Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Continuous administration of polyphenols from aqueous rooibos (Aspalathus linearis) extract ameliorates dietary-induced metabolic disturbances in hyperlipidemic mice

The incidence of obesity and related metabolic diseases is increasing globally. Current medical treatments often fail to halt the progress of such disturbances, and plant-derived polyphenols are increasingly being investigated as a possible way to provide safe and effective complementary therapy. Rooibos (Aspalathus linearis) is a rich source of polyphenols without caloric and/or stimulant components. We have tentatively characterized 25 phenolic compounds in rooibos extract and studied the effects of continuous aqueous rooibos extract consumption in mice. The effects of this extract, which contained 25% w/w of total polyphenol content, were negligible in animals with no metabolic disturbance but were significant in hyperlipemic mice, especially in those in which energy intake was increased via a Western-type diet that increased the risk of developing metabolic complications. In these mice, we found hypolipemiant activity when given rooibos extract, with significant reductions in serum cholesterol, triglyceride and free fatty acid concentrations. Additionally, we found changes in adipocyte size and number as well as complete prevention of dietary-induced hepatic steatosis. These effects were not related to changes in insulin resistance. Among other possible mechanisms, we present data indicating that the activation of AMP-activated protein kinase (AMPK) and the resulting regulation of cellular energy homeostasis may play a significant role in these effects of rooibos extract. Our findings suggest that adding polyphenols to the daily diet is likely to help in the overall management of metabolic diseases.     

Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Uncovering the post-embryonic functions of gametophytic- and embryonic-lethal genes

An estimated 500-1 000 Arabidopsis (Arabidopsis thaliana) genes mutate to embryonic lethality. In addition, several hundred mutations have been identified that cause gametophytic lethality. Thus, a significant fraction of the ～25,000 protein-coding genes in Arabidopsis are indispensable to the early stages of the diploid phase or to the haploid gametophytic phase. The expression patterns of many of these genes indicate that they also act later in development but, because the mutants die at such early stages, conventional methods limit the study of their roles in adult diploid plants. Here, we describe the toolset that allows researchers to assess the post-embryonic functions of plant genes for which only gametophytic- and embryonic-lethal alleles have been isolated.     

Nutritional value of meat: the influence of nutrition and physical activity on vitamin B12 concentrations in ruminant tissues

An important nutritional characteristic of ruminant meat is its high content in vitamin B12. The variability of these contents is not known. Three studies were been set up in order to test the influence of the animal species (2 studies on Charolais steers slaughtered at 30-32 months of age, n = 24 and n = 30 and a third one on lambs slaughtered at 4.5 months of age, n = 21), of the nature of the diet (grass vs. maize silage, lucerne or concentrate diets) and of physical activity (without or with walking) on the vitamin B12 contents of different muscle types (rather oxidative (Rectus Abdominis, RA), intermediate (Longissimus Dorsi, LD), or glycolytic (Semi Tendinosus, ST)) and on the liver. The animals were supplemented in macro and trace minerals according to usual feeding practices in France in order to theoretically avoid any risk of deficiency. For this reason, cobalt allowances, which are necessary for the ruminal synthesis of vitamin B12, could differ among treatments. The results indicate the following: (1) cobalt allowances varied widely among treatments, from (sub-)deficient to plethoric allowances, influencing vitamin B12 contents of the liver, and muscles (only in case of deficiency), (2) the effects of dietary treatments or of physical exercise were essentially related to differences in cobalt allowances, (3) the oxidative type muscle (RA) showed contents which were double those in glycolytic type muscle (RA 10.8 vs. ST 5.0 ng.g(-1)) and (4) vitamin B12 contents of raw muscles were lower than the values indicated in tables of feed composition for humans for cooked meat (0.5 to 1 vs. 2 to 3 microg.100 g(-1)).     

Integrated Evaluation of Environmental Parameters Influencing <Emphasis Type="Italic">Vibrio</Emphasis> Occurrence in the Coastal Northern Adriatic Sea (Italy) Facing the Venetian Lagoon

In the marine environment, the persistence and abundance of Vibrio are related to a number of environmental parameters. The influence of the different environmental variables in determining the Vibrio occurrence could be different in the specific geographic areas around the world. Moreover, oceanographic parameters are generally interdependent and should not be considered separately when their influence on bacterial presence and concentration is tested. In this study, an integrated approach was used to identify key parameters determining the abundance of Vibrio spp in marine samples from the Venetian Lagoon in Italy, which is an important area for fish farming and tourism. Multivariate techniques have been adopted to analyze the dataset: using PCA, it was shown that a relatively high proportion of the total variance in this area was mainly due to two independent variables, namely salinity and temperature. Using cluster analysis, it was possible to categorize different groups with homogeneous features as regards space (“stations”) and time (“seasons”) distribution, as well as to quantify the values of environmental variables and the Vibrio abundances in each category. Furthermore, integrating key environmental factors and bacterial concentration values, it was possible to identify levels of salinity and sea surface temperature which were optimal for Vibrio concentration in water, plankton, and sediment samples. The identification of key environmental variables conditioning Vibrio occurrence should facilitate ocean monitoring, making it possible to predict unexpected variations in marine microflora which determine possible public health risks in coastal areas.     

Effects of the forage-to-concentrate ratio of the diet on feeding behaviour in young Blond d'Aquitaine bulls

The activities of bulls, their feeding behaviour and their ruminal pH were examined at several stages during the finishing period, according to the forage-to-concentrate ratio of their diet. Twenty-four bulls of the Blond d'Aquitaine breed (initial body weight = 326 ± 21 kg) were assigned to six balanced pens with a space allowance of 9.4 m2 per bull during the finishing period. They were fed three different diets with achieved forage-to-concentrate ratios of (i) 8% straw and 92% concentrate, (ii) 44% hay and 56% concentrate and (iii) 57% maize silage and 43% concentrate. Bulls had ad libitum access to feed dispensed once daily. Offered and refusals were weighed on 5 consecutive days per week. The bulls were slaughtered at the common final live weight of 650 kg and the finishing period lasted 138, 181 and 155 days for straw-concentrate, hay-concentrate and maize silage-concentrate diets, respectively. The time budget was estimated four times by scan sampling with a 10-min interval. Feeding behaviour was appraised using data from electronic feeding gates. Ruminal pH was measured from a ruminal fluid sample collected by rumenocentesis. On average, the bulls spent 78% of the time lying or standing still, and 11% of the time eating. The forage-to-concentrate ratio of the diet influenced only those activities directly linked to feeding, i.e. eating and drinking. Bulls fed a high-concentrate diet spent less time eating than the other bulls (47 min v. >2 h) and took shorter meals (7 min v. 17 min). The bulls fed the straw-concentrate diet spread their meals over the entire day, whereas the others maintained two major peaks of eating activity, the main one in the morning after feed dispensing, the other one at the end of the diurnal period. Intake rate ranged widely between diets, from 58 g/min on average for the diets based on hay or maize silage up to 173 g/min for the high-concentrate diet. The concentrate-diet bulls also had a lower ruminal pH during the first 2 months of the finishing period. The dispersion of meals based on a high-acidosis-risk diet may be a way to limit the decrease in ruminal pH.