Characterization of the amino acids involved in substrate specificity of methionine sulfoxide reductase A

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.     

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and allowed us to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

Gut microbiota modulate the metabolism of brown adipose tissue in mice

A two by two experimental study has been designed to determine the effect of gut microbiota on energy metabolism in mouse models. The metabolic phenotype of germ-free (GF, n = 20) and conventional (n = 20) mice was characterized using a NMR spectroscopy-based metabolic profiling approach, with a focus on sexual dimorphism (20 males, 20 females) and energy metabolism in urine, plasma, liver, and brown adipose tissue (BAT). Physiological data of age-matched GF and conventional mice showed that male animals had a higher weight than females in both groups. In addition, conventional males had a significantly higher total body fat content (TBFC) compared to conventional females, whereas this sexual dimorphism disappeared in GF animals (i.e., male GF mice had a TBFC similar to those of conventional and GF females). Profiling of BAT hydrophilic extracts revealed that sexual dimorphism in normal mice was absent in GF animals, which also displayed lower BAT lactate levels and higher levels of (D)-3-hydroxybutyrate in liver, plasma, and BAT, together with lower circulating levels of VLDL. These data indicate that the gut microbiota modulate the lipid metabolism in BAT, as the absence of gut microbiota stimulated both hepatic and BAT lipolysis while inhibiting lipogenesis. We also demonstrated that (1)H NMR metabolic profiles of BAT were excellent predictors of BW and TBFC, indicating the potential of BAT to fight against obesity.     

Stress physiologique et état de santé des plus anciens Hommes anatomiquement modernes du sud-est de l’Europe (données dentaires,couche 6-1, Buran-Kaya III,Crimée)

Buran-Kaya III site, in Crimea, provides an exceptional stratigraphic sequence, extending from Middle Paleolithic to Neolithic. Among the archaeological layers allocated to the Gravettian (sensu lato), more than 170 anthropological remains have been discovered in the layer 6-1. They represent the oldest Upper Paleolithic modern humans from Eastern Europe in a well-documented archaeological context. The aim of this article, based on dental remains and enamel dental hypoplasias in particular, is to evaluate the health of the modern humans, which lived more than 32,000 years BP ago in Crimea. This study puts into the light the low frequency of hypoplasias (occurring in the two youngest individuals). This article complete the rare studies concerning the presence of this physiological stress during the Early Upper Paleolithic and confirm the scarcity of enamel dental hypoplasias and dental pathologies, such as caries, at this period.     

Petrosal and inner ear anatomy and allometry amongst specimens referred to Litopterna (Placentalia)

New isolated petrosals from the Itaboraí beds of Brazil (late Palaeocene or early Eocene) are here described and referred to the early diverging litoptern Miguelsoria parayirunhor, based on phylogenetic, size, and abundance arguments. Both the external and internal anatomy of these specimens were investigated, which for the first time document many details of the auditory region of a Palaeogene litoptern. Our cladistic analysis, which included our new observations, failed to recover a monophyletic Litopterna but did not exclude it. A constrained analysis for the monophyly of this order showed that several features such as a (sub)quadrangular and anteroposteriorly elongated tensor tympani fossa and a large notch in the vicinity of the external opening of the cochlear canaliculus may constitute synapomorphies for Litopterna. The evolution of several other auditory characters amongst Litopterna is discussed and the relative dimensions of the inner ear and surrounding petrosal in the group were also investigated. This allowed detection of negative allometry of the bony labyrinth within the petrosal, which was confirmed by measurements and regression analysis across a larger sample of placental mammals. This scaling effect probably has an important influence on several characters of the bony labyrinth and petrosal, amongst which are the length of the vestibular aqueduct and cochlear canaliculus. It demonstrates that many aspects of the morphological variation of the bony labyrinth need to be thoroughly investigated before being incorporated into phylogenetic analyses. © 2015 The Linnean Society of London     

Orderly pattern of development of the autoantibody response in (New Zealand White x BXSB)F1 lupus mice: characterization of target antigens and antigen spreading by two-dimensional gel electrophoresis and mass spectrometry

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White x BXSB)F(1) (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, alpha-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-alpha-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.