Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid

Main conclusion

Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ^{9cis,11trans,13cis} ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ¹²-double bond of linoleic acid (18:2Δ^9cis,12cis ) into conjugated Δ^11trans and Δ^13cis -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1?^9cis ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.     

Pulp partitioning and worker specialization in polistine wasps (Hymenoptera,Vespidae, Polistinae)

The character of pulp partitioning was studied in three Palaearctic polistine wasps (Polistes dominula and P. gallicus in the Black Sea Biosphere Reserve, and Polistes nimpha in the Lugansk Nature Reserve, Ukraine) and in five Neotropical species (P. lanio, P. versicolor, Mischocyttarus injucundus, M. alfkeni, Metapolybia cingulata, and Protopolybia exigua in Trinidad). In independent-founding Polistes and Mischocyttarus species, all cases of pulp partitioning were connected with aggression on the part of the foundresses. An experimental increase of nesting density of P. dominula led to changes in the relative abundance of workers in the polyethic task groups. Some workers practically stopped hunting and switched over to pulp delivery; some of these workers possessed well-developed ovaries and were able to lay eggs. The experimentally removed part of the nest envelope in the swarm-founding wasp Metapolybia cingulata was restored only in colonies that included young workers. The specific features of nest building activity in independent-founding and swarm-founding polistine wasps are discussed.     

Quantifying killing of orangutans and human-orangutan conflict in Kalimantan, Indonesia

Human-orangutan conflict and hunting are thought to pose a serious threat to orangutan existence in Kalimantan, the Indonesian part of Borneo. No data existed prior to the present study to substantiate these threats. We investigated the rates, spatial distribution and causes of conflict and hunting through an interview-based survey in the orangutan's range in Kalimantan, Indonesia. Between April 2008 and September 2009, we interviewed 6983 respondents in 687 villages to obtain socio-economic information, assess knowledge of local wildlife in general and orangutan encounters specifically, and to query respondents about their knowledge on orangutan conflicts and killing, and relevant laws. This survey revealed estimated killing rates of between 750 and 1800 animals killed in the last year, and between 1950 and 3100 animals killed per year on average within the lifetime of the survey respondents. These killing rates are higher than previously thought and are high enough to pose a serious threat to the continued existence of orangutans in Kalimantan. Importantly, the study contributes to our understanding of the spatial variation in threats, and the underlying causes of those threats, which can be used to facilitate the development of targeted conservation management.     

Examination of the Cytotoxic and Embryotoxic Potential and Underlying Mechanisms of Next-Generation Synthetic Trioxolane and Tetraoxane Antimalarials

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria.     

Selection of Fruit by Gibbons (Hylobates muelleri × agilis) in the Rain Forests of Central Borneo

Gibbons (Hylobates spp.) are among the main frugivorous primates in Southeast Asia, yet little is known about the criteria by which they select fruit for consumption. We studied two gibbon groups for 14 mo in the lowland dipterocarp forests of Central Borneo to determine their selectivity for different fruit species and traits. Ideal gibbon fruit were yellow, large, with a juicy-soft pulp, thin skin and available in large crops. Gibbons ultimately sought seedless fruit, but when seeds were present they selected fruit with a single, well-protected seed. Given that few fruit exhibited all the desired traits, we also carried out a multiple regression using the selection ratios of the various fruit species and their associated fruit traits to determine which traits ultimately determined gibbon choice. The analysis was stratified to account for differences in fruit availability. Selection was strongest when fruit were abundant in the forest and was based on seed width (<21 mm), color (yellow-orange), and fruit weight (1–5 g). No selection is apparent when food abundance was intermediate, but when fruit were scarce they preferentially ate larger fruit (6–30 g).     

A nomenclature for restriction enzymes,DNA methyltransferases,homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.     

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.     

Gametic interactions promote inbreeding avoidance in house mice

Reproduction among related individuals is generally maladaptive. Inbreeding imposes significant costs on individual reproductive success, and can decrease population fitness. Theory predicts that polyandrous females can avoid inbreeding by exploiting paternity‐biasing mechanisms that enable differential sperm ‘use’. Evidence of sperm selection is difficult to demonstrate because patterns of non‐random paternity can be generated by a variety of different mechanisms. Here, using in vitro fertilisation in mice, we provide evidence of sperm selection at the gametic level. We mixed the sperm of sibling and non‐sibling males, and observed a fertilisation bias towards the sperm of non‐sibling males. The number of motile sperm and sperm swimming performance did not differ between competitors among the replicate assays. Therefore, our result can only be ascribed to egg‐driven sperm selection against related sperm. We conclude that the expression or secretion of gametic proteins could provide the molecular basis for this mechanism of cryptic female choice.