Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

NMR analyses on N-hydroxymethylated nucleobases - implications for formaldehyde toxicity and nucleic acid demethylases.

Formaldehyde is produced in cells by enzyme-catalysed demethylation reactions, including those occurring on N-methylated nucleic acids. Formaldehyde reacts with nucleobases to form N-hydroxymethylated adducts that may contribute to its toxicity/carcinogenicity when added exogenously, but the chemistry of these reactions has been incompletely defined. We report NMR studies on the reactions of formaldehyde with canonical/modified nucleobases. The results reveal that hydroxymethyl hemiaminals on endocyclic nitrogens, as observed with thymidine and uridine monophosphates, are faster to form than equivalent hemiaminals on exocyclic nitrogens; however, the exocyclic adducts, as formed with adenine, guanine and cytosine, are more stable in solution. Nucleic acid demethylase (FTO)-catalysed hydroxylation of (6-methyl)adenosine results in (6-hydroxymethyl)adenosine as the major observed product; by contrast no evidence for a stable 3-hydroxymethyl adduct was accrued with FTO-catalysed oxidation of (3-methyl)thymidine. Collectively, our results imply N-hydroxymethyled adducts of nucleic acid bases, formed either by reactions with formaldehyde or via demethylase catalysis, have substantially different stabilities, with some being sufficiently stable to have functional roles in disease or the regulation of nucleic acid/nucleobase activity.

Betti reaction enables efficient synthesis of 8-hydroxyquinoline inhibitors of 2-oxoglutarate oxygenases.

There is interest in developing potent, selective, and cell-permeable inhibitors of human ferrous iron and 2-oxoglutarate (2OG) oxygenases for use in functional and target validation studies. The 3-component Betti reaction enables efficient one-step C-7 functionalisation of modified 8-hydroxyquinolines (8HQs) to produce cell-active inhibitors of KDM4 histone demethylases and other 2OG oxygenases; the work exemplifies how a template-based metallo-enzyme inhibitor approach can be used to give biologically active compounds.

Disordered insulin secretion in the development of insulin resistance and Type 2 diabetes.

For many years, the development of insulin resistance has been seen as the core defect responsible for the development of Type 2 diabetes. However, despite extensive research, the initial factors responsible for insulin resistance development have not been elucidated. If insulin resistance can be overcome by enhanced insulin secretion, then hyperglycaemia will never develop. Therefore, a ß-cell defect is clearly required for the development of diabetes. There is a wealth of evidence to suggest that disorders in insulin secretion can lead to the development of decreased insulin sensitivity. In this review, we describe the potential initiating defects in Type 2 diabetes, normal pulsatile insulin secretion and the effects that disordered secretion may have on both ß-cell function and hepatic insulin sensitivity. We go on to examine evidence from physiological and epidemiological studies describing ß-cell dysfunction in the development of insulin resistance. Finally, we describe how disordered insulin secretion may cause intracellular insulin resistance and the implications this concept has for diabetes therapy. In summary, disordered insulin secretion may contribute to development of insulin resistance and hence represent an initiating factor in the progression to Type 2 diabetes.

Decreasing amputation rates in patients with diabetes-a population-based study.

AIMS: To assess the changing rate of amputation in patients with diabetes over a 7-year period. METHODS: All patients undergoing lower extremity amputation in Tayside, Scotland between 1 January 2000 and 31 December 2006 were identified. Temporal linkage of cases to the diabetes database was used to ascertain which amputations were in patients with diabetes. RESULTS: The incidence of major amputations fell from 5.1 [95% confidence interval (CI) 3.8-6.4] to 2.9 (95% CI 1.9-3.8) per 1000 patients with diabetes (P < 0.05). There is a clear linear trend in the adjusted incidence of major amputation (P = 0.023 and 0.027 for age- and sex-adjusted, and duration- and sex-adjusted incidences, respectively). The adjusted incidence of total amputations followed decreased linear regression trend over the whole study period when adjusted for age and sex or diabetes duration and sex (P = 0.002). CONCLUSIONS: There has been a significant reduction in the incidence of major lower extremity amputation in patients with diabetes over the 7-year period.

Classification, evolution, and species groups within the Triatominae.

Classification of the Triatominae has become a complex balance between traditional approaches and a wide variety of evolutionary interpretations. On the one hand is the need for a stable classification of practical use for those involved in vector surveillance and control. On the other is the desire to adequately reflect evolutionary theory derived from a range of molecular, cytogenetic and morphometric comparisons, with additional complications raised by current interpretations of the subfamily as a recently derived polyphyletic assemblage. Here we review key aspects of triatomine systematics and evolution, to derive a pragmatic classification that seeks to build on traditional morphological concepts within the context of current evolutionary theories.

Mechanisms and structures of crotonase superfamily enzymes--how nature controls enolate and oxyanion reactivity.

Structural and mechanistic studies on the crotonase superfamily (CS) are reviewed with the aim of illustrating how a conserved structural platform can enable catalysis of a very wide range of reactions. Many CS reactions have precedent in the 'carbonyl' chemistry of organic synthesis; they include alkene hydration/isomerization, aryl-halide dehalogenation, (de)carboxylation, CoA ester and peptide hydrolysis, fragmentation of beta-diketones and C-C bond formation, cleavage and oxidation. CS enzymes possess a canonical fold formed from repeated betabetaalpha units that assemble into two approximately perpendicular beta-sheets surrounded by alpha-helices. CS enzymes often, although not exclusively, oligomerize as trimers or dimers of trimers. Two conserved backbone NH groups in CS active sites form an oxyanion 'hole' that can stabilize enolate/oxyanion intermediates. The range and efficiency of known CS-catalyzed reactions coupled to their common structural platforms suggest that CS variants may have widespread utility in biocatalysis.

Macular oedema is not predicted by perifoveal single blot haemorrhages.

AIMS: To ascertain which perifoveal changes on digital retinal screening in diabetes predict the need for subsequent macular grid or focal laser therapy. METHODS: Between 1 January 2004 and 31 December 2005, all consecutive retinal images where any lesion was within one disc diameter of the fovea were reviewed. Patients were categorized by lesion at screening as having microaneurysm, single blot haemorrhage, multiple blot haemorrhages and exudates or circinate exudates within one disc diameter of the fovea. We compared these retinal images with the findings on slit lamp examination and the related decision for laser photocoagulation. RESULTS: Four hundred and twenty-four retinal images were identified. Of these, 52 were excluded, principally because of an interval between photography and clinic attendance of greater than 120 days, leaving 372 retinal images in the study group (313 patients). No patients with a single blot haemorrhage required immediate laser therapy at ophthalmology review compared with 13 (23%) of those with multiple blot haemorrhages and 36 (16%) of those with exudates or circinate lesions (P < 0.001). Thirty-nine patients with a single blot haemorrhage who did not require laser therapy underwent ongoing follow-up. None of these underwent laser therapy for maculopathy within the study time frame (9 months from initial screening event). CONCLUSIONS: In this study, no patients with a single blot haemorrhage within one disc diameter of the fovea on digital retinal screening required laser treatment.

Strategy review of traps and targets for tsetse and african trypanosomiasis control

It presents informations about strategic for tsetse and african trypanosomiasis control. Also brings informations about transmission, historical aspects, diagnosis and treatment and eradition strategies. Document in PDF format, required Acrobat Reader.

Structural and mechanistic studies on the peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PhyH).

Phytanic acid (PA) is an epimeric metabolite of the isoprenoid side chain of chlorophyll. Owing to the presence of its epimeric beta-methyl group, PA cannot be metabolized by beta-oxidation. Instead, it is metabolized in peroxisomes via alpha-oxidation to give pristanic acid, which is then oxidized by beta-oxidation. PhyH (phytanoyl-CoA 2-hydroxylase, also known as PAHX), an Fe(II) and 2OG (2-oxoglutarate) oxygenase, catalyses hydroxylation of phytanoyl-CoA. Mutations of PhyH ablate its role in alpha-oxidation, resulting in PA accumulation and ARD (adult Refsum's disease). The structure and function of PhyH is discussed in terms of its clinical importance and unusual selectivity. Most point mutations of PhyH causing ARD cluster in two distinct groups around the Fe(II)- and 2OG-binding sites. Therapaeutic possibilities for the treatment of Refsum's disease involving PhyH are discussed.

Competitive inhibitors of the CphA metallo-beta-lactamase from Aeromonas hydrophila.

Various inhibitors of metallo-beta-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-beta-lactamases.

Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling.

The 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures. Recently, it has been discovered that they act as sensors in the hypoxic response in humans and other animals. Substrate oxidation is coupled to conversion of 2OG to succinate and carbon dioxide. Kinetic, spectroscopic and structural studies are consistent with a consensus mechanism in which ordered binding of (co)substrates enables control of reactive intermediates. Binding of the substrate to the active site triggers the enzyme for ligation of dioxygen to the metal. Oxidative decarboxylation of 2OG then generates the ferryl species thought to mediate substrate oxidation. Structural studies reveal a conserved double-stranded beta-helix core responsible for binding the iron, via a 2His-1carboxylate motif and the 2OG side chain. The rigidity of this core contrasts with the conformational flexibility of surrounding regions that are involved in binding the substrate. Here we discuss the roles of 2OG oxygenases in terms of the generic structural and mechanistic features that render the 2OG oxygenases suited for their functions.

Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors.

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.

A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases.

A widely used generic assay for 2-oxoglutarate-dependent oxygenases relies upon monitoring the release of 14CO2 from labeled [1-14C]-2-oxoglutarate. We report an alternative assay in which depletion of 2-oxoglutarate is monitored by its postincubation derivatization with o-phenylenediamine to form a product amenable to fluorescence analysis. The utility of the procedure is demonstrated by assays with hypoxia-inducible factor hydroxylases where it was shown to give results similar to those reported with the radioactive assay, but it is more efficient and readily adapted to a multiwell format. The process should be amenable to the assay of other 2-oxoglutarate-consuming enzymes and to the discovery of inhibitors.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco.

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 +/- 3.1 mg a.i./m2 in walls and 11.9 +/- 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 +/- 6.9 mg a.i./m2 and 34.7 +/- 10.4 mg a.i./m2 in walls and 28.8 +/- 19.2 mg a.i./m2 and 24.9 +/- 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.

Factor inhibiting hypoxia-inducible factor (FIH) and other asparaginyl hydroxylases.

FIH (Factor inhibiting hypoxia-inducible factor), an asparaginyl beta-hydroxylase belonging to the super-family of 2-oxoglutarate and Fe(II)-dependent dioxygenases, catalyses hydroxylation of Asn-803 of hypoxia-inducible factor, a transcription factor that regulates the mammalian hypoxic response. Only one other asparaginyl beta-hydroxylase, which catalyses hydroxylation of both aspartyl and asparaginyl residues in EGF (epidermal growth factor)-like domains, has been characterized. In the light of recent crystal structures of FIH, we compare FIH with the EGFH (EGF beta-hydroxylase) and putative asparagine/asparaginyl hydroxylases. Sequence analyses imply that EGFH does not contain the HXD/E iron-binding motif characteristic of most of the 2-oxoglutarate oxygenases.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 ± 3.1 mg a.i./m2 in walls and 11.9 ± 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 ± 6.9 mg a.i./m2 and 34.7 ± 10.4 mg a.i./m2 in walls and 28.8 ± 19.2 mg a.i./m2 and 24.9 ± 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.

Modulating the hypoxia-inducible factor signaling pathway: applications from cardiovascular disease to cancer.

Humans, like other complex aerobic organisms, possess highly evolved systems for the delivery of dioxygen to all the cells of the body. These systems are regulated since excessive levels of dioxygen are toxic. In animals hypoxia causes an increase in the transcription levels of specific genes, including those encoding for vascular endothelial growth factor and erythropoietin. At the transcriptional level, the hypoxic response is mediated by hypoxia-inducible factor (HIF), an alpha,beta-heterodimeric protein. HIF-beta is constitutively present, but HIF-alpha levels are regulated by dioxygen. Under hypoxic conditions, levels of HIF-alpha rise, allowing its dimerization with HIF-beta and enabling transcriptional activation. Under normoxic conditions both the level of HIF-alpha and its ability to enable transcription are directly controlled by its post-translational oxidation by oxygenases. Hydroxylation of HIF-alpha at either of two conserved prolyl residues enables its recognition by the von Hippel-Lindau tumour suppressor protein which targets it for proteasomal degradation. Hydroxylation of an asparaginyl residue in the C-terminal transactivation domain of HIF-alpha directly prevents its interaction with the coactivator p300 from the transcription complex. Hydroxylation of HIF-alpha is catalysed by members of the iron (II) and 2-oxoglutarate dependent oxygenase family. In humans, three prolyl-hydroxylase isozymes (PHD1-3, for prolyl hydroxylase domain enzymes) and an asparaginyl hydroxylase (FIH, for factor inhibiting HIF) have been identified. Recent studies have identified additional post-translational modifications of HIF-alpha including acetylation and phosphorylation. Modulation of the HIF mediated hypoxic response is of potential use in a wide range of disease states including cardiovascular disease and cancer. Here we review current knowledge of the HIF pathway focusing on its regulation by dioxygen and discussion of potential targets and challenges in attempts to modulate the pathway for medicinal application.

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5 percent suspension concentrace (SC), lambda-cyhalothrin 10 percent wettable powder (WP), deltamethrin 2.5 percent SC, and 2.5 percent WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5 percent of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50 percent) were observed for deltamethrin 2.5 percent SC and lambda-cyhalothrin 10 percent WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.

The role of iron and 2-oxoglutarate oxygenases in signalling.

Sensing of ambient dioxygen levels and appropriate feedback mechanisms are essential processes for all multicellular organisms. In animals, moderate hypoxia causes an increase in the transcription levels of specific genes, including those encoding vascular endothelial growth factor and erythropoietin. The hypoxic response is mediated by hypoxia-inducible factor (HIF), an alphabeta heterodimeric transcription factor in which both the HIF subunits are members of the basic helix-loop-helix PAS (PER-ARNT-SIM) domain family. Under hypoxic conditions, levels of HIFalpha rise, allowing dimerization with HIFbeta and initiating transcriptional activation. Two types of dioxygen-dependent modification to HIFalpha have been identified, both of which inhibit the transcriptional response. Firstly, HIFalpha undergoes trans -4-hydroxylation at two conserved proline residues that enable its recognition by the von Hippel-Lindau tumour-suppressor protein. Subsequent ubiquitinylation, mediated by an ubiquitin ligase complex, targets HIFalpha for degradation. Secondly, hydroxylation of an asparagine residue in the C-terminal transactivation domain of HIFalpha directly prevents its interaction with the co-activator p300. Hydroxylation of HIFalpha is catalysed by enzymes of the iron(II)- and 2-oxoglutarate-dependent dioxygenase family. In humans, three prolyl hydroxylase isoenzymes (PHD1-3) and an asparagine hydroxylase [factor inhibiting HIF (FIH)] have been identified. The role of 2-oxoglutarate oxygenases in the hypoxic and other signalling pathways is discussed.