Aminolaevulinic acid diffusion characteristics in 'in vitro' normal human skin and actinic keratosis: implications for topical photodynamic therapy

Background: The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT.

Neuropeptide signalling systems in flatworms

Two distinct families of neuropeptides are known to endow platyhelminth nervous systems-the FMRFamide-like peptides (FLPs) and the neuropepticle Fs (NPFs). Flatworm FLPs are strusturally simple, each 4-6 amino acids in length with a carboxy terminal aromatic-hydropliobic-Arg-Phe-amide motif. Thus far, four distinct flatworm FLPs have been characterized, with only one of these from a parasite. They have a widespread distribution within the central and peripheral nervous system of every flatworm examined, including neurones serving the attachment organs, the somatic Musculature and the reproductive system. The only physiological role that has been identified for flatworm FLPs is myoexcitation. Flatworm NPFs are believed to be invertebrate homologues of the vertebrate neuropeptide Y (NPY) family of peptides. Flatworm NPFs are 36-39 amino acids in length and are characterized by a caboxy terminal GRPRFarnide signature and conserved tyrosine residues at positions 10 and 17 from the carboxy terminal. Like FLPs, NPF occurs throughout flatworm nervous systems, although less is known about its biological role. While there is some evidence for a myoexcitatory action in cestodes and flukes, more compelling physiological data indicate that flatworm NPF inhibits cAMP levels in a manner that is characteristic of NPY action in vertebrates. The widespread expression of these neuropeptides in flanworm parasites highlights the potential of these signalling systems to yield new targets for novel anthelmintics. Although platyhelminth FLP and NPF receptors await identification, other molecules that play pivotal roles in neuropeptide signalling have been uncovered. These enzymes, involved in the biosynthesis and processing of flatworm neuropeptides, have recently been described and offer other distinct and attractive targets for therapeutic interference.

Twenty-eight divergent polysaccharide loci specifying within and amongst strain capsule diversity in three strains of Bacteroides fragilis

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

Multiplex analysis of age-related protein and lipid modifications in human Bruch's membrane

Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related(1). These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae(2-5), closely related to the kelps(6,7) (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic(2) approaches to explore these and other(4,5) aspects of brown algal biology further.

A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an

Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy

A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of similar to 55 angstrom and a thickness of similar to 37 angstrom. It is predominantly helical, comprising 13 alpha helices interspersed by six 3(10) helices and 11 beta-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes. (C) 2010 Elsevier Ltd. All rights reserved.

Persistent Inflammation Subverts Thrombospondin-1–Induced Regulation of Retinal Angiogenesis and Is Driven by CCR2 Ligation

Neovascular retinal disease is a leading cause of blindness orchestrated by inflammatory responses. Although noninfectious uveoretinitis is mediated by CD4(+) T cells, in the persistent phase of disease, angiogenic responses are observed, along with degeneration of the retina. Full clinical manifestation relies on myeloid-derived cells, which are phenotypically distinct from, but potentially sharing common effector responses to age-related macular degeneration. To interrogate inflammation-mediated angiogenesis, we investigated experimental autoimmune uveoretinitis, an animal model for human uveitis. After the initial acute phase of severe inflammation, the retina sustains a persistent low-grade inflammation with tissue-infiltrating leukocytes for over 4 months. During this persistent phase, angiogenesis is observed as retinal neovascular membranes that arise from inflamed venules and postcapillary venules, increase in size as the disease progresses, and are associated with infiltrating arginase-1(+) macrophages. In the absence of thrombospondin-1, retinal neovascular membranes are markedly increased and are associated with arginase-1(-) CD68(+) macrophages, whereas deletion of the chemokine receptor CCR2 resulted in reduced retinal neovascular membranes in association with a predominant neutrophil infiltrate. CCR2 is important for macrophage recruitment to the retina in experimental autoimmune uveoretinitis and promotes chronicity in the form of a persistent angiogenesis response, which in turn is regulated by constitutive expression of angiogenic inhibitors like thrombospondin-1. This model offers a new platform to dissect the molecular and cellular pathology of inflammation-induced ocular angiogenesis.

The Structural and Molecular Biology of Type I Galactosemia: Enzymology of Galactose 1-phosphate Uridylyltransferase

Reduced galactose 1-phosphate uridylyltransferase (GAIT) activity is associated with the genetic disease type 1 galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GAIT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (11) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GAIT is required to assist greater understanding of the effects of disease-associated mutations. (C) 2011 IUBMB IUBMB Life, 63(9): 694-700, 2011

Relative metabolomic flux: Predicting changes in the environmental metabolome from the metagenome

Background: The world's oceans are home to a diverse array of microbial life whose metabolic activity helps to drive the earth's biogeochemical cycles. Metagenomic analysis has revolutionized our access to these communities, providing a system-scale perspective of microbial community interactions. However, while metagenome sequencing can provide useful estimates of the relative change in abundance of specific genes and taxa between environments or over time, this does not investigate the relative changes in the production or consumption of different metabolites.
Results: We propose a methodology, Predicted Relative Metabolic Turnover (PRMT) that defines and enables exploration of metabolite-space inferred from the metagenome. Our analysis of metagenomic data from a time-series study in the Western English Channel demonstrated considerable correlations between predicted relative metabolic turnover and seasonal changes in abundance of measured environmental parameters as well as with observed seasonal changes in bacterial population structure.
Conclusions: The PRMT method was successfully applied to metagenomic data to explore the Western English Channel microbial metabalome to generate specific, biologically testable hypotheses. Generated hypotheses linked organic phosphate utilization to Gammaproteobactaria, Plantcomycetes, and Betaproteobacteria, chitin degradation to Actinomycetes, and potential small molecule biosynthesis pathways for Lentisphaerae, Chlamydiae, and Crenarchaeota. The PRMT method can be applied as a general tool for the analysis of additional metagenomic or transcriptomic datasets.

PEPTIDES RELATED TO THE DIPLOPTERA-PUNCTATA ALLATOSTATINS IN NONARTHROPOD INVERTEBRATES - AN IMMUNOCYTOCHEMICAL SURVEY

The allatostatins are a family of peptides isolated originally from the cockroach, Diploptera punctata. Related peptides have been identified in Periplaneta americana and the blowfly, Calliphora vomitoria. These peptides have been shown to be potent inhibitors of juvenile hormone synthesis in these species. A peptide inhibitor of juvenile hormone biosynthesis has also been isolated from the moth, Manduca sexta; however, this peptide has no structural homology with the D. punctata-type allatostatins. Investigations of the phylogeny of the D. punctata allatostatin peptide family have been started by examining a number of nonarthropod invertebrates for the presence of allatostatin-like molecules using immunocytochemistry with antisera directed against the conserved C-terminal region of this family. Allatostatin-like immunoreactivity (ALIR) was demonstrated in the nervous systems of Hydra oligactis (Hydrozoa), Moniezia expansa (Cestoda), Schistosoma mansoni (Trematoda), Artioposthia triangulata (Turbellaria), Ascaris suum (Nematoda), Lumbricus terrestris (Oligochaeta), Limax pseudoflavus (Gastropoda), and Eledone cirrhosa (Cephalopoda). ALIR could not be demonstrated in Ciona intestinalis (Ascidiacea). These results suggest that molecules related to the allatostatins may play an important role in nervous system function in many invertebrates as well as in insects and that they also have an ancient evolutionary lineage. (C) 1994 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.

Common Themes in Glycoconjugate Assembly Using the Biogenesis of O-Antigen Lipopolysaccharide as a Model System

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Burkholderia cenocepacia requires the RpoN sigma factor for biofilm formation and intracellular trafficking within macrophages

Chronic respiratory infections by Burkholderia cenocepacia in cystic fibrosis patients are associated with increased morbidity and mortality, but virulence factors determining the persistence of the infection in the airways are not well characterized. Using a chronic pulmonary infection model, we previously identified an attenuated mutant with an insertion in a gene encoding an RpoN activator protein, suggesting that RpoN and/or components of the RpoN regulon play a role in B. cenocepacia virulence. In this study, we demonstrate that a functional rpoN gene is required for bacterial motility and biofilm formation in B. cenocepacia K56-2. Unlike other bacteria, RpoN does not control flagellar biosynthesis, as evidenced by the presence of flagella in the rpoN mutant. We also demonstrate that, in macrophages, the rpoN mutant is rapidly trafficked to lysosomes while intracellular wild-type B. cenocepacia localizes in bacterium-containing vacuoles that exhibit a pronounced delay in phagolysosomal fusion. Rapid trafficking to the lysosomes is also associated with the release of red fluorescent protein into the vacuolar lumen, indicating loss of bacterial cell envelope integrity. Although a role for RpoN in motility and biofilm formation has been previously established, this study is the first demonstration that the RpoN regulon in B. cenocepacia is involved in delaying phagolysosomal fusion, thereby prolonging bacterial intracellular survival within macrophages.