Stress induction and antimicrobial properties of a lipid transfer protein in germinating sunflower seeds

Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose function in vivo remains unknown. In this research, we studied a LTP previously isolated from sunflower seeds (Ha-AP10), which displays strong antimicrobial activity against a model fungus. The protein is present during at least the first 5 days of germination, and tissue printing experiments revealed the homogeneous distribution of the protein in the cotyledons. Here we report that Ha-AP10 exerts a weak inhibitory effect on the growth of Alternaria alternata, a fungus that naturally attacks sunflower seeds. These data put into question the contribution of Ha-AP10 as an antimicrobial protein of direct effect on pathogenic fungus, and rather suggest a function related to the mobilization of lipid reserves. We also show that the levels of Ha-AP10 in germinating seeds increase upon salt stress, fungal infection and ABA treatment, indicating that it somehow participates in the adaptative responses of germinating sunflower seeds.     

Challenging the inbreeding hypothesis in a eusocial mammal: population genetics of the naked mole‐rat,Heterocephalus glaber

The role of genetic relatedness in the evolution of eusociality has been the topic of much debate, especially when contrasting eusocial insects with vertebrates displaying reproductive altruism. The naked mole‐rat, Heterocephalus glaber, was the first described eusocial mammal. Although this discovery was based on an ecological constraints model of eusocial evolution, early genetic studies reported high levels of relatedness in naked mole‐rats, providing a compelling argument that low dispersal rates and consanguineous mating (inbreeding as a mating system) are the driving forces for the evolution of this eusocial species. One caveat to accepting this long‐held view is that the original genetic studies were based on limited sampling from the species’ geographic distribution. A growing body of evidence supports a contrary view, with the original samples not representative of the species—rather reflecting a single founder event, establishing a small population south of the Athi River. Our study is the first to address these competing hypotheses by examining patterns of molecular variation in colonies sampled from north and south of the Athi and Tana rivers, which based on our results, serve to isolate genetically distinct populations of naked mole‐rats. Although colonies south of the Athi River share a single mtDNA haplotype and are fixed at most microsatellite loci, populations north of the Athi River are considerably more variable. Our findings support the position that the low variation observed in naked mole‐rat populations south of the Athi River reflects a founder event, rather than a consequence of this species’ unusual mating system.     

Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

When exploring immersed surfaces the cypris larvae of barnacles employ a tenacious and rapidly reversible adhesion mechanism to facilitate their characteristic ‘walking’ behaviour. Although of direct relevance to the fields of marine biofouling and bio-inspired adhesive development, the mechanism of temporary adhesion in cyprids remains poorly understood. Cyprids secrete deposits of a proteinaceous substance during surface attachment and these are often visible as ‘footprints’ on previously explored surfaces. The attachment structures, the antennular discs, of cyprids also present a complex morphology reminiscent of both the hairy appendages used by some terrestrial invertebrates for temporary adhesion and a classic ‘suction cup’. Despite the numerous analytical approaches so-far employed, it has not been possible to resolve conclusively the respective contributions of viscoelastic adhesion via the proteinaceous ‘temporary adhesive’, ‘dry’ adhesion via the cuticular villi present on the disc and the behavioural contribution by the organism. In this study, high-speed photography was used for the first time to capture the behaviour of cyprids at the instant of temporary attachment and detachment. Attachment is facilitated by a constantly sticky disc surface – presumably due to the presence of the proteinaceous temporary adhesive. The tenacity of the resulting bond, however, is mediated behaviourally. For weak attachment the disc is constantly moved on the surface, whereas for a strong attachment the disc is spread out on the surface. Voluntary detachment is by force, requiring twisting or peeling of the bond – seemingly without any more subtle detachment behaviours. Micro-bubbles were observed at the adhesive interface as the cyprid detached, possibly an adaptation for energy dissipation. These observations will allow future work to focus more specifically on the cyprid temporary adhesive proteins, which appear to be fundamental to adhesion, inherently sticky and exquisitely adapted for reversible adhesion underwater.     

Clathrin-mediated endocytosis is essential in Trypanosoma brucei

In Trypanosoma brucei, the plasma membrane is dominated by glycosylphosphatidylinositol (GPI)-anchored proteins. Endocytic activity correlates with expression levels of the clathrin heavy chain TbCLH, and additional evidence suggests that rapid endocytosis may play a role in evasion of the immune response. TbCLH is present on both endocytic vesicles and post-Golgi elements, suggesting a similar range of functions in trypanosomes to higher eukaryotes. We have assessed the role of TbCLH using RNA interference (RNAi). Suppression of TbCLH expression results in rapid lethality in the bloodstream stage, the form most active for endocytosis. The flagellar pocket, the site of both endocytosis and exocytosis, becomes massively enlarged, suggesting that membrane delivery is unaffected but removal is blocked. Endocytosis in TbCLHRNAi cells is essentially undetectable, suggesting that clathrin-mediated mechanisms are the major route for endocytosis in T.brucei and hence that GPI-anchored proteins are endocytosed by clathrin-dependent pathways in trypanosomes. In contrast, a massive internal accumulation of vesicles and significant alterations to trafficking of a lysosomal protein were observed in the procyclic stage, indicating developmental variation in clathrin function in trypanosomes.     

Studies of the M15 β-Galactosidase Complementation Process

M15 -Galactosidase was activated by heat-denatured wild-type -galactosidase, urea, and heat-denatured wild-type -galactosidase, a peptide made up of residues 6–44 of -galactosidase and CB2, the peptide that is normally used for complementation (residues 3–92 of -galactosidase). In each case roughly equal activation levels were attained. Heat-denatured wild-type -galactosidase was present as a finely divided visible white precipitate both before and after complementation. The heat-denatured protein by itself did not migrate on native PAGE and both the protein and the activity that occurred as a result of the complementation also remained at the point of application. The N-terminal ends of the heat-denatured wild-type -galactosidase must have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. -Galactosidase denatured by both urea and heat resulted in a streak of interacting protein on the native PAGE. Upon activation, a streak (indicating that interaction was still occurring) was still present, but it moves more slowly. Complementation using a peptide called XP (made up of residues 6–44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 -galactosidase monomer of less than 1:1. The fastest migrating of the three bands predominated at ratios near 1:1. A single active tetrameric form of M15 -galactosidase was formed with CB2. In both of these last two cases an active slow-moving diffuse band also formed (possibly a dimer of the tetramer). A quantitation of the amount of peptide bound to M15 -galactosidase by titration with XP and with CB2 and by using gel filtration after an excess of fluorescent-labeled XP was added showed that peptide bound in a 1:1 ratio (peptide/monomer) when full activity was achieved. These fluorescent studies also showed that peptide initially bound to dimer and that the tetramer was then formed.     

Experimental production of “septa” and apparent subdivision of muscle mitochondria

The rapid formation (in less than 45 min) of internal septa and the apparent subdivisionin situ of mitochondria from cardiac and skeletal muscle are described following a variety of experimental treatments. For example, the ionophore A23187, caffeine, DNP, ruthenium red, and the insecticide lindane have been applied to intact, glycerinated, and chemically skinned skeletal muscle fibers and to cardiac muscle strips from both amphibians and mammals. In some mitochondria, the two compartments are in the same configuration; in others they are different. The significance of these mitochondrial septa is discussed, and it is suggested that the findings are consistent with the hypothesis that a variety of experimental procedures can promote rapid mitochondrial division.