Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

Dendritic cell-mediated induction of mucosal cytotoxic responses following intravaginal immunization with the nontoxic B subunit of cholera toxin

The use of the nontoxic B subunit of cholera toxin (CTB) as mucosal adjuvant and carrier-delivery system for inducing secretory Ab responses has been documented previously with different soluble Ags. In this study, we have evaluated this approach for inducing CTL responses against a prototype Ag, OVA, in the female genital mucosa. We report here the ability of an immunogen comprised of CTB conjugated to OVA (CTB-OVA) given by intravaginal (ivag) route to induce genital OVA-specific CTLs in mice. Using adoptive transfer models, we demonstrate that ivag application of CTB-OVA activates OVA-specific IFN-gamma-producing CD4 and CD8 T cells in draining lymph nodes (DLN). Moreover, ivag CTB induces an expansion of IFN-gamma-secreting CD8+ T cells in DLN and genital mucosa and promotes Ab responses to OVA. In contrast, ivag administration of OVA alone or coadministered with CTB failed to induce such responses. Importantly, we demonstrate that ivag CTB-OVA generates OVA-specific CTLs in DLN and the genital mucosa. Furthermore, genital CD11b+ CD11c+ dendritic cells (DCs), but not CD8+ CD11c+ or CD11c- APCs, present MHC class I epitopes acquired after ivag CTB-OVA, suggesting a critical role of this DC subset in the priming of genital CTLs. Inhibition studies indicate that the presentation of OVA MHC class I epitopes by DCs conditioned with CTB-OVA involves a proteasome-dependent and chloroquine-sensitive mechanism. These results demonstrate that CTB is an efficient adjuvant-delivery system for DC-mediated induction of genital CTL responses and may have implications for the design of vaccines against sexually transmitted infections.     

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis. This work was supported by INSERM, CNRS, ARC, French Ministry of Research (ACI), Japanese Ministry of Education, Culture, Sports, Science, and Technology, and Niigata University Research Projects.     

Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation

Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5–10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l⁻¹ was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.     

Comparison of penetratin and other homeodomain-derived cell-penetrating peptides: interaction in a membrane-mimicking environment and cellular uptake efficiency

Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn(2+) as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI-TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.     

Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry

We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.     

Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression

Microspore embryogenesis (ME) is a process in which the gametophytic pollen programme of the microspore is reorientated towards a new embryo sporophytic programme. This process requires a stress treatment, usually performed in the anther or isolated microspores for several days. Despite the universal use of stress to induce ME, very few studies have addressed the physiological processes that occur in the anther during this step. To further understand the processes triggered by stress treatment, we followed the response of anthers by measuring the expression of stress-related genes in two barley (Hordeum vulgare L.) cultivars differing in their ME response. Genes encoding enzymes involved in oxidative stress (glutathione-S-transferase, GST; oxalate oxidase, OxO), in the synthesis of jasmonic acid (13-lipoxygenase, Lox; allene oxide cyclase, AOC; allene oxide synthase, AOS) and in the phenylpropanoid pathway (phenylalanine ammonia lyase, PAL), as well as those encoding PR proteins (Barwin, chitinase 2b, Chit 2b; glucanase, Gluc; basic pathogenesis-related protein 1, PR1; pathogenesis-related protein 10, PR10) were up-regulated in whole anthers upon stress treatment, indicating that anther perceives stress and reacts by triggering general plant defence mechanisms. In particular, both OxO and Chit 2b genes are good markers of anther reactivity owing to their high level of induction during the stress treatment. The effect of copper sulphate appeared to limit the expression of defence-related genes, which may be correlated with its positive effect on the yield of microspore embryos.