New polymorphic microsatellite loci developed and characterized from edible dormouse (Glis glis)

Eight new polymorphic microsatellite loci were isolated and characterized from the edible dormouse (Glis glis). All loci were polymorphic in the 70 individuals tested. The number of alleles per locus ranged from 2 to 6, with a mean of 4. Observed and expected heterozygosities ranged from 0.10 to 0.71 and 0.15 to 0.67, respectively. No evidence of linkage disequilibrium between loci has been found. These eight new loci, plus previously characterized markers, now make it possible to undertake more detailed population genetic studies of G. glis, which is relevant to the preservation of this species.     

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

A novel approach for boosting antimicrobial peptides through end tagging with hydrophobic oligopeptide stretches is demonstrated. Focusing on two peptides derived from kininogen, GKHKNKGKKNGKHNGWK (GKH17) and HKHGHGHGKHKNKGKKN (HKH17), tagging resulted in enhanced killing of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and fungal Candida albicans. Microbicidal potency increased with tag length, also in plasma, and was larger for Trp and Phe stretches than for aliphatic ones. The enhanced microbicidal effects correlated to a higher degree of bacterial wall rupture. Analogously, tagging promoted peptide binding to model phospholipid membranes and liposome rupture, particularly for anionic and cholesterol-void membranes. Tagged peptides displayed low toxicity, particularly in the presence of serum, and resisted degradation by human leukocyte elastase and by staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo and in vivo in porcine S. aureus skin infection models. The generality of end tagging for facile boosting of antimicrobial peptides without the need for post-synthesis modification was also demonstrated.     

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

Background

Cellular prion-related protein (PrP^c) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP^c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypotesize that PrP^c could exert antimicrobial activity.

Methodology and Principal Findings

Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-α in vitro.

Conclusions

The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.     

Extinction debt on oceanic islands

Habitat destruction is the leading cause of species extinctions. However, there is typically a time‐lag between the reduction in habitat area and the eventual disappearance of the remnant populations. These “surviving but ultimately doomed” species represent an extinction debt. Calculating the magnitude of such future extinction events has been hampered by potentially inaccurate assumptions about the slope of species–area relationships, which are habitat‐ and taxon‐specific. We overcome this challenge by applying a method that uses the historical sequence of deforestation in the Azorean Islands, to calculate realistic and ecologically‐adjusted species–area relationships. The results reveal dramatic and hitherto unrecognized levels of extinction debt, as a result of the extensive destruction of the native forest:>95%, in<600 yr. Our estimations suggest that more than half of the extant forest arthropod species, which have evolved in and are dependent on the native forest, might eventually be driven to extinction. Data on species abundances from Graciosa Island, where only a very small patch of secondary native vegetation still exists, as well as the number of species that have not been found in the last 45 yr, despite the extensive sampling effort, offer support to the predictions made. We argue that immediate action to restore and expand native forest habitat is required to avert the loss of numerous endemic species in the near future.     

Plant fecundity and pre-dispersal reproductive losses in a common and a rare Euphorbia species (Euphorbiaceae)

Comparative studies on the reproductive biology of closely related plant species have provided valuable information to understand the causes and consequences of common-rare differences with possible applications for the management of threatened populations. The magnitude and spatiotemporal variability of seed production and pre-dispersal reproductive losses were studied for 3 years in the rare endemic Euphorbia welwitschii and in its widespread congener E. characias. The factors responsible for a decrease in potential seed production in these species were the lack of a functional ovary in the cyathium, ovary and fruit abortion, seed predation by insects and seed abortion. In E. characias, the moth Acroclita subsequana was also responsible for minor reproductive losses. The proportion of male cyathia varied significantly between seasons, populations and species, being consistently higher in E. characias than in E. welwitschii. Reproductive losses that resulted in ovary, fruit and seed abortion affected mostly the endemic species and were heavier in the populations located near the sea due to local adverse climatic conditions. Seed predators inflicted higher reproductive losses to the endemic species than to its widespread congener and their impact was particularly heavy at Risco. The two Euphorbia species differed markedly in cyathia production, with E. welwitschii producing systematically a lower number of cyathia than its widespread congener and this, together with higher levels of ovary, fruit and seed abortion, seemed to be the main reasons for the low reproductive output of this rare species.     

Repair of the mutagenic DNA oxidation product, 5-formyluracil

The oxidation of the thymine methyl group can generate 5-formyluracil (FoU). Template FoU residues are known to miscode, generating base substitution mutations. The repair of the FoU lesion is therefore important in minimizing mutations induced by DNA oxidation. We have studied the repair of FoU in synthetic oligonucleotides when paired with A and G. In E. coli cell extract, the repair of FoU is four orders of magnitude lower than the repair of U and is similar for both FoU:A and FoU:G base pairs. In HeLa nuclear extract, the repair of FoU:A is similarly four orders of magnitude lower than the repair of uracil, although the FoU:G lesion is repaired 10 times more efficiently than FoU:A. The FoU:G lesion is shown to be repaired by E. coli mismatch uracil DNA glycosylase (Mug), thermophile mismatch thymine DNA glycosylase (Tdg), mouse mismatch thymine DNA glycosylase (mTDG) and human methyl-CpG-binding thymine DNA glycosylase (MBD4), whereas the FoU:A lesion is repaired only by Mug and mTDG. The repair of FoU relative to the other pyrimidines examined here in human cell extract differs from the substrate preferences of the known glycosylases, suggesting that additional, and as yet unidentified glycosylases exist in human cells to repair the FoU lesion. Indeed, as observed in HeLa nuclear extract, the repair of mispaired FoU derived from misincorporation of dGMP across from template FoU could promote rather than minimize mutagenesis. The pathways by which this important lesion is repaired in human cells are as yet unexplained, and are likely to be complex.     

Nitric oxide and cyclooxygenase may participate in the analgesic and anti-inflammatory effect of the cucurbitacins fraction from Wilbrandia ebracteata

Wilbrandia ebracteata is a medicinal plant from South America used in folk medicine for the treatment of chronic rheumatic diseases. We have shown that the high performance liquid chromatography-characterized (HPLC) dichloromethane fraction isolated from Wilbrandia ebracteata (WEDC) inhibits the parameters observed in experimental models of inflammation in vivo and in vitro. In the present study, we extend our previous observations on the analgesic effects of WEDC by investigating its actions using the hot plate test and zymosan-induced writhing test in mice, as well as zymosan-induced arthritis in rats evaluating articular inflammatory pain, cell migration and determination of NO release into the joint exudate. The effect of WEDC on the activity of COX-1 and COX-2 in vitro and its ulcerogenic capacity in vivo were also investigated. The oral treatment of the animals with WEDC (1-10 mg/kg) produced a significant, dose-dependent reduction of articular incapacitation and abdominal contortions in the writhing test. The same effect was not observed in the hot plate and rota-rod tests. WEDC also reduced nitrite release into the zymosan-inflamed joints. In the evaluation of COX activity, we observed that WEDC was able to selectively inhibit COX-2 but not COX-1 activity in COS-7 cells. Moreover, WEDC treatment did not show gastrointestinal toxicity. Our data confirm the anti-nociceptive activities of the WEDC and indicate that this effect could be associated with inhibition of cyclooxygenase-2 (COX-2) and nitric oxide release. The effects could be attributed to cucurbitacins since several of these were isolated from the WEDC.     

The effect of DMA level on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I--control -- fresh undiluted semen (12 males) and three experimental groups (20 males each) - semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 microl of fresh semen, in the experimental groups with 40 microl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest--in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher (P相似文献   

Detergent Formulations for Wool Domestic Washings Containing Immobilized Enzymes

The stability of immobilized and native Esperase, a commercial serine protease, was studied by incubating the enzymes in four formulations containing the same amount of anionic and non-ionic surfactants. The results show that the activity of the immobilized enzyme is not affected by the presence of detergents while the native enzyme lost 50% of activity after 20 min of incubation in these four formulations. The washing performance of the detergents prepared with the immobilized Esperase was studied on cotton and wool fabric samples stained with human blood and egg yolk, using as control the detergent containing native Esperase. The best stain removal for cotton samples stained with human blood was achieved using the detergent with immobilized Esperase. Several physical tests confirmed that wool keratin was not degraded by the immobilized Esperase, validating the ability to use formulated detergents containing this immobilized enzyme for safe wool domestic washing.