Long‐term evolution of the natural isolate of Escherichia coli 536 in the mouse gut colonized after maternal transmission reveals convergence in the constitutive expression of the lactose operon

In vitro experimental evolution has taught us many lessons on the molecular bases of adaptation. To move towards more natural settings, evolution in the mice gut has been successfully performed. Yet, these experiments suffered from the use of laboratory strains as well as the use of axenic or streptomycin‐treated mice to maintain the inoculated strains. To circumvent these limitations, we conducted a one‐year experimental evolution in vivo using a natural isolate of E. coli, strain 536, in conditions mimicking as much as possible natural environment with mother‐to‐offspring microbiota transmission. Mice were then distributed in 24 independent cages and separated into two different diets: a regular one (chow diet, CD) and high‐fat and high‐sugar one (Western Diet, WD). Genome sequences revealed an early and rapid selection during the breastfeeding period that selected the constitutive expression of the well‐characterized lactose operon. E. coli was lost significantly more in CD than WD; however, we could not detect any genomic signature of selection, nor any diet specificities during the later part of the experiments. The apparently neutral evolution presumably due to low population size maintained nevertheless at high frequency the early selected mutations affecting lactose regulation. The rapid loss of lactose operon regulation challenges the idea that plastic gene expression is both optimal and stable in the wild.     

Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA(3)(Lys) into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.     

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family.     

Enhanced Renewal of Erythroid Progenitors in Myelodysplastic Anemia by Peripheral Serotonin

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aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species

Background  

Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B₂ variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B₁ variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene.     

Mechanism of Lysine Oxidation in Human Lens Crystallins during Aging and in Diabetes

Oxidative mechanisms during nuclear sclerosis of the lens are poorly understood, in particular metal-catalyzed oxidation. The lysyl oxidation product adipic semialdehyde (allysine, ALL) and its oxidized end-product 2-aminoadipic acid (2-AAA) were determined as a function of age and presence of diabetes. Surprisingly, whereas both ALL and 2-AAA increased with age and strongly correlated with cataract grade and protein absorbance at 350 nm, only ALL formation but not 2-AAA was increased by diabetes. To clarify the mechanism of oxidation, rabbit lenses were treated with hyperbaric oxygen (HBO) for 48 h, and proteins were analyzed by gas and liquid chromatography mass spectrometry for ALL, 2-AAA, and multiple glycation products. Upon exposure to HBO, rabbit lenses were swollen, and nuclei were yellow. Protein-bound ALL increased 8-fold in the nuclear protein fractions versus controls. A dramatic increase in methyl-glyoxal hydroimidazolone and carboxyethyl-lysine but no increase of 2-AAA occurred, suggesting more drastic conditions are needed to oxidize ALL into 2-AAA. Indeed the latter formed only upon depletion of glutathione and was catalyzed by H₂O₂. Neither carboxymethyl-lysine nor glyoxal hydroimidazolone, two markers of glyco-/lipoxidation, nor markers of lenticular glycemia (fructose-lysine, glucospane) were elevated by HBO, excluding significant lipid peroxidation and glucose involvement. The findings strongly implicate dicarbonyl/metal catalyzed oxidation of lysine to allysine, whereby low GSH combined with ascorbate-derived H₂O₂ likely contributes toward 2-AAA formation, since virtually no 2-AAA formed in the presence of methylglyoxal instead of ascorbate. An important translational conclusion is that chelating agents might help delay nuclear sclerosis.     

Positive Regulatory Control Loop between Gut Leptin and Intestinal GLUT2/GLUT5 Transporters Links to Hepatic Metabolic Functions in Rodents

Background and Aims

The small intestine is the major site of absorption of dietary sugars. The rate at which they enter and exit the intestine has a major effect on blood glucose homeostasis. In this study, we determine the effects of luminal leptin on activity/expression of GLUT2 and GLUT5 transporters in response to sugars intake and analyse their physiological consequences.

Methodology

Wistar rats, wild type and AMPKα₂ ^−/− mice were used. In vitro and in vivo isolated jejunal loops were used to quantify transport of fructose and galactose in the absence and the presence of leptin. The effects of fructose and galactose on gastric leptin release were determined. The effects of leptin given orally without or with fructose were determined on the expression of GLUT2/5, on some gluconeogenesis and lipogenic enzymes in the intestine and the liver.

Principal Findings

First, in vitro luminal leptin activating its receptors coupled to PKCβII and AMPKα, increased insertion of GLUT2/5 into the brush-border membrane leading to enhanced galactose and fructose transport. Second in vivo, oral fructose but not galactose induced in mice a rapid and potent release of gastric leptin in gastric juice without significant changes in plasma leptin levels. Moreover, leptin given orally at a dose reproducing comparable levels to those induced by fructose, stimulated GLUT5-fructose transport, and potentiated fructose-induced: i) increase in blood glucose and mRNA levels of key gluconeogenesis enzymes; ii) increase in blood triglycerides and reduction of mRNA levels of intestinal and hepatic Fasting-induced adipocyte factor (Fiaf) and iii) increase in SREBP-1c, ACC-1, FAS mRNA levels and dephosphorylation/activation of ACC-1 in liver.

Conclusion/Significance

These data identify for the first time a positive regulatory control loop between gut leptin and fructose in which fructose triggers release of gastric leptin which, in turn, up-regulates GLUT5 and concurrently modulates metabolic functions in the liver. This loop appears to be a new mechanism (possibly pathogenic) by which fructose consumption rapidly becomes highly lipogenic and deleterious.     

Investigating the establishment of primary cell culture from different abalone (<Emphasis Type="Italic">Haliotis midae</Emphasis>) tissues

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.     

Ciliary Neurotrophic Factor Protects Striatal Neurons against Excitotoxicity by Enhancing Glial Glutamate Uptake

Ciliary neurotrophic factor (CNTF) is a potent neuroprotective cytokine in different animal models of glutamate-induced excitotoxicity, although its action mechanisms are still poorly characterized. We tested the hypothesis that an increased function of glial glutamate transporters (GTs) could underlie CNTF-mediated neuroprotection. We show that neuronal loss induced by in vivo striatal injection of the excitotoxin quinolinic acid (QA) was significantly reduced (by ∼75%) in CNTF-treated animals. In striatal slices, acute QA application dramatically inhibited corticostriatal field potentials (FPs), whose recovery was significantly higher in CNTF rats compared to controls (∼40% vs. ∼7%), confirming an enhanced resistance to excitotoxicity. The GT inhibitor dl-threo-β-benzyloxyaspartate greatly reduced FP recovery in CNTF rats, supporting the role of GT in CNTF-mediated neuroprotection. Whole-cell patch-clamp recordings from striatal medium spiny neurons showed no alteration of basic properties of striatal glutamatergic transmission in CNTF animals, but the increased effect of a low-affinity competitive glutamate receptor antagonist (γ-d-glutamylglycine) also suggested an enhanced GT function. These data strongly support our hypothesis that CNTF is neuroprotective via an increased function of glial GTs, and further confirms the therapeutic potential of CNTF for the clinical treatment of progressive neurodegenerative diseases involving glutamate overflow.