Synthesis and evaluation of arylalkoxy- and biarylalkoxy-phenylamide and phenylimidazoles as potent and selective sphingosine-1-phosphate receptor subtype-1 agonists

In pursuit of potent and selective sphingosine-1-phosphate receptor agonists, we have utilized previously reported phenylamide and phenylimidazole scaffolds to explore extensive side-chain modifications to generate new molecular entities. A number of designed molecules demonstrate good selectivity and excellent in vitro and in vivo potency in both mouse and rat models. Oral administration of the lead molecule 11c (PPI-4667) demonstrated potent and dose-responsive lymphopenia.     

Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca²⁺ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca²⁺, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca²⁺ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca²⁺, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.Actin is crucial to such processes as cell movement, cell division, and apoptosis, which are regulated by numerous actin-binding proteins, including gelsolin, Arp2/3, and profilin (for review, see Ref. 1). Gelsolin, the most potent actin filament-severing protein known, can bind to, sever, cap, and nucleate actin filaments in a calcium-, pH-, ATP-, and phospholipid-dependent manner (for review, see Ref. 2). Villin, found in microvilli of absorptive epithelium, is a second member of the gelsolin family of actin-binding proteins. In addition to standard gelsolin-type activities, villin is able to bundle actin filaments and is subject to regulation by tyrosine phosphorylation as well as by Ca²⁺ and phosphatidylinositol 4,5-bisphosphate (for review, see Ref. 3). Many comparisons have been made between gelsolin and villin. The two share 50% amino acid sequence identity and show similar proteolytic cleavage patterns (4). Both contain six similarly folded domains, but villin possesses a seventh domain at its C terminus, the headpiece (HP)² domain, which folds into a compact structure that introduces a second F-actin-binding site into the protein. Recent studies indicate that villin uses the HP F-actin-binding sites to achieve bundling (5). In an environment devoid of free Ca²⁺, gelsolin and villin assume inactive conformations. After binding Ca²⁺, both undergo conformational rearrangements that expose their binding sites for F-actin. In villin, this includes revealing the HP actin-binding site through a “hinge mechanism” (6).Biochemical and structural studies have revealed eight Ca²⁺-binding sites of two types in gelsolin (for review, see Ref. 7). Each of the six domains contains a complete and evolutionarily conserved site, termed type 2, whereas G1 and G4 provide partial Ca²⁺ coordination at interfaces with actin through sites termed type 1. Sequential mutagenesis of these sites in villin has identified six functional Ca²⁺-binding sites (8): two major sites, one each of type 1 and type 2, in V1, plus four type 2 sites in V2–V6. The type 1 site in V1 regulates F-actin-capping and F-actin-severing activities, whereas the lower affinity type 2 site in V1 only affects severing (9). The other four sites are involved in stabilizing villin conformation, but they do not directly influence actin-severing activity. NMR studies of a fragment of villin that consists of V6 and the HP domain have implicated V6 residues Asn⁶⁴⁷, Asp⁶⁴⁸, and Glu⁶⁷⁰ in binding Ca²⁺ (10). These experiments also revealed the first 80 residues of V6 to undergo significant conformational change as a result of Ca²⁺ binding.Nanomolar to micromolar concentrations of free Ca²⁺ govern the actin-binding activities of gelsolin. In contrast, micromolar and millimolar concentrations of calcium ions are required for villin to exhibit capping and severing, respectively. However, after tyrosine phosphorylation, villin can sever actin filaments even at nanomolar Ca²⁺ concentrations (11). Furthermore, although the actin-severing ability of the N-terminal half of villin is calcium-dependent, that by the N-terminal half of gelsolin is not. In contrast, the binding of G-actin of the C-terminal half of both villin and gelsolin requires Ca²⁺. Creation of hybrid proteins demonstrated that the domains of villin and gelsolin are not interchangeable (12).Abundant x-ray crystallographic structural information exists for gelsolin, including the calcium ion-free (Ca²⁺-free), inactive structure of the intact protein (13), the activated N- and C-terminal halves, each in a bimolecular complex with actin (7, 14), and the activated C-terminal half on its own (15, 16). Structural data for intact villin are unavailable and are limited to fragment V1 (17), solved using NMR methods, and the HP domain, solved by NMR and x-ray crystallography (18, 19). NMR experiments also indicate that HP is connected to V6 by a 40-residue disordered linker. As a result, HP has been proposed to bind actin independently of the remainder of the protein (10).In this report, we present the structure of Ca²⁺-free, isolated villin V6, which exhibits a typical gelsolin domain fold. The long helix in V6 in this structure is straight, unlike the corresponding helix in G6 of intact Ca²⁺-free gelsolin, which is bent, and only straightens on calcium activation of the intact protein. Hence, V6 appears to be in an active conformation in the absence of Ca²⁺. Molecular dynamics simulations indicate that the preferred state of the long helix is also straight for isolated G6 in the absence of Ca²⁺. Furthermore, they suggest a bistable mechanism of helix conformational change regulated by the presence of the remaining domains, by calcium ions, and by other interactants. We therefore propose a mechanism for the gelsolin family proteins whereby Ca²⁺ triggers the straightening of the domain 6 helix in the native conformation of the inactive proteins to propagate more widespread conformational changes.     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Sequence diversity of the OprD protein of environmental Pseudomonas strains

OprD has been widely described for Pseudomonas aeruginosa at both structural and functional levels. Here, we describe the sequence diversity of the OprD proteins from other fluorescent Pseudomonads. We analysed the sequence of the oprD gene in each of the 49 Pseudomonas isolates, mostly putida and fluorescens species, obtained from various environmental sources, including soil, rhizosphere and hospitals. Phylogeny based on OprD sequences distinguished three well-separated clusters in the P. fluorescens species whereas P. putida isolates formed only one cluster. The OprD sequences were generally well conserved within each cluster whereas on the opposite, they were highly variable from one cluster to another and particularly with regards to the cluster of P. aeruginosa. Predicted secondary structures, based on the topological model elaborated for P. aeruginosa, suggest signatures in the large extracellular loops of OprD, which are linked to the OprD-based clusters. Correlations between these OprD-based clusters and ecological niches, growth on various carbon sources and antibiotic sensitivity were investigated.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed,using the Arabidopsis genome sequence and radish genetic mapping

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H. C. BeckerS. Giancola and S. Marhadour contributed equally to this work     

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.     

Notch signaling in intestinal homeostasis across species: the cases of Drosophila, Zebrafish and the mouse

Notch signaling has been recently shown to have a fundamental role in stem cell maintenance and control of proper homeostasis in the intestine of different species. Here, we briefly review the current literature on Notch signals in the intestine of Drosophila, Zebrafish and the mouse, and try to highlight conserved and divergent Notch functions across species. Notch signals show a remarkably conserved role in skewing cell fate choices in intestinal lineages throughout evolution. Genetic analysis demonstrates that loss of Notch signaling invariably leads to increased numbers of secretory cells and loss of enterocytes, while gain of Notch function will completely block secretory cell differentiation. Finally, we discuss the potential contribution of Notch signaling to the initiation of colorectal cancer by controlling the maintenance of the undifferentiated state of intestinal neoplastic cells and speculate on the therapeutic consequences of affecting cancer stem cells.     

Petunia phospholipase c1 is involved in pollen tube growth

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.