmata. Situated geographically between the other two, the Choele-Choel population has the highest frequency of inversions and many of them are homozygous. Its members have a higher total chiasma frequency than that observed in specimens from Sierra de la Ventana, and a greatly reduced number of interstitial chiasmata, similar to that observed in individuals from the latter population.     

Novel Tools to Analyze the Function of Salmonella Effectors Show That SvpB Ectopic Expression Induces Cell Cycle Arrest in Tumor Cells

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.     

Evolutionary conservation of DNA methylation in CpG sites within ultraconserved noncoding elements

Ultraconserved noncoding elements (UCNEs) constitute less than 1 Mb of vertebrate genomes and are impervious to accumulating mutations. About 4000 UCNEs exist in vertebrate genomes, each at least 200 nucleotides in length, sharing greater than 95% sequence identity between human and chicken. Despite extreme sequence conservation over 400 million years of vertebrate evolution, we show both ordered interspecies and within-species interindividual variation in DNA methylation in these regions. Here, we surveyed UCNEs with high CpG density in 56 species finding half to be intermediately methylated and the remaining near 0% or 100%. Intermediately methylated UCNEs displayed a greater range of methylation between mouse tissues. In a human population, most UCNEs showed greater variation than the LINE1 transposon, a frequently used epigenetic biomarker. Global methylation was found to be inversely correlated to hydroxymethylation across 60 vertebrates. Within UCNEs, DNA methylation is flexible, conserved between related species, and relaxed from the underlying sequence selection pressure, while remaining heritable through speciation.