Alloparental feeding in Adélie penguins: why is it uncommon?

We investigated alloparental interactions and conditions which could facilitate or prevent the expression of alloparental behaviours in Adélie penguins (Pygoscelis adeliae), a long-lived seabird which nests in high-density colonies around Antarctica. Observation sessions were carried out during the crèche stage on 48 identified pairs and 50 identified chicks in a 217-nest subcolony. As the season progressed, young were fed less often by their own parents because these were increasingly absent from the breeding site and less responsive to their offspring’s solicitations. As a consequence, young and particularly those with a low body mass, coming from a two-chick brood, opted for gradually soliciting more from other adults to obtain food, preferentially those nesting in their direct vicinity. Unsuccessful breeders represented a low and constant part of the adult population and were not specifically solicited by unrelated young. Despite the increasing chick demand, only 4.1% (3 out of 73) of alloparental solicitations resulted in feeding, which is negligible compared to parental feeding. To investigate factors that could trigger the appearance of alloparental care, we carried out comparisons with king (Aptenodytes patagonicus) and emperor penguins (Aptenodytes forsteri) which represent the closest species for which data on alloparental behaviour were available. Our results show different trends to those observed in these species and three factors may explain the low occurrence of alloparental behaviour in Adélie penguins: (1) the low and constant proportion of unsuccessful breeders, (2) the absence of chick selectivity towards unsuccessful breeders, and (3) the late period of chick accessibility for potential alloparents.     

Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.     

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells

Ultraviolet A (UVA) radiation represents more than 90% of the UV spectrum reaching Earth's surface. Exposure to UV light, especially the UVA part, induces the formation of photoexcited states of cellular photosensitizers with subsequent generation of reactive oxygen species (ROS) leading to damages to membrane lipids, proteins and nucleic acids. Although UVA, unlike UVC and UVB, is poorly absorbed by DNA, it inhibits cell cycle progression, especially during S-phase. In the present study, we examined the role of the DNA damage checkpoint response in UVA-induced inhibition of DNA replication. We provide evidence that UVA delays S-phase in a dose dependent manner and that UVA-irradiated S-phase cells accumulate in G2/M. We show that upon UVA irradiation ATM-, ATR- and p38-dependent signalling pathways are activated, and that Chk1 phosphorylation is ATR/Hus1 dependent while Chk2 phosphorylation is ATM dependent. To assess for a role of these pathways in UVA-induced inhibition of DNA replication, we investigated (i) cell cycle progression of BrdU labelled S-phase cells by flow cytometry and (ii) incorporation of [methyl-(3)H]thymidine, as a marker of DNA replication, in ATM, ATR and p38 proficient and deficient cells. We demonstrate that none of these pathways is required to delay DNA replication in response to UVA, thus ruling out a role of the canonical S-phase checkpoint response in this process. On the contrary, scavenging of UVA-induced reactive oxygen species (ROS) by the antioxidant N-acetyl-l-cystein or depletion of vitamins during UVA exposure significantly restores DNA synthesis. We propose that inhibition of DNA replication is due to impaired replication fork progression, rather as a consequence of UVA-induced oxidative damage to protein than to DNA.     

Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Medicago truncatula is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant’s efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N₂ fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for M. truncatula was identification of two epistatic interactions in controlling plant N₂ fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions.     

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001