Pathways to improving skin regeneration

A significant proportion of the human population suffers from some form of skin disorder, whether it be from burn injury or inherited skin anomalies. The ideal treatment for skin disorders would be to regrow skin tissue from stem cells residing in the individual patient's skin. Locating these adult stem cells and elucidating the molecules involved in orchestrating the production of new skin cells are important steps in devising more-efficient methods of skin production and wound healing via the ex vivo expansion of patient keratinocytes in culture. This review focuses on the structure of the skin, the identification of skin stem cells, and the role of Notch, Wnt and Hedgehog signalling cascades in regulating the fate of epidermal stem cells.     

AnovArray: a set of SAS macros for the analysis of variance of gene expression data

Background  

Analysis of variance is a powerful approach to identify differentially expressed genes in a complex experimental design for microarray and macroarray data. The advantage of the anova model is the possibility to evaluate multiple sources of variation in an experiment.     

Chemical synthesis of the novel Ca2+ messenger NAADP

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (beta-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (p-NAADP) 1 and the identity of this material confirmed by biological evaluation. This flexible synthetic route offers new opportunities for the generation of NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.     

Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular modeling substantiated by site-specific mutagenesis and biochemical analyses

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDP-glucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.     

Characterisation and biodistribution of two neutral 99mTc(CO)3 complexes with a tridentate ligand

N-(2-Mercapto-propyl)-1,2-phenylenediamine (MPPDA) and N-beta-aminoethylglycine (AEG) were labelled with 99mTc(CO)3(+) to form the neutral complexes [99mTc(CO)3(MPPDA)] and [99mTc(CO)3(AEG)]. Both complexes were formed in excellent yields and their identity was confirmed by LC-MS. In mice, none of the new tracer agents showed brain uptake. [(99m)Tc(CO)3(MPPDA)] was trapped mainly in the liver and excreted via the hepatobiliary system, whereas [99mTc(CO)3(AEG)] was excreted rapidly via the kidneys to the urine.     

8-O-Azeloyl-14-benzoylaconine: a new alkaloid from the roots of Aconitum karacolicum Rapcs and its antiproliferative activities

A new alkaloid of Aconitum karacolicum Rapcs, from the Ranunculaceae family, collected in Kirghizstan, was isolated from the roots of this plant, using a purification scheme based upon its in vitro antiproliferative properties against three human tumour cell lines in culture. Structural identification was performed using high resolution MS-MS mass spectrometry and (1)H, (13)C, 2D NOESY NMR spectroscopy analysis. This compound consists of a 14-benzoylaconine moiety substituted on C-8 by an azeloyl chain. It presents in vitro cytotoxicity with an IC(50) of about 10-20 microM, which warrants further investigation on its possible interest in cancer chemotherapy.     

Molecular modeling and site-directed mutagenesis of plant chloroplast monogalactosyldiacylglycerol synthase reveal critical residues for activity

Monogalactosyldiacylglycerol (MGDG), the major lipid of plant and algal plastids, is synthesized by MGD (or MGDG synthase), a dimeric and membrane-bound glycosyltransferase of the plastid envelope that catalyzes the transfer of a galactosyl group from a UDP-galactose donor onto a diacylglycerol acceptor. Although this enzyme is essential for biogenesis, and therefore an interesting target for herbicide design, no structural information is available. MGD monomers share sequence similarity with MURG, a bacterial glycosyltransferase catalyzing the transfer of N-acetyl-glucosamine on Lipid 1. Using the x-ray structure of Escherichia coli MURG as a template, we computed a model for the fold of Spinacia oleracea MGD. This structural prediction was supported by site-directed mutagenesis analyses. The predicted monomer architecture is a double Rossmann fold. The binding site for UDP-galactose was predicted in the cleft separating the two Rossmann folds. Two short segments of MGD (beta2-alpha2 and beta6-beta7 loops) have no counterparts in MURG, and their structure could not be determined. Combining the obtained model with phylogenetic and biochemical information, we collected evidence supporting the beta2-alpha2 loop in the N-domain as likely to be involved in diacylglycerol binding. Additionally, the monotopic insertion of MGD in one membrane leaflet of the plastid envelope occurs very likely at the level of hydrophobic amino acids of the N-terminal domain.     

AP2 clathrin adaptor complex, but not AP1, controls the access of the major histocompatibility complex (MHC) class II to endosomes

Newly synthesized MHC II alpha- and beta-chains associated with the invariant chain chaperone (Ii) enter the endocytic pathway for Ii degradation and loading with peptides before transport to the cell surface. It is unclear how alphabetaIi complexes are sorted from the Golgi apparatus and directed to endosomes. However, indirect evidence tends to support direct transport involving the AP1 clathrin adaptor complex. Surprisingly, we show here that knocking down the production of AP1 by RNA interference did not affect the trafficking of alphabetaIi complexes. In contrast, AP2 depletion led to a large increase in surface levels of alphabetaIi complexes, inhibited their rapid internalization, and strongly delayed the appearance of mature MHC II in intracellular compartments. Thus, in the cell systems studied here, rapid internalization of alphabetaIi complexes via an AP2-dependent pathway represents a key step for MHC II delivery to endosomes and lysosomes.     

Mycobacteria use their surface-exposed glycolipids to infect human macrophages through a receptor-dependent process

Two subfamilies of the polar glycopeptidolipids (GPLs) located on the surface of Mycobacterium smegmatis, along with unknown phospholipids, were recently shown to participate in the nonopsonic phagocytosis of mycobacteria by human macrophages (Villeneuve, C., G. Etienne, V. Abadie, H. Montrozier, C. Bordier, F. Laval, M. Daffe, I. Maridonneau-Parini, and C. Astarie-Dequeker. 2003. Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids. J. Biol. Chem. 278: 51291-51300). As demonstrated herein, a phospholipid mixture that derived from the methanol-insoluble fraction inhibited the phagocytosis of M. smegmatis. Inhibition was essentially attributable to phosphatidylinositol mannosides (PIMs), namely PIM2 and PIM6, because the purified phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were inactive. This was further confirmed using purified PIM2 and PIM6 from M. bovis BCG that decreased by half the internalization of M. smegmatis. Both compounds also inhibited the uptake of M. tuberculosis and M. avium but had no effect on the internalization of zymosan used as a control particle of the phagocytic process. When coated on latex beads, PIM2 and polar GPL (GPL III) favored the particle entry through complement receptor 3. GPL III, but not PIM2, also directed particle entry through the mannose receptor. Therefore, surface-exposed mycobacterial PIM and polar GPL participate in the receptor-dependent internalization of mycobacteria in human macrophages.