Critical evaluation of different mass transfer equations to model N2O emissions from water resource recovery facilities with diffuse aeration.

N2O measurements by liquid sensors in aerated tanks are an input to gas-liquid mass-transfer models for the prediction of N2O off-gas emissions. The prediction of N2O emissions from Water Resource Recovery Facilities (WRRFs) was evaluated by three different mass-transfer models using Benchmark Simulation Model 1 (BSM1) as a reference model. Inappropriate selection of mass-transfer model may result in miscalculation of carbon footprints based on soluble N2O online measurements. The film theory considers a constant mass-transfer expression, while more complex models suggest that emissions are affected by the aeration type, efficiency, and tank design characteristics. The differences among model predictions were 10-16% at dissolved oxygen (DO) concentration of 0.6 g/m3, when biological N2O production was the highest, while the flux of N2O was 20.0-24 kg N2O-N/d. At lower DO, the nitrification rate was low, while at DO higher than 2 g/m3, the N2O production was reduced leading to higher rates of complete nitrification and a flux of 5 kg N2O-N/d. The differences increased to 14-26% in deeper tanks, due to the pressure assumed in the tanks. The predicted emissions are also affected by the aeration efficiency when KLaN2O depends on the airflow instead of the KLaO2. Increasing the nitrogen loading rate under DO concentration of 0.50-0.65 g/m3 increased the differences in predictions by 10-20% in both alpha 0.6 and 1.2. A sensitivity analysis indicated that the selection of different mass-transfer models did not affect the selection of biochemical parameters for N2O model calibration.

Correlation between nitrous oxide (N2O) emission and carbon to nitrogen (COD/N) ratio in denitrification process: a mitigation strategy to decrease greenhouse gas emission and cost of operation.

The reliability and accuracy of in-situ ion selective electrode and ultraviolet (NOx) probes have been investigated at four different treatment plants with different operational conditions. This study shows that the mentioned probes tend to compromise their accuracy and trending stability at lower NOx of <1.0 mg N/L, which if used as a measuring variable for PI feedback controller for denitrification (biological reduction of nitrate to nitrogen gas), would cause overfeeding the external carbon source. In-situ Clark-type N2O sensors, recently introduced for industrial scale use (Unisense Environment) could potentially open a new horizon in the automation of biological processes and particularly denitrification. To demonstrate the applicability of such probes for automation, two in-situ N2O probes were used in two treatment plants in parallel with NOx-N probes. The effects of operational conditions such as COD/N ratios and the correlation between NOx and N2O were investigated at those plants. N2O production at non-detect dissolved oxygen concentrations and pH of 7-7.2 were found to be a function of influent nitrogen load or the ratio of COD/NINFLUENT. Finally, using an N2O probe as a proxy sensor for nitrates is proposed as a measured variable in the PI feedback in the automation of the denitrification process with a NOx set point of <1.2 mg N/L).

C-terminal modification of osteopontin inhibits interaction with the αVß3-integrin.

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the α(V)ß(3)-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the α(V)ß(3)-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the α(V)ß(3)-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the α(V)ß(3)-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the α(V)ß(3)-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the α(V)ß(3)-integrin by modification of the highly conserved C-terminal region of the protein.

Selection of a novel and highly specific tumor necrosis factor alpha (TNFalpha) antagonist: insight from the crystal structure of the antagonist-TNFalpha complex.

Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.

Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice.

OBJECTIVE: Uremia markedly increases the risk of atherosclerosis. Thus, effective anti-atherogenic treatments are needed for uremic patients. This study examined effects of non-lipidated recombinant human apoA-I (h-apoA-I) and a recombinant trimeric apoA-I molecule (TripA-I) on lipid metabolism and atherosclerosis in uremic apoE-/- mice. METHODS AND RESULTS: Upon intraperitoneal injection, h-apoA-I and TripA-I rapidly associated with plasma HDL and reduced mouse apoA-I plasma levels without affecting total or HDL cholesterol concentrations. The plasma half-life was approximately 36 h for TripA-I and approximately 16 h for h-apoA-I. Injection of h-apoA-I (100mg/kg) or TripA-I (100mg/kg) twice weekly for 7 weeks did not affect the cross-sectional area of atherosclerotic lesions in the aortic root, or the en face lesion area and cholesterol content in the thoracic aorta in uremic apoE-/- mice. Also, the treatments did not affect expression of selected inflammatory genes in the thoracic aorta or plasma concentrations of soluble ICAM-1 and VCAM-1. However, h-apoA-I-treated mice had larger smooth muscle cell-staining areas in aortic root plaques than PBS-treated mice (4.8+/-0.8% vs. 2.5+/-0.6%, P<0.05). CONCLUSIONS: The data suggest that long-term treatment with non-lipidated h-apoA-I or TripA-I might affect plaque composition but does not reduce atherosclerotic lesion size in uremic apoE-/- mice.

Regulation of insulin-like growth factor (IGF) bioactivity by sequential proteolytic cleavage of IGF binding protein-4 and -5.

The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.

Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator.

In the search for new ligands for the plasminogen kringle 4 binding-protein tetranectin, it has been found by ligand blot analysis and ELISA that tetranectin specifically bound to the plasminogen-like hepatocyte growth factor and tissue-type plasminogen activator. The dissociation constants of these complexes were found to be within the same order of magnitude as the one for the plasminogen-tetranectin complex. The study also revealed that tetranectin did not interact with the kindred proteins: macrophage-stimulating protein, urokinase-type plasminogen activator and prothrombin. In order to examine the function of tetranectin, a kinetic analysis of the tPA-catalysed plasminogen activation was performed. The kinetic parameters of the tetranectin-stimulated enhancement of tPA were comparable to fibrinogen fragments, which are so far the best inducer of tPA-catalysed plasminogen activation. The enhanced activation was suggested to be caused by tetranectin's ability to bind and accumulate tPA in an active conformation.

Annexin-V binds to the intracellular part of the beta(5) integrin receptor subunit.

Bovine lactadherin binds to the alpha(v)beta(3) and alpha(v)beta(5) integrins in an RGD-dependent manner and also to anionic phospholipids. During the affinity purification of lactadherin binding receptors, a 35-kDa protein persistently coeluted with the alpha(v)beta(5) integrin receptor. Subsequently, peptide mapping, amino acid sequencing, and mass spectrometry analysis identified this protein as bovine annexin-V. Annexin-V accompanied the integrin receptor eluted with either RGD peptide or with EDTA suggesting that annexin-V bound specifically to the alpha(v)beta(5) integrin. To further investigate this putative interaction of annexin-V with the alpha(v)beta(5) integrin receptor, human annexin-V and intracellular domains of the human alpha(v)beta(5) integrin subunits were used in ligand blotting assays. Radiolabeled annexin-V showed weak binding to the intracellular part of beta(5) integrin subunit. However, by adding the aminophospholipid, phosphatidyl serine, the interaction with the beta(5) cytoplasmic peptide was enhanced many fold. Furthermore, the interaction was shown to be independent of phosphorylation, as annexin-V bound to unphosphorylated beta(5) peptide at a similar level to the phosphorylated peptide. Since binding of annexin-V to the alpha(v) integrin subunit tail was not detected, annexin-V was shown to associate specifically with the beta(5) cytoplasmic tail. Together these findings suggest a novel link between annexins and the integrin receptor family.