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Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.

Duckweeds, the fastest growing angiosperms, are gaining increasing attention with respect to their practical applications. Different clones of the same duckweed species vary in their physiological properties. Hence, screening of suitable clones of a species is very important. To enable the identification of clones, a clear taxonomic classification and barcoding at different taxonomic levels, i.e. genera, species, and clones is a pre-requisite. In the present project, we have focused on the genera Spirodela and Landoltia. Spirodela polyrhiza (L.) Schleid. (42 clones), Spirodela intermedia W. Koch (14 clones), and Landoltia punctata (G. Meyer) Les & Crawford (15 clones) were characterized using three plastidic sequences (rpl16, rps16, atpF-atpH) and AFLP fingerprinting. Genome size determination showed significant differences between the two genera. The genetic variability is lowest in S.polyrhiza and highest in S.intermedia. Although the resolution of phenetic variability by AFLP fingerprinting is much higher than the sequence variation of the selected plastidic regions, not all clones could be identified unequivocally. However, without any exception, all clones were strictly categorized into the three species as defined by the morphological markers. The results do not justify the separation of some clones as Spirodela biperforata from S. intermedia.

We have previously identified pyloric pressures and plasma cholecystokinin (CCK) concentrations as independent determinants of energy intake following administration of intraduodenal lipid and intravenous CCK. We evaluated in healthy men whether these parameters also determine energy intake in response to intraduodenal protein, and whether, across the nutrients, any predominant gastrointestinal (GI) factors exist, or many factors make small contributions. Data from nine published studies, in which antropyloroduodenal pressures, GI hormones, and GI /appetite perceptions were measured during intraduodenal lipid or protein infusions, were pooled. In all studies energy intake was quantified immediately after the infusions. Specific variables for inclusion in a mixed‐effects multivariable model for determination of independent predictors of energy intake were chosen following assessment for collinearity, and within‐subject correlations between energy intake and these variables were determined using bivariate analyses adjusted for repeated measures. In models based on all studies, or lipid studies, there were significant effects for amplitude of antral pressure waves, premeal glucagon‐like peptide‐1 (GLP‐1) and time‐to‐peak GLP‐1 concentrations, GLP‐1 AUC and bloating scores (P

BACKGROUND Branched-chain amino acids (BCAAs), particularly leucine, act as nutrient signals regulating protein synthesis and degradation as well as glucose metabolism. In addition, leucine has been demonstrated in animal experiments to modulate eating and energy homeostasis. OBJECTIVE We aimed to characterize the effects of physiologic and supraphysiologic loads of intraduodenal leucine on eating, gut hormone and motor functions, and blood glucose in humans. DESIGN Twelve lean men were studied on 3 occasions in a randomized, double-blind order. Antropyloroduodenal motility, plasma ghrelin, cholecystokinin, glucagon-like peptide 1, peptide YY, insulin, glucagon, blood glucose, appetite perceptions, and gastrointestinal symptoms were measured during 90-min intraduodenal infusions of leucine at 0.15 kcal/min (total 3.3 g, 13.5 kcal), 0.45 kcal/min (total 9.9 g, 40.5 kcal), or saline (control). Ad libitum eating from a buffet lunch was quantified immediately after the infusions. RESULTS Leucine at 0.45 kcal/min inhibited eating (energy intake by ∼13%, P < 0.05), increased plasma cholecystokinin, slightly reduced blood glucose and increased plasma insulin, and decreased antral pressures (all P < 0.05). Leucine at 0.15 kcal/min had no effect on food intake, blood glucose, or antral pressures but also slightly increased plasma cholecystokinin (P < 0.05). Neither dose affected plasma ghrelin, glucagon, glucagon-like peptide 1 and peptide YY, or pyloric and duodenal pressures. Plasma leucine concentrations were related to the dose of intraduodenal leucine, with substantial increases during both 0.15 and 0.45 kcal/min. CONCLUSIONS The effects of intraduodenal infusions of free leucine on eating are probably not primarily mediated by changes in gut motor and hormone functions, with perhaps the exception of cholecystokinin. Instead, increased plasma leucine concentrations may be a potential signal mediating the eating-inhibitory effect of leucine. The study was registered as a clinical trial with the Australia and New Zealand Clinical Trial Registry (www.anzctr.org.au) as ACTRN12613000899741.

FOXO transcription factors regulate several aspects of the cellular stress response and antioxidant defence. It has been known for some time that FOXO cysteine residues are essential for oxidative stress-induced interactions with coregulators such as CBP/p300, which reversibly interacts with FOXO4 upon exposure to H2O2. Here, we investigated whether cysteine residues are essential for FOXO1 activity under basal conditions. To this end, we generated epitope-tagged cysteine-deficient mutants of human FOXO1 (all 7 cysteines were replaced by serines). Cysteine deficiency did not impair FOXO1 DNA binding, as determined using EMSAs, nor was nucleocytoplasmic distribution and shuttling affected in HEK293 or HepG2 cells overexpressing WT or Cys-deficient FOXO1. In contrast, stimulation of FOXO-responsive promoter constructs by overexpression of FOXO1 was far less prominent in cells overexpressing Cys-deficient FOXO1 as compared to those transfected with WT-FOXO1. Similarly, FOXO1 overexpression-induced increases in FOXO target gene mRNA levels was impaired in Cys-deficient FOXO1 relative to WT-FOXO1. A series of single-Cys mutation experiments led us to conclude that, of all 7 cysteines, only C612 is required for full FOXO1 transactivation activity under basal, non-stressed conditions. The exact role of C612 in controlling FOXO1 activity remains to be established.

Cysteine-S-glutathionylation is a reversible post-translational modification occurring under oxidative stress. Here, we tested how transcription factor FoxO1a activity is affected by the absence of its cysteine residues and whether glutathionylation of FoxO1a might occur. A V5-tagged cysteine-deficient mutant of human FoxO1a (all 7 cysteines were replaced by serines) was generated by site directed mutagenesis, followed by transfection of HepG2 human hepatoma cells or HEK293 human embryonic kidney cells to overexpress either wild type (WT) or Cys-deficient V5-FoxO1a. Neither insulin-induced phosphorylation of FoxO1a nor its nucleocytoplasmic shuttling was attenuated in cysteine-deficient FoxO1a. Moreover, no alteration of basal FoxO1a DNA binding activity (EMSA) was noticed in the Cys-deficient version versus WT under normal culture conditions. However, exposure to diamide, a thiol-oxidizing agent, revealed that, while FoxO1a-DNA interaction was attenuated in the WT form, this oxidant-induced attenuation was less prominent in the cysteine-deficient mutant. Furthermore, transactivation of a FoxO-responsive element-driven reporter gene was less prominent in cells overexpressing Cys-deficient FoxO1a than in those transfected with WT FoxO1a. Immunoprecipitation of glutathionylated proteins in cells exposed to diamide co-precipitated WT-FoxO1a more efficiently than Cys-deficient FoxO1a. Moreover, Western blotting analyses (reducing/non-reducing) of diamide-exposed cells overexpressing WT or mutant FoxO1a suggest that oxidant-induced FoxO1a interaction with cofactors is attenuated in the Cys-deficient mutant. In summary, these data suggest that cysteine residues in FoxO1a, while not affecting insulin-induced phosphorylation and nucleocytoplasmic shuttling, are important mediators of FoxO1a/DNA interaction under conditions of oxidative stress. Moreover, these data point to glutathionylation of FoxO1a and/or yet to be identified cofactors under exposure to oxidants, suggesting a regulation of FoxO activity through S-glutathionylation.