Attenuation of cardiovascular remodeling in DOCA-salt rats by the vasopeptidase inhibitor, omapatrilat

Omapatrilat, a vasopeptidase inhibitor, inhibits both neutral endopeptidase and angiotensin-converting enzyme with similar potency. The aim of this study was to investigate whether omapatrilat prevents or reverses cardiovascular remodeling and hypertension in deoxycorticosterone acetate (DOCA)-salt rats. Male Wistar rats (313 2 g, n=114) were uninephrectomized (UNX) with or without further treatment with DOCA and 1% NaCl in the drinking water. Compared with UNX control rats, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, perivascular and interstitial cardiac fibrosis and inflammation, endothelial dysfunction, and the prolongation of ventricular action potential duration within four weeks. The administration of omapatrilat (40 mg/kg/day po) for two weeks commencing two weeks after surgery attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation. In contrast, omapatrilat treatment did not lower systolic blood pressure nor improve endothelial dysfunction. This study concludes that the renin-angiotensin-aldosterone, natriuretic peptide, and bradykinin systems are directly involved in the pathogenesis of cardiovascular remodeling in the DOCA-salt model of hypertension in rats, which may be independent of their effects on blood pressure.

Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) alpha-amylase inhibitor transgene

The nutritive value of transgenic peas expressing an a-amylase inhibitor (alpha-Ail) was evaluated with broiler chickens. The effects of feeding transgenic peas on the development of visceral organs associated with digestion and nutrient absorption were also examined. The chemical composition of the conventional and the transgenic peas used in this study were similar. In the two feeding trials, that were conducted normal and transgenic peas were incorporated into a maize-soybean diet at concentrations up to 500 g kg(-1). The diets were balanced to contain similar levels of apparent metabolisable energy (AME) and amino acids. In the first trial, the birds were fed the diets from 3 to 17days post-hatching and with levels of transgenic peas at 250 g kg(-1) or greater there was a significant reduction in body weight but an increase in feed intake resulting in deceased feed conversion efficiency. In the second trial, in which the birds were fed diets containing 300 g kg(-1) transgenic peas until 40 days of age, growth performance was significantly reduced. It was also demonstrated that the ileal starch digestibility coefficient (0.80 vs 0.42) was significantly reduced in the birds fed transgenic peas. Determination of AME and ileal digestibility of amino acids in 5-week-old broilers demonstrated a significant reduction in AME (12.12 vs 5.08 MJ kg(-1) DM) in the birds fed the transgenic peas. The AME value recorded for transgenic peas reflected the lower starch digestibility of this line. Real digestion of protein and amino acids was unaffected by treatment. Expression of a-Ail in peas did not appear to affect bird health or the utilisation of dietary protein. However, the significant reduction in ileal digestion of starch in transgenic peas does reduce the utility of this feedstuff in monogastric diets where efficient energy utilisation is required. (c) 2006 Society of Chemical Industry.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3) d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L-1. Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosonlonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662. (c) 2005 Elsevier Ltd. All rights reserved.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N2O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. NO accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as NO. After mixing SNDPR sludge with other denitrifying sludge, N2O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N2O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source. (C) 2005 Elsevier B.V. All rights reserved.

Occurrence and simulation of nitrification in two contrasting sugarcane soils from the Australian wet tropics

The concentration of ammonium-nitrogen (NH4+-N) frequently exceeds that of nitrate-N (NO3--N) in Australian wet tropical sugarcane soils. The amount of mineral N in soil is the net result of complex processes in the field, so the objective of this experiment was to investigate nitrification and ammonification in these soils under laboratory conditions. Aerobic and saturated incubations were performed for 1 week on 2 wet tropical soils. Net NO3--N increased significantly in both soils during both types of incubation. A second series of aerobic incubations of these soils treated with NH4+-N and inoculated with subtropical nitrifying soils was conducted for 48 days. Nitrification in the wet tropical soils was not significantly affected by inoculation, and virtually all added N was nitrified during the incubation period. Mineral N behaviour of the 48-day incubations was captured with the APSIM-SoilN model. As nitrification proceeded under laboratory conditions and was able to be captured by the model, it was concluded that nitrification processes in the wet tropical soils studied were not different from those in the subtropical soils. Processes that remove NO3- from the soil, such as leaching and denitrification, may therefore be important factors affecting the proportions of NH4+-N and NO3--N measured under field conditions.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated for the enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N center dot L-1 (3.4 mu M), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacter at approximately 0.011 mg HNO2-N center dot L-1 (0.8 mu M), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N center dot L-1 (1.6 mu M). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Integration of sulphate reduction, autotrophic denitrification and nitrification to achieve low-cost excess sludge minimisation for Hong Kong sewage

An integrated anaerobic-aerobic treatment system of sulphate-laden wastewater was proposed here to achieve low sludge production, low energy consumption and effective sulphide control. Before integrating the whole system, the feasibility of autotrophic denitrification utilising dissolved sulphide produced during anaerobic treatment of sulphate rich wastewater was studied here. An upflow anaerobic sludge blanket reactor was operated to treat sulphate-rich synthetic wastewater (TOC = 100 mg/L and sulphate = 500 mg/L) and its effluent with dissolved sulphide and external nitrate solution were fed into an anoxic biofilter. The anaerobic reactor was able to remove 77-85% of TOC at HRT of 3 h and produce 70-90 mg S/L sulphide in dissolved form for the subsequent denitrification. The performance of anoxic reactor was stable, and the anoxic reactor could remove 30 mg N/L nitrate at HRT of 2 h through autotrophic denitrification. Furthermore, sulphur balance for the anoxic filter showed that more than 90% of the removed sulphide was actually oxidised into sulphate, thereby there was no accumulation of sulphur particles in the filter bed. The net sludge productions were approximately 0.15 to 0.18 g VSS/g COD in the anaerobic reactor and 0.22 to 0.31 g VSS/g NO3--N in the anoxic reactor. The findings in this study will be helpful in developing the integrated treatment system to achieve low-cost excess sludge minimisation.

Feasibility of controlling nitrification in predenitrification plants using DO, pH and ORP sensors

Experimental studies were carried out on a bench-scale nitrogen removal system with a predenitrification configuration to gain insights into the spatial and temporal variations of DO, pH and ORP in such systems. It is demonstrated that these signals correlate strongly with the operational states of the system, and could therefore be used as system performance indicators. The DO concentration in the first aerobic zone, when receiving constant aeration, and the net pH change between the last and first aerobic zones display strong correlations with the influent ammonia concentration for the domestic wastewater used in this study. The pH profile along the aerobic zones gives good indication on the extent of nitrification. The experimental results also showed a good correlation between ORP values in the last aerobic zone and effluent ammonia and nitrate concentrations, provided that DO in this zone is controlled at a constant level. These results suggest that the DO, pH and ORP sensors could potentially be used as alternatives to the on-line nutrient sensors for the control of continuous systems. An idea of using a fuzzy inference system to make an integrated use of these signals for on-line aeration control is presented and demonstrated on the bench-scale system with promising results. The use of these sensors has to date only been demonstrated in intermittent systems, such as sequencing batch reactor systems.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1 beta (IL-1 beta) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from 106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p

Perindopril, an angiotensin converting enzyme inhibitor, in pulmonary hypertensive rats: comparative effects on pulmonary vascular structure and function

1 Hypoxic pulmonary hypertension in rats (10% O-2, 4 weeks) is characterized by changes in pulmonary vascular structure and function. The effects of the angiotensin converting enzyme inhibitor perindopril (oral gavage, once daily for the 4 weeks of hypoxia) on these changes were examined. 2 Perindopril (30 mg kg(-1) d(-1)) caused an 18% reduction in pulmonary artery pressure in hypoxic rats. 3 Structural changes (remodelling) in hypoxic rats included increases in (i) critical closing pressure in isolated perfused lungs (remodelling of arteries (50 mu m 0.d.) and (ii) medial wall thickness of intralobar pulmonary arteries, assessed histologically (vessels 30-100 and 101-500 mu m o.d.). Perindopril 10 and 30 mg kg(-1) d(-1) attenuated remodelling in vessels less than or equal to 100 mu m (lungs and histology), 30 mg kg(-1) d(-1) was effective in vessels 101-500 mu m but neither dose prevented hypertrophy of main pulmonary artery. 3 mg kg(-1) d(-1) was without effect. 4 Perindopril (30 mg kg(-1) d(-1)) prevented the exaggerated hypoxic pulmonary vasoconstrictor response seen in perfused lungs from hypoxic rats but did not prevent any of the functional changes (i.e. the increased contractions to 5-HT, U46619 (thromboxane-mimetic) and K+ and diminished contractions to angiotensins I and II) seen in isolated intralobar or main pulmonary arteries. Acetylcholine responses were unaltered in hypoxic rats. 5 We conclude that, in hypoxic rats, altered pulmonary vascular function is largely independent of remodelling. Hence any drug that affects only remodelling is unlikely to restore pulmonary vascular function to normal and, like perindopril, may have only a modest effect on pulmonary artery pressure.