The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.     

The use of molecularly imprinted sol-gels in pharmaceutical separations

This paper illustrates the potential of the sol-gel process to imprint the pharmaceutical active--N-[N-[(1S)-1-carboxssy-3-phenylpropyl]-l-lysyl]-L-proline, (lisinopril dihydrate). This template exhibits unique difficulties such as limited solubility in non-polar and most polar porogens with multiple functionality evident in its 4 pKa values. Selectivity for this template was achieved using a 3-monomer sol-gel system utilising solid phase extraction (SPE). Analysis of the template and its related substances was achieved using HPLC. The effect of solvent polarity on the rebinding of the template was studied. Through optimisation of porogen and extraction solvent, the imprinted material (MIP) demonstrated enhanced selectivity, for the template, over a non-imprinted material (NIP). Selectivity was also illustrated for the original template over two of its related substances. The effect of starting monomer ratio on selectivity was studied to determine the interactions, which could best be exploited to further enhance selectivity.     

PTH-1R responses to PTHrP and regulation by vitamin D in keratinocytes and adjacent fibroblasts

Vitamin D and PTHrP are essential for the differentiation of keratinocytes and epidermal development. The action of PTHrP on skin is mediated via its PTH-1R receptors present in both epidermal and dermal cells. This suggests that PTHrP may have a paracrine/autocrine role, and its receptors may act in association or in negative cooperativity. We compared the intracellular signaling pathways in response to PTHrP (1-34) and to various PTHrP peptides, the N-terminal (1-34), Mid region (67-89), and C-terminal (107-139) fragments, and the possible modulation of PTHrP and its receptor mRNA expressions by vitamin D. Adjacent dermal fibroblasts as freshly isolated keratinocytes expressed both PTHrP and PTH-1R mRNAs, and responded to the various PTHrP fragments. bPTH and PTHrP(1-34) increased both cellular cAMP and [Ca(2+)]i in keratinocytes and fibroblasts. In contrast, PTHrP (107-139) increased [Ca(2+)]i but not cAMP in the two cell types. PTHrP (67-89) had no effect in keratinocytes, and only increased [Ca(2+)]i in fibroblasts. Vitamin D deficiency in weaned rats increased the expression of PTHrP mRNA in keratinocytes, and decreased it in fibroblasts and kidneys. Vitamin D deficiency increased PTH-1R mRNA expression in keratinocytes and kidneys, but not in fibroblasts. Although keratinocytes and skin fibroblasts are target cells for PTHrP and express PTH-1R, the two adjacent cell types differ as regards their intracellular signaling in response to PTHrP peptides. Moreover vitamin D regulates PTHrP and PTH-1R in a cell-specific manner.     

Increased Na+ intake during gestation in rats is associated with enhanced vascular reactivity and alterations of K+ and Ca2+ function

Gestation is associated with decreased blood pressure and resistance to the effects of vasoconstrictor agents. A recent study showed that pregnant rats, on increased sodium intake, present physiological changes that resemble those observed in preeclampsia. We investigated the effects of sodium supplementation on reactivity and on potassium and Ca(2+) channel activity in blood vessels during gestation. Sodium supplements, 0.9% or 1.8% NaCl as drinking water, were given to nonpregnant and pregnant rats for 7 days (last week of gestation). Reactivity to phenylephrine (PE), KCl, arginine vasopressin (AVP), and tetraethylammonium (TEA) was measured in aortic rings under modulation of potassium and calcium channels. TEA, a nonselective K(+) channel inhibitor, induced concentration-dependent responses in aortic rings from nonpregnant but not in those from pregnant rats. The response to TEA was restored in rings from pregnant rats after preincubation with 10 mmol/l KCl. Sodium supplementation did not affect the response to TEA in the aortas of pregnant animals. After sodium supplementation, maximum responses to PE and AVP were decreased and increased in aortic rings from nonpregnant and pregnant rats, respectively. Cromakalim (an ATP-sensitive K(+) channel activator)-induced inhibition of the responses to the three vasoconstrictors was more striking in aorta from nonpregnant than pregnant rats on regular diet, whereas it produced similar inhibition in tissues from both groups of animals on 0.9% and 1.8% NaCl. NS-1619 (a Ca(2+)-sensitive K(+) activator) elicited heightened effects in the aortas of pregnant animals receiving 0.9% NaCl supplementation. Nifedipine (a Ca(2+) channel blocker) caused greater inhibition of the contractile responses in tissues from nonpregnant rats on regular diet, and its action was increased in pregnant rats on sodium-supplemented diets. These data demonstrate that augmented sodium intake during gestation in the rat is linked with the reversal of gestational-associated resistance to vasopressors and indicate that this is an experimental model showing some features of gestational hypertension.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Effect of hypercholesterolemia and of oxidative stress on the nitric oxide-cGMP pathway

Soluble guanylyl cyclase (sGC) is a key enzyme of the NO-cGMP pathway which is believed to mediate vasoprotective actions. In cardiovascular diseases such as hypercholesterolemia and atherosclerosis, these important functions of the vascular endothelium are strongly impaired. One of the major reasons for this so-called endothelial dysfunction is the increased vascular generation of reactive oxygen species such as superoxide and peroxynitrite. We aimed to investigate whether superoxide and peroxynitrite impacts on the expression and function of sGC and if such a mechanism occurs in a hypercholestemia-induced atherosclerosis. Our experiments with isolated rat aortic rings showed that extracellular superoxide has no effect on expression and function of sGC, while subjection of these rings to continuously generated extracellular peroxynitrite reduced sGC activity. Furthermore, intracellular superoxide as generated by LY85385 almost completely inhibited sGC-activity and increased its expression. In the cholesterol-fed White New Zealand rabbit, we found a 3.5-fold upregulation of sGC, while basal and NO-stimulated sGC-activities were only slightly enhanced and the vasodilator potency of SNAP was decreased by 10-fold. A great portion of the overexpressed dysfunctional sGC is located in intimal lesions. Finally, platelet sGC-activity and the anti-aggregatory effect of SNAP were not changed. These data suggest that endothelial dysfunction in hypercholesterolemia is associated with an oxidative stress-dependent and reversible overexpression of a dysfunctional vascular sGC, while inhibition of platelet sGC-activity is most likely not involved in hypercholesterolemia-induced platelet hyperreactivity.     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Lead optimization of antifungal peptides with 3D NMR structures analysis

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.