Development of food intake controls: Neuroendocrine and environmental regulation of food intake during early life

This article is part of a Special Issue "Energy Balance".     

The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis

Background

The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.).

Results

An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1.

Conclusions

The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1175) contains supplementary material, which is available to authorized users.     

Copper binding to the Alzheimer’s disease amyloid precursor protein

Alzheimer’s disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called Aβ by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce Aβ levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in Aβ production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer’s disease. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Characterization of eight microsatellite loci in the Galápagos endemic land snail Bulimulus reibischi, and their cross-species amplification

Variation in and amplification conditions for eight polymorphic microsatellite loci initially identified from Bulimulus akamatus, a pulmonate land snail from Galápagos, are described. Intraspecific polymorphism and heterozygosity of the eight markers were studied in 19 populations of Bulimulus reibischi, a closely related species of B. akamatus. Furthermore, the eight loci were also cross-amplified in six other closely related bulimulid species. The number of alleles across populations of B. reibischi at six loci is moderate (three to 10), but considerable for two other loci (19 and 20). There is no strong evidence for linkage among any of the loci examined.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.     

Selection of mitotic Chinese hamster ovary cells from microcarriers. Cell cycle-dependent induction of mutation by 5-bromo-2'-deoxyuridine and ethyl methanesulfonate

Large quantities of mitotic cells may be collected by mitotic detachment from a population of Chinese hamster ovary cells growing on positively charged dextran microcarriers in suspension culture. Exponentially growing cells are treated for 2.5 h with colcemid and mitotic cells are detached from the microcarriers by increasing the stirring speed. A yield of 4-6% of the total population is obtained and, of the cells collected, 85-95% are arrested in metaphase. Using this means to synchronize cells we have determined the cell cycle dependence of the toxic and mutagenic effects of 5-bromo-2'-deoxyuridine (BUdR) and ethyl methanesulfonate (EMS). Mutation was measured at two independent loci: resistance to 6-thioguanine and resistance to ouabain. Both mutagens were more toxic during S phase as compared to G1 or G2 or mitosis. BUdR induced significant mutation only during S phase. The maximum induction of 6-thioguanine resistance was observed in cultures treated 10 h after plating of mitotic cells (2 h into S phase), while the maximum induction of ouabain resistance was observed in cultures treated 10-12 h after plating of mitotic cells (2-4 h into S phase). EMS induced significant mutation at all points in the cell cycle. Mutation induction reached a minimum during S phase but the magnitude of difference between any two points in the cell cycle was found to be less than two-fold.