Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2 alpha gain-of-function mutation

The hypoxia-inducible factors (HIFs; isoforms HIF-1 alpha, HIF-2 alpha, HIF-3 alpha) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2 alpha gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2 alpha gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2 alpha gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2 alpha gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1 alpha is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.-Formenti, F., Beer, P. A., Croft, Q. P. P., Dorrington, K. L., Gale, D. P., Lappin, T. R. J., Lucas, G. S., Maher, E. R., Maxwell, P. H., McMullin, M. F., O'Connor, D. F., Percy, M. J., Pugh, C. W., Ratcliffe, P. J., Smith, T. G., Talbot, N. P., Robbins, P. A. Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2 alpha gain-of-function mutation. FASEB J. 25, 2001-2011 (2011). www.fasebj.org

Alternating ring-opening metathesis copolymerization of bicyclo[2.2.1]hept-2-ene and cyclopentene

Almost alternating copolymers of bicyclo[2.2.1]hept-2-ene and cyclopentene have been formed by ring-opening metathesis polymerization using a RuCl3-phenol catalyst system; this highly novel result is attributed to differential steric influences exerted by a hydrogen-bonded solvent cage which encloses the catalyst site.

Dioxygenase-catalysed cis-dihydroxylation of meta-substituted phenols to yield cyclohexenone cis-diol and derived enantiopure cis-triol metabolites

cis-Dihydroxylation of meta-substituted phenol (m-phenol) substrates, to yield the corresponding cyclohexenone cis-diol metabolites, was catalysed by arene dioxygenases present in mutant and recombinant bacterial strains. The presence of cyclohexenone cis-diol metabolites and several of their cyclohexene and cyclohexane cis-triol derivatives was detected by LC-TOFMS analysis and confirmed by NMR spectroscopy. Structural and stereochemical analyses of chiral ketodiol bioproducts, was carried out using NMR and CD spectroscopy and stereochemical correlation methods. The formation of enantiopure cyclohexenone cis-diol metabolites is discussed in the context of postulated binding interactions of the m-phenol substrates at the active site of toluene dioxygenase (TDO).

The Kinetics of Semiconductor Photocatalysis in Activity-Indicator Films

The kinetics of dye reduction, in photocatalyst indicator ink films on self cleaning glass, is studied with respect to dye concentration. The water-based, photocatalyst indicator inks comprised a redox dye, D-ox, a sacrificial electron donor (glycerol) and a polymer, hydroxyethyl cellulose. The dyes used were: Resazurin (Rz), dichloroindo-phenol (DCIP) and methylene blue (MB), although the latter required acidification of the ink (0.01M HCl) to make it work effectively under ambient conditions. Under anaerobic conditions, the photoreduction of each of the dyes, in an otherwise identical ink formulation, on Activ (TM) self-cleaning glass is zero-order with respect to [D-ox]. Seven commercial samples of Rz, each in a typical ink formulation, were tested on the same piece of self-cleaning glass under aerobic conditions and produced a striking range (over 280%) of different apparent activities for the glass, when there should have been none. The underlying cause of this variation in assessed activity is shown to be due to the combination of a variation in the purity of the commercial samples and the zero-order nature of the kinetics of indicator dye reduction. The relevance of this work and the latter observation, in particular to future use of these films for the rapid assessment of the activities of new and established photocatalytic films, is briefly discussed.

Effect of plasticizer-polymer compatibility on the response characteristics of optical thin CO2 and O-2 sensing films

A homologous family of dialkyl phthalates has been used to investigate the effect of plasticizer/polymer compatibility on the response characteristics of transparent, plastic, thin optical gas sensing films for CO2 and oxygen. Plasticizer/polymer compatibilities were determined through the value of the difference in solubility parameter, i.e. Delta delta, for the plasticizer and polymer with a Delta delta value of zero indicating high compatibility. A strong correlation was found between plasticizer/polymer compatibility and sensitivity in phenol red/ethyl cellulose CO2-sensitive films and this relationship extended to CO2-sensitive films based on other polymers such as polystyrene and poly(methyl methacrylate). It extended also to optical O-2-sensitive films implying that the relationship is general for thin-film optical sensors. Other results from the CO2-sensitive films in different polymers indicated that the film sensitivity is largely independent of the polymer matrix regardless of its inherent gas permeability, when a sufficient quantity of compatible plasticizer is present. (C) 1998 Elsevier Science B.V.

Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6) M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF2-alpha-VHL Axis as the Major Regulator of Erythropoietin Production

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.

Physico-chemical characterization of a recombinant cytoplasmic form of lysine:N6-hydroxylase

A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates. Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

A universal measure of chaotropicity and kosmotropicity

Diverse parameters, including chaotropicity, can limit the function of cellular systems and thereby determine the extent of Earth's biosphere. Whereas parameters such as temperature, hydrophobicity, pressure, pH, Hofmeister effects, and water activity can be quantified via standard scales of measurement, the chao-/kosmotropic activities of environmentally ubiquitous substances have no widely accepted, universal scale. We developed an assay to determine and quantify chao-/kosmotropicity for 97 chemically diverse substances that can be universally applied to all solutes. This scale is numerically continuous for the solutes assayed (from +361kJkg^-1mol^-1 for chaotropes to -659kJkg^-1mol^-1 for kosmotropes) but there are key points that delineate (i) chaotropic from kosmotropic substances (i.e. chaotropes =+4; kosmotropes =-4kJkg^-1mol^-1); and (ii) chaotropic solutes that are readily water-soluble (log P<1.9) from hydrophobic substances that exert their chaotropic activity, by proxy, from within the hydrophobic domains of macromolecular systems (log P>1.9). Examples of chao-/kosmotropicity values are, for chaotropes: phenol +143, CaCl₂ +92.2, MgCl₂ +54.0, butanol +37.4, guanidine hydrochloride +31.9, urea +16.6, glycerol [>6.5M] +6.34, ethanol +5.93, fructose +4.56; for kosmotropes: proline -5.76, sucrose -6.92, dimethylsulphoxide (DMSO) -9.72, mannitol -6.69, trehalose -10.6, NaCl -11.0, glycine -14.2, ammonium sulfate -66.9, polyethylene glycol- (PEG-)1000 -126; and for relatively neutral solutes: methanol, +3.12, ethylene glycol +1.66, glucose +1.19, glycerol [<5M] +1.06, maltose -1.43 (kJkg^-1mol^-1). The data obtained correlate with solute interactions with, and structure-function changes in, enzymes and membranes. We discuss the implications for diverse fields including microbial ecology, biotechnology and astrobiology.

Micro-organic compounds in the Humber rivers

The concentrations of a range of micro-organic compounds in the Humber rivers have been measured at weekly intervals over a period of 1 year. The compounds include the triazine herbicides (simazine, atrazine, propazine, desmetryn and prometryn), selected organophosphorus insecticides (fenitrothion, malathion and parathion), phenylurea (chlorotoluron, diuron, isoproturon and linuron) and phenoxyacid herbicides (2,4-D, MCPA, MCPB and mecoprop), phenol derivatives (phenol, 2-ethylphenol, 2-chlorophenol, 4-nitrophenol, 2-methylphenol, 4-ethylphenol and 2,4-dichlorophenol), organochlorine insecticides (HEOD, DDT, TDE, DDE, HCB, alpha-BHC and lindane), PCB's and some synthetic pyrethroid insecticides (cis/trans-permethrin, fenvalerate and cypermethrin). The results indicate the high frequency of occurrence of many compounds in the southern Humber rivers Aire, Calder, Trent and Don compared with the more occasional concentrations found in the upland rivers with catchments dominated by low intensity agriculture. The more water soluble herbicides, atrazine, simazine, isoproturon and diuron are detected frequently in the southern rivers with the highest concentrations and abundance in the rivers Aire, Calder and Trent. The most abundant phenolic compound is 2,4-dichlorophenol usually occurring at concentrations <1 mu g/l. The organochlorine insecticides and PCB's are generally at concentrations <0.01 mu g/l, cis/trans-permethrin are the only synthetic pyrethroids detected and these are found in the rivers Aire and Calder at about equal concentrations of between 0.01 and 0.11 mu g/l. The results of the occurrence of simazine and atrazine in the rivers Trent, Don and Aire illustrate peaks in concentration in the spring and then later in the year during the early autumn coinciding with the first major storm after the summer. In the rivers Trent and Don, the annual exports (March 1994-95) of atrazine are lower than for simazine whereas in the R. Calder the yield of atrazine is higher than for simazine suggesting differences in use in these catchments. The maximum triazine concentration observed, i.e. 8 mu g/l of atrazine in the R. Calder, could have inhibiting effects on the phytoplankton and algal growth, although because of the transient nature of the peaks, recovery is expected to be rapid. The only other compounds measured at concentrations likely to produce detrimental ecotoxicological effects are cis/trans permethrin in the rivers Aire and Calder. (C) 1997 Elsevier Science B.V.

Dry gel conversion of organosilane templated mesoporous silica: from amorphous to crystalline catalysts for benzene oxidation

Mesoporous silica grown using [3-(trimethoxysilyl)propyl]octadecyldimethylammonium chloride as the mesoporogen in the presence of Fe and Al is X-ray amorphous, but contains very small domains with features of MFI zeolite as evidenced by IR and Raman spectroscopy. When applied as a catalyst, this amorphous sample shows good performance in the selective oxidation of benzene using nitrous oxide. Addition of tetrapropylammonium as structure directing agent to the as-synthesized mesoporous silica and subsequent dry gel conversion results in the formation of hierarchical Fe/ZSM-5 zeolite. During dry gel conversion the wormhole mesostructure of the initial material is completely lost. A dominant feature of the texture after crystallization is the high interconnectivity of micropores and mesopores. Substantial redistribution of low-dispersed Fe takes place during dry gel conversion towards highly dispersed isolated Fe species outside the zeolite framework. The catalytic performance in the oxidation of benzene to phenol of these highly mesoporous zeolites is appreciably higher than that of the parent material.

Hierarchical zeolites prepared by organosilane templating: A study of the synthesis mechanism and catalytic activity

The crystallization of hierarchical ZSM-5 in the presence of the organosilane octadecyl-dimethyl-(3-trimethoxysilyl-propyl)-ammonium chloride as the mesoporogen was investigated as a function of time and temperature. The synthesis by this method proceeds in two steps. The rapid formation of a predominantly amorphous disordered mesoporous aluminosilicate precursor phase is followed by the formation of globular highly mesoporous zeolite particles involving dissolution of the precursor phase. It is difficult to completely convert the initial phase into the final hierarchical zeolite. This limits the amount of aluminium built into the MFI network and the resulting Bronsted acidity. In the presence of iron, more crystalline hierarchical zeolite is obtained. These Fe-containing zeolites are excellent catalysts for the selective oxidation of benzene to phenol. Their hierarchical pore structure leads to higher reaction rates due to increased mass transfer and increased catalyst longevity despite more substantial coke formation. (C) 2011 Elsevier B.V. All rights reserved.

Microwave-assisted Cu-catalyzed Ullmann ether synthesis in a continuous-flow milli-plant

The combination of milli-scale processing and microwave heating has been investigated for the Cu-catalyzed Ullmann etherification in fine-chemical synthesis, providing improved catalytic activity and selective catalyst heating. Wall-coated and fixed-bed milli-reactors were designed and applied in the Cu-catalyzed Ullmann-type CO coupling of phenol and 4-chloropyridine. In a batch reactor the results show clearly increased yields for the microwave heated process at low microwave powers, whereas high powers and catalyst loadings reduced the benefits of microwave heating. Slightly higher yields were found in the Cu/ZnO wall-coated as compared to the Cu/TiO fixed-bed flow-reactor. The benefit here is that the reaction occurs at the surface of the metal nanoparticles confined within a support film making the nano-copper equally accessible. Catalyst deactivation was mainly caused by Cu oxidation and coke formation; however, at longer process times leaching played a significant role. Catalyst activity could partially be recovered by removal of deposited by-product by means of calcination. After 6h on-stream the reactor productivities were 28.3 and 55.1kgprod/(mR3h) for the fresh Cu/ZnO wall-coated and Cu/TiO fixed-bed reactor, respectively. Comparison of single- and multimode microwaves showed a threefold yield increase for single-mode microwaves. Control of nanoparticles size and loading allows to avoid high temperatures in a single-mode microwave field and provides a novel solution to a major problem for combining metal catalysis and microwave heating. Catalyst stability appeared to be more important and provided twofold yield increase for the CuZn/TiO catalyst as compared to the Cu/TiO catalyst due to stabilized copper by preferential oxidation of the zinc. For this catalyst a threefold yield increase was observed in single-mode microwaves which, to the best of our knowledge, led to a not yet reported productivity of 172kgprod/(mR3h) for the microwave and flow Ullmann CO coupling. © 2012 Elsevier B.V.

A more direct way to make catalysts: one-pot ligand-assisted aerobic stripping and electrodeposition of copper on graphite

Electrodeposition of metals onto conductive supports such as graphite potentially provides a lower-waste method to form heterogeneous catalysts than the standard methods such as wet impregnation. Copper electrodeposition onto pressed graphite disc electrodes was investigated from aqueous CuSO4-ethylenediamine solutions by chronoamperometry with scanning electron microscopy used to ascertain the particle sizes obtained by this method. The particle size was studied as a function of pH, CuSO4-ethylenediamine concentration, and electrodeposition time. It was observed that decreasing the pH, copper-ethylenediamine concentration and time each decreased the size of the copper particles observed, with the smallest obtained being around 5-20 nm. Furthermore, electroless aerobic oxidation of copper metal in the presence of ethylenediamine was successfully coupled with the electrodeposition in the same vessel. In this way, deposition was achieved sequentially on up to twenty different graphite discs using the same ethylenediamine solution, demonstrating the recyclability of the ligand. The materials thus prepared were shown to be catalytically active for the mineralisation of phenol by hydrogen peroxide. Overall, the results provide a proof-of-principle that by making use of aerobic oxidation coupled with electrochemical deposition, elemental base metals can be used directly as starting materials to form heterogeneous catalysts without the need to use metal salts as catalyst precursors.