Limbal stem cell deficiency and ocular phenotype in ectrodactyly-ectodermal dysplasia-clefting syndrome caused by p63 mutations

Objective: To describe the ocular phenotype in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (MIM#604292) and to determine the pathogenic basis of visual morbidity. Design: Retrospective case series. Participants: Nineteen families (23 patients) affected by EEC syndrome from the United Kingdom, Ireland, and Italy. Methods: General medical examination to fulfill the diagnostic criteria for EEC syndrome and determine the phenotypic severity. Mutational analysis of p63 was performed by polymerase chain reaction-based bidirectional Sanger sequencing. All patients with EEC syndrome underwent a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency (LSCD) was diagnosed clinically on the basis of corneal conjunctivalization and anatomy of the limbal palisades of Vogt. Impression cytology using immunofluorescent antibodies was performed in 1 individual. Histologic and immunohistochemical analyses were performed on a corneal button and corneal pannus from 2 EEC patients. Main Outcome Measures: The EEC syndrome phenotypic severity (EEC score), best-corrected Snellen visual acuity (decimal fraction), slit-lamp biomicroscopy, tear function index, tear breakup time, LSCD, p63 DNA sequence variants, impression cytology, and corneal histopathology. Results: Eleven heterozygous missense mutations in the DNA binding domain of p63 were identified in all patients with EEC syndrome. All patients had ocular involvement and the commonest was an anomaly of the meibomian glands and lacrimal drainage system defects. The major cause of visual morbidity was progressive LSCD, which was detected in 61% (14/23). Limbal stem cell deficiency was related to advancing age and caused a progressive keratopathy, resulting in a dense vascularized corneal pannus, and eventually leading to visual impairment. Histologic analysis and impression cytology confirmed LSCD. Conclusions: Heterozygous p63 mutations cause the EEC syndrome and result in visual impairment owing to progressive LSCD. There was no relationship of limbal stem cell failure with the severity of EEC syndrome, as classified by the EEC score, or the underlying molecular defect in p63. Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. © 2012 American Academy of Ophthalmology.

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, Fasciolidae)

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. Ln addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic. (C) 2001 Wiley-Liss, Inc.

The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase

Polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferases (PNPTs) constitute a family of eukaryotic and prokaryotic membrane proteins that catalyze the transfer of a sugar-1-phosphate to a phosphoisoprenyl lipid carrier. All PNPT members share a highly conserved 213-Valine-Phenylalanine-Methionine-Glycine-Aspartic acid-217 (VFMGD) motif. Previous studies using the MraY protein suggested that the aspartic acid residue in this motif, D267, is a nucleophile for a proposed double-displacement mechanism involving the cleavage of the phosphoanhydride bond of the nucleoside. Here, we demonstrate that the corresponding residue in the E. coli WecA, D217, is not directly involved in catalysis, as its replacement by asparagine results in a more active enzyme. Kinetic data indicate that the D217N replacement leads to more than twofold increase in V(max) without significant change in the K(m) for the nucleoside sugar substrate. Furthermore, no differences in the binding of the reaction intermediate analog tunicamycin were found in D217N as well as in other replacement mutants at the same position. We also found that alanine substitutions in various residues of the VFMGD motif affect to various degrees the enzymatic activity of WecA in vivo and in vitro. Together, our data suggest that the highly conserved VFMGD motif defines a common region in PNPT proteins that contributes to the active site and is likely involved in the release of the reaction product.

Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus

Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.

Undecaprenyl phosphate recycling comes out of age

The recycling of the lipid carrier undecaprenyl-phosphate (Und-P) requires the dephosphorylation of Und-PP, a reaction proposed to occur at the external or periplasmic side of the bacterial cell membrane. In this issue of Molecular Microbiology, experiments based on the analysis of lipopolysaccharide modifications in Escherichia coli demonstrate that the phosphorylation of lipid A at position 1 is catalysed by the membrane enzyme LpxT (formerly YeiU). This enzyme specifically transfers the distal phosphate group from Und-PP to lipid A 1-phosphate to produce lipid A 1-diphosphate. Furthermore, this reaction requires a functionally intact MsbA protein, which catalyses the transfer of lipid A across the membrane, confirming that the LpxT-mediated lipid A modification occurs on the periplasmic side of the membrane. These observations provide a novel and unexpected link between periplasmic lipid A modifications and the Und-PP recycling pathway.

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.

An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling

Undecaprenyl phosphate (Und-P) is a universal lipid carrier of glycan biosynthetic intermediates for carbohydrate polymers that are exported to the bacterial cell envelope. Und-P arises from the dephosphorylation of undecaprenyl pyrophosphate (Und-PP) molecules produced by de novo synthesis and also from the recycling of released Und-PP after the transfer of the glycan component to other acceptor molecules. The latter reactions take place at the periplasmic side of the plasma membrane, while cytoplasmic enzymes catalyse the de novo synthesis. Four Und-PP pyrophosphatases were recently identified in Escherichia coli. One of these, UppP (formerly BacA), accounts for 75 % of the total cellular Und-PP pyrophosphatase activity and has been suggested to participate in the Und-P de novo synthesis pathway. Unlike UppP, the other three pyrophosphatases (YbjG, YeiU and PgpB) have a typical acid phosphatase motif also found in eukaryotic dolichyl-pyrophosphate-recycling pyrophosphatases. This study shows that double and triple deletion mutants in the genes uppP and ybjG, and uppP, ybjG and yeiU, respectively, are supersensitive to the Und-P de novo biosynthesis inhibitor fosmidomycin. In contrast, single or combined deletions including pgpB have no effect on fosmidomycin supersensitivity. Experimental evidence is also presented that the acid phosphatase motifs of YbjG and YeiU face the periplasmic space. Furthermore, the quadruple deletion mutant DeltauppP-DeltaybjG-DeltayeiU-DeltawaaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.