Differential expression of particulate methane monooxygenase genes in the verrucomicrobial methanotroph 'Methylacidiphilum kamchatkense' Kam1.

Methane monooxygenases (MMOs) are oxygen-dependent enzymes that catalyze the oxidation of methane to methanol in the methanotrophic bacteria. The thermoacidophilic verrucomicrobial methanotroph 'Methylacidiphilum kamchatkense' Kam1 contains three complete and phylogenetically distinct copies of the pmoCAB gene cluster apparently organized as operons, each encoding all three subunits of particulate MMO (pMMO), and a truncated pmoCA cluster encoding only two of the subunits. Two of the clusters are present as a tandem array, but the other clusters occur in isolation.

Complement C3 is a novel plasma clot component with anti-fibrinolytic properties.

Background And Method: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes.

Results: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components.

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation.

Male accessory glands (MAGs) of insects are responsible for the production of many of the seminal fluid proteins and peptides that elicit physiological and behavioral responses in the post-mated female. In the yellow fever mosquito, Aedes aegypti, seminal fluid components are responsible for stimulating egg production, changing female behavior away from host-seeking toward egg-laying and mating refractoriness, but hitherto no behavior-modifying molecule from the MAGs has been structurally characterized. We now show using mass spectrometry and HPLC/ELISA that the MAG is a major site of synthesis of the biologically active decapeptide, Aea-HP-1 (pERPhPSLKTRFamide) that was first characterized by Matsumoto and colleagues in 1989 from mosquito head extracts and shown to have host-seeking inhibitory properties.

Efficacy of hydrosurgical debridement and nanocrystalline silver dressings for infection prevention in type II and III open injuries.

The aim of this study was to retrospectively evaluate the clinical and culture-positive infection rates of open Gustilo/Anderson type II and III fractures using a protocol nanocrystalline silver wound dressing and hydrosurgical debridement. Retrospective case series through chart review on all type II and III open fractures were treated using a novel protocol from December 2005 to March 2008 (N = 17). All Gustilo/Anderson grade II and III open fractures were treated with a novel protocol at a Level I trauma centre.

An analysis of the phosphoproteome of immune cell lines exposed to the immunomodulatory mycotoxin deoxynivalenol.

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.

Proteomic analysis of the effects of the immunomodulatory mycotoxin deoxynivalenol.

The mycotoxin deoxynivalenol (DON) contaminates cereals worldwide and is a common contaminant in the Western European diet. At high doses, DON induces acute gastrointestinal toxicity; chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, 2-DE was used to identify proteomic changes in human B (RPMI1788) and T (JurkatE6.

An analysis of the Caenorhabditis elegans lipid raft proteome using geLC-MS/MS.

Lipid rafts are microdomains of the phospholipid bilayer, proposed to form semi-stable "islands" that act as a platform for several important cellular processes; major classes of raft-resident proteins include signalling proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteomic studies into lipid rafts have been mainly carried out in mammalian cell lines and single cell organisms. The nematode Caenorhabditis elegans, the model organism with a well-defined developmental profile, is ideally suited for the study of this subcellular locale in a complex developmental context.

A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator.

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A.

Allosteric communication between cAMP binding sites in the RI subunit of protein kinase A revealed by NMR.

The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIalpha-(98-381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIalpha become structured upon cAMP binding, including the interdomain alphaC:A and alphaC':A helices that connect CNB domains A and B and are primary recognition sites for the C subunit.

Proteomic analysis of IgE-mediated secretion by LAD2 mast cells.

The LAD2 cell line is a relatively recent addition to the range of mast cell analogues and is of particular importance as it is the only human analogue which can be stimulated to degranulate in an IgE-dependent manner. Mast cells are tissue-based effector cells which have historically been shown to play an important role in the adaptive immune response, though there is now gathering evidence of their significance as a component of the innate immune system. These functions can be attributed to the ability of mast cells to regulate secretion of a wide variety of potent biologically active mediators through immediate and delayed responses.

Electrophoretic field gradient focusing with on-column detection by fluorescence quenching.

Native, uncoloured, proteins can be focused in a column containing a fluorescent packing material, using hydrodynamic flow and a counteracting non-linear electric field, and imaged along the length of the channel by fluorescence quenching.

Proteomic screening of a cell line model of esophageal carcinogenesis identifies cathepsin D and aldo-keto reductase 1C2 and 1B10 dysregulation in Barrett's esophagus and esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer.

Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers.

PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor.

PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP).

Identification of a locus (LCA9) for Leber's congenital amaurosis on chromosome 1p36.

Leber's congenital amaurosis (LCA) is the most common cause of inherited childhood blindness and is characterised by severe retinal degeneration at or shortly after birth. We have identified a new locus, LCA9, on chromosome 1p36, at which the disease segregates in a single consanguineous Pakistani family. Following a whole genome linkage search, an autozygous region of 10 cM was identified between the markers D1S1612 and D1S228.

Identification of a locus on chromosome 2q11 at which recessive amelogenesis imperfecta and cone-rod dystrophy cosegregate.

A consanguineous Arab pedigree in which recessive amelogenesis imperfecta (AI) and cone-rod dystrophy cosegregate, was screened for linkage to known retinal dystrophy and tooth abnormality loci by genotyping neighbouring microsatellite markers. This analysis resulted in linkage with a maximum lod score of 7.03 to the marker D2S2187 at the achromatopsia locus on chromosome 2q11, and haplotype analysis placed the gene(s) involved in a 2 cM/5 Mb interval between markers D2S2209 and D2S373.

Peptidyl dipeptidases (Ance and Acer) of Drosophila melanogaster: major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme.

Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro substrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length.

Mutations in a protein target of the Pim-1 kinase associated with the RP9 form of autosomal dominant retinitis pigmentosa.

The RP9 form of autosomal dominant retinitis pigmentosa (adRP) maps to a locus on human chromosome 7p14. We now report two different disease associated mutations in a previously unidentified human gene, the mouse orthologue of which has been characterised by its interaction with the Pim-1 oncogene. In the original linked family we identified the missense mutation H137L.

Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3).

The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent.

Inactivation of a tachykinin-related peptide: identification of four neuropeptide-degrading enzymes in neuronal membranes of insects from four different orders.

Tachykinin-related peptides (TRP) are widely distributed in the CNS of insects, where they are likely to function as transmitters/modulators. Metabolic inactivation by membrane ecto-peptidases is one mechanism by which peptide signalling is terminated in the CNS. Using locustatachykinin-1 (LomTK-1, GPSGFYGVRamide) as a substrate and several selective peptidase inhibitors, we have compared the types of membrane associated peptidases present in the CNS of four insects, Locusta migratoria, Leucophaea maderae, Drosophila melanogaster and Lacanobia oleracea.

Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP), the commonest form of inherited retinal dystrophies is a clinically and genetically heterogeneous disorder. It is characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of peripheral visual field. RP is inherited either in an autosomal dominant, autosomal recessive or X-linked mode.