Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4 +/-5.4%; P

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7)M native GIP, with an IC50 value of 2.6 muM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P <0.05 to P <0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P <0.001) and lower the glycemic excursion (1.5-fold decrease; P <0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes. (C) 2002 Elsevier Science (USA).

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C-P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C-P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling.

Use of a batch rotating photocatalytic contactor for the degradation of organic pollutants in wastewater

Aqueous solutions of a chlorinated VOC, 3,4-dichlorobut-1-ene, as well as other pollutants, may be mineralised to carbon dioxide, water and hydrochloric acid using a sealed rotating photocatalytic reactor. The effect of pH, dissolved oxygen concentration, light intensity, pollutant concentration and rotation speed on the degradation rate have been investigated as well as competition kinetics with methanol. This reactor may be optimised to minimise competition effects in mixed solutions. (C) 2001 Elsevier Science B.V. All rights reserved.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

A series of ten cis-dihydro-diol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and H-1 NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Enzyme-catalysed oxygenation and deoxygenation routes to chiral thiosulfinates

Enantioenriched thiosulfinates have been obtained by dioxygenase- and chloroperoxidase-catalysed oxidation of 1,2-disulfides and dimethyl sulfoxide reductase-catalysed deoxygenation.

Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6 of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.

Decreased levels of secretory leucoprotease inhibitor in the Pseudomonas-infected cystic fibrosis lung are due to neutrophil elastase degradation

Secretory leucoprotease inhibitor (SLPI) is a neutrophil serine protease inhibitor constitutively expressed at many mucosal surfaces, including that of the lung. Originally identified as a serine protease inhibitor, it is now evident that SLPI also has antimicrobial and anti-inflammatory functions, and therefore plays an important role in host defense. Previous work has shown that some host defense proteins such as SLPI and elafin are susceptible to proteolytic degradation. Consequently, we investigated the status of SLPI in the cystic fibrosis (CF) lung. A major factor that contributes to the high mortality rate among CF patients is Pseudomonas aeruginosa infection. In this study, we report that P. aeruginosa-positive CF bronchoalveolar lavage fluid, which contains lower SLPI levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective at cleaving recombinant human SLPI. Additionally, we found that only NE inhibitors were able to prevent SLPI cleavage, thereby implicating NE in this process. NE in excess was found to cleave recombinant SLPI at two novel sites in the NH(2)-terminal region and abrogate its ability to bind LPS and NF-kappaB consensus binding sites but not its ability to inhibit activity of the serine protease cathepsin G. In conclusion, this study provides evidence that SLPI is cleaved and inactivated by NE present in P. aeruginosa-positive CF lung secretions and that P. aeruginosa infection contributes to inactivation of the host defense screen in the CF lung.

Type XVIII collagen degradation products in acute lung injury

Introduction In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in patients with lung injury. The aims of the study were to determine if endostatin is elevated in the plasma/bronchoalveolar lavage fluid of patients with acute lung injury and ascertain whether the levels reflect the severity of injury and alveolar inflammation, and to assess if endostatin changes occur early after the injurious lung stimuli of one lung ventilation and lipopolysaccharide (LPS) challenge.

The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required for G1-S Progression

Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G1 and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion significantly impaired G1-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. Moreover, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, known downstream targets of Ras and RhoA signaling. These observations clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target. Cancer Res; 70(8); 3329–39. ©2010 AACR.

The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane Trafficking and Activation but Leaves K-Ras Unaffected

The proto-oncogenic Ras isoforms (H, N, and K) have a C-terminal CAAX motif and undergo the same post-translational processing steps, although they traffic to the plasma membrane through different routes. Previously, we have shown that overexpression of the deubiquitinating enzyme USP17 inhibits H-Ras localization to the plasma membrane. Now we report that whereas H-Ras and N-Ras were unable to localize to the plasma membrane in the presence of USP17, K-Ras4b localization was unaffected. EGF stimulation was unable to induce N-Ras membrane localization in USP17-expressing cells. In addition, N-Ras activity and downstream signaling through the MAPK MEK/ERK and PI3K/JNK pathways were blunted. However, we still detected abundant N-Ras localization at the ER and Golgi in USP17-expressing cells. Collectively, our data showed that the deubiquitinating enzyme USP17 blocks EGF-induced N-Ras membrane trafficking and activation, but left K-Ras unaffected.

The replacement of a native freshwater amphipod by an invader: roles for environmental degradation and intraguild predation

We assessed the extent to which an invader, Gammarus pulex (Crustacea: Amphipoda), has replaced a native, Gammarus duebeni celticus, over a 13-year period in a European river system and some of the abiotic and biotic factors that could account for this. Between 1988 and 2001, 56% of mixed-species sites had become invader-only sites, whereas no mixed sites had become native only again. The native dominated areas of higher dissolved oxygen and water quality, with the reciprocal true for the invader. Field transplant experiments revealed that native survivorship was lower in areas where it had been replaced than in areas where the invader does not yet occur. In invader-only areas, native survivorship was lower than that of the invader when kept separately and lowest when both species were kept together. We also observed predation of the native by the invader. Laboratory oxygen manipulation experiments revealed that at 30% saturation, the native's survivorship was two thirds that of the invader. We conclude that decreasing water quality favours replacement of the native by the invader.