Discovery of novel and orally active FXR agonists for the potential treatment of dyslipidemia & diabetes

Herein we describe the synthesis and structure activity relationship of a new class of FXR agonists identified from a high-throughput screening campaign. Further optimization of the original hits led to molecules that were highly active in an LDL-receptor KO model for dyslipidemia. The most promising candidate is discussed in more detail.     

Fibrinogen and fibrin interaction with concanavalin a dimer and its influence on coagulation

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies shows that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can be bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, Bβ and γ plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.     

Aleglitazar,a new,potent, and balanced dual PPARα/γ agonist for the treatment of type II diabetes

Design, synthesis, and SAR of novel α-alkoxy-β-arylpropionic acids as potent and balanced PPARαγ coagonists are described. One representative thereof, Aleglitazar ((S)-2Aa), was chosen for clinical development. Its X-ray structure in complex with both receptors as well as its high efficacy in animal models of T2D and dyslipidemia are also presented.     

Dissecting sensor functions in cell wall integrity signaling in Kluyveromyces lactis

KlWSC1, KlWSC2/3 and KlMID2, which encode putative plasma membrane sensors for cell wall integrity signaling in Kluyveromyces lactis, were cloned and characterized. Double and triple deletion mutants show severe cell integrity defects, indicating overlapping functions. The Klwsc1 Klmid2 double deletion phenotype can be suppressed by overexpression of the downstream components KlROM2, KlPKC1 and KlBCK1. KlWsc1 sensor domain analyses showed that an amino-terminal elongation as well as an extension within the cytoplasmic domain are dispensable for function. Heterologous complementation by KlMID2 and KlWSC1 in Saccharomyces cerevisiae is only achieved upon overexpression. In contrast to ScMID2, ScWSC1 complements in K. lactis. Functional studies with chimeric Mid2 constructs indicate that species specificity is mainly conferred by the extracellular domain. Sensor-GFP fusions localize to the plasma membrane, with a cell cycle dependent distribution of KlWsc1-GFP. Both Wsc-type sensors concentrate in discrete spots within the plasma membrane.     

Genetic and biochemical characterization of the yeast spo12 protein

We have performed a genetic and biochemical analysis of the SPO12 gene, which regulates meiotic nuclear divisions in budding yeast. When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II. We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation. Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p. We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, and SPO13, whose mutant phenotype is like that of spo12, can partially suppress the meiotic defect of spo12 mutants when overexpressed. We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M. We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation in HCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.     

A possible non-sexual origin of mate preference: are male guppies mimicking fruit?

In most animals, the origins of mating preferences are not clear. The "sensory-bias" hypothesis proposes that biases in female sensory or neural systems are important in triggering sexual selection and in determining which male traits will become elaborated into sexual ornaments. Subsequently, other mechanisms can evolve for discriminating between high- and low-quality mates. Female guppies (Poecilia reticulata) generally show a preference for males with larger, more chromatic orange spots. It has been proposed that this preference originated because it enabled females to obtain high-quality mates. We present evidence for an alternative hypothesis, that the origin of the preference is a pleiotropic effect of a sensory bias for the colour orange, which might have arisen in the context of food detection. In field and laboratory experiments, adult guppies of both sexes were more responsive to orange-coloured objects than to objects of other colours, even outside a mating context. Across populations, variation in attraction to orange objects explained 94% of the inter-population variation in female mate preference for orange coloration on males. This is one of the first studies to show both an association between a potential trigger of a mate-choice preference and a sexually selected trait, and also that an innate attraction to a coloured inanimate object explains almost all of the observed variation in female mate choice. These results support the "sensory-bias" hypothesis for the evolution of mating preferences.     

Cache pilfering in a granivore guild: Implications for reintroduction management

Reintroduction programs that release endangered species back into areas from which they have been extirpated rarely take competitive interactions between species into account. The endangered Pacific pocket mouse (Perognathus longimembris pacificus) is being reintroduced to parts of its former range where multiple species of native rodents have overlapping diets. The granivorous species in this foraging guild compete for seeds exploitatively and through direct interference interactions, and pocket mice are the smallest and least dominant species in the guild. Repeated aggressive interactions from resident heterospecifics could lower the chances of reintroduced pocket mice establishing burrows during the post-release settlement period. As such, temporarily reducing the density of competing species through exclusionary fencing might be an advisable reintroduction strategy, in combination with other interventions, such as predator exclusion. The presence of other members of the seed-foraging guild, however, could have a net benefit for pocket mice, if the pocket mice pilfer from the other species’ seed caches more than the other species pilfer from their caches. To test the frequency of cache pilfering between species, we conducted a field experiment with fluorescent dyed seeds. Two of 10 pocket mice pilfered from heterospecifics, but only 1 of 33 heterospecifics pilfered from pocket mice. In a field-enclosure experiment, we could not conclude that any of the 4 species tested used heterospecific scent to find (or avoid) seed caches, and pocket mice were less efficient in pilfering from artificial caches, recovering fewer seeds than the larger species. We did not find strong evidence that Pacific pocket mice benefit from living in sympatry with larger, dominant species. Although further research is needed to elucidate the relationship between heterospecific density and the prevalence of cache pilfering, a conservative reintroduction approach would be to select receiver sites with low densities of known competitors to benefit pocket mice during the critical post-release establishment phase. © 2019 The Wildlife Society.     

Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed. presenilin 1-associated protein-mitochondrial carrier homolog 1; mitochondria; apoptosis; presenilin; Alzheimer's disease     

Effect of cooking and legume species upon calcium, iron and zinc uptake by Caco-2 cells

An in vitro system, consisting of simulated gastrointestinal digestion and Caco-2 cell culture, was used to estimate the uptake of calcium, iron and zinc from white beans, chickpeas and lentils, and the effect of cooking upon uptake, with the ultimate aim of evaluating legumes as a dietary source of the aforementioned minerals. In raw products, differences were observed in the uptake percentages by Caco-2 cells of a same mineral from different legumes, although these were not related to the total mineral content. In the three elements studied, the highest uptake values corresponded to chickpeas. Traditional cooking significantly (p<0.05) increased the uptake (%) of calcium, iron and zinc from white beans, and of calcium from lentils. This effect can be partially ascribed to the conversion of inositol hexaphosphate to its lower phosphate forms. When mineral uptakes from raw, traditionally cooked, and ready-to-eat lentils were compared, the highest uptake values corresponded to the ready-to-eat product, which could be attributed to the combined effect of EDTA soaking, the cooking under pressure process, and citric and ascorbic acid addition.