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Biomedical subjects

P A Wright

Publications and source records attributed to P A Wright.

At least 19 recordsLinked to original sources
PubMed2006

Rapid increase in the partial pressure of NH3 on the cutaneous surface of air-exposed mangrove killifish, Rivulus marmoratus.

Mangrove killifish, Rivulus marmoratus, are tolerant of prolonged periods of air exposure (>30 days). Air-exposed R. marmoratus eliminate more than 40% of their total ammonia through NH(3) volatilization; however, the sites and mechanisms are unclear. We hypothesized that the cutaneous surface is an important site of NH(3) volatilization in air-exposed R. marmoratus. Ion-selective microelectrodes were used to measure the NH(4)(+) concentration and pH in the boundary layer on the cutaneous surface of fish in water or air (acute: 1 h, chronic: 11 days). Following acute and chronic air exposure, there was a approximately 18-fold increase in the NH(4)(+) concentration and a 0.3-0.6 pH unit increase on the cutaneous surface of R. marmoratus. In air-exposed fish, the calculated cutaneous partial pressure (P(NH(3))) was 608-1,251 microTorr, representing a 33- to 75-fold increase over control (immersed) fish. The P(NH(3)) on the cutaneous surface water film was more than sufficient to account for the rate of NH(3) volatilization under terrestrial conditions. Together, these data indicate that during air exposure, R. marmoratus utilize the cutaneous surface as a key site of NH(3) volatilization.

Adaptation, Physiological↗
PubMed2004

Dogmas and controversies in the handling of nitrogenous wastes: osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity.

Marine elasmobranchs retain relatively high levels of urea to counterbalance the osmotic strength of seawater. Oviparous species, such as the little skate Raja erinacea, release encapsulated embryos that hatch after about 9 months on the seafloor. To study the ureosmotic capability of skate embryos, we measured a variety of possible osmolytes and ornithine-urea cycle (OUC) enzyme activities in little skate embryos, and determined their physiological response to dilute seawater (75% SW) exposure relative to controls (100% SW). The urea:trimethylamine oxide (TMAO) + other osmolytes ratio was 2.3-2.7:1. At the earliest stage of development investigated (4 months), there were significant levels of the key OUC enzyme, carbamoyl phosphate synthetase III, as well as ornithine transcarbamoylase, arginase and glutamine synthetase, providing evidence for a functional OUC. Embryos (4 and 8 months) survived and recovered from exposure to 5 days of 75% SW. There was a significant increase in the rate of urea excretion (five- to tenfold), no change in OUC enzyme activities, and significant decreases in the tissue content of urea, TMAO and other osmolytes in embryos exposed to 75% SW compared to 100% SW. Taken together, the data indicate that little skate embryos synthesize and retain urea, as well as a suite of other osmolytes, in order to regulate osmotic balance with the external environment. Interestingly, these ureosmotic mechanisms are in place as early as 4 months, around the time at which the egg capsule opens and the embryo is in more direct contact with the external environment.

Analysis of Variance↗
PubMed2004

Assessment of pasteurisation of milk and cream produced by on-farm dairies using a fluorimetric method for alkaline phosphatase activity.

The alkaline phosphatase test is used as an indicator of adequate pasteurisation of milk and cream. A proprietary fluorimetric technique (Fluorophos) is a sensitive and quantitative method for the determination of alkaline phosphatase (ALP) activity in milk products. Currently, adequate pasteurisation of milk products is regarded as confirmed in samples that contain a residual bovine ALP activity of < or =500 mU/litre. This is equivalent to the statutory acceptable level of 4ug phenol/ml required by the EC analytical method. The purpose of the present study was to assess the effectiveness of pasteurisation of milk and cream produced by on-farm dairies. In a longitudinal study over a four-year period, 4,999 samples of milk and cream were collected from 130 on-farm dairies and from two large commercial dairies in NW England for comparison. Bovine ALP activity of >500 mU/litre was deemed as a failure and was found in 3.5% of whole milk, 2.4% semiskimmed milk, 5.0% of skimmed milk, and 39% of cream samples from on-farm dairies. Bovine ALP activity of >100 and <500 mU/litre was found in 18.4% of whole milk, 9.3% of semi-skimmed milk, 13.2% skimmed milk and 44.5% of cream samples from on-farm dairies. Results with skimmed milk samples showed significantly lower bovine ALP activity than whole milk. All 409 milk and cream samples from two large commercial dairies passed the fluorimetric test at less than 500 mU/litre of bovine ALP, and 99% of these milk and cream samples had bovine ALP activity of less than 100 mU/litre. The presence of residual bovine phosphatase indicates a failure and may be due to either inadequate pasteurisation or post pasteurisation contamination with raw milk. Residual bovine phosphatase was demonstrated in 108/114 (94.7%) of milk samples with a bovine ALP activity greater than 500 mU/litre, i.e. true failures. Of more concern is that residual bovine phosphatase was found in 395/401 (98.5%) of samples that gave bovine ALP activity greater than 100 mU/litre but equal to or less than 500 mU/litre. Residual bovine phosphatase was demonstrated in 37/108 (30.2%) of cream samples with bovine ALP activity greater than 500 mU/litre. Presence of reactivated bovine phosphatase is not an indication of a failure but can mask the presence of residual bovine phosphatase. Reactivated bovine phosphatase was found in 74/106 (69.8%) of cream samples. Our results confirm that the more sensitive fluorimetric method is suitable for testing pasteurised whole milk and semiskimmed milk, but for statutory purposes the acceptable level of residual bovine phosphatase should be <100 mU/litre. Our findings have highlighted a potential problem when testing skimmed milk and cream samples from on-farm dairies. To ensure public safety we need more stringent standards for the ALP test and new methods that will accurately confirm that pasteurisation of these products has been achieved.

Alkaline Phosphatase↗
PubMed2002

Phenotypic diversity of Campylobacter isolates from sporadic cases of human enteritis in the UK.

AIMS: The aim of this study was to identify and subtype a large collection of isolates of Campylobacter spp. to quantify diversity among strains causing human disease from geographically diverse sources in the United Kingdom. METHODS AND RESULTS: Isolates were characterized by the Penner serotyping scheme, Preston phage typing and biotyping methods. The diversity index calculated from the combined results of all three methods was 0.997 and indicated that isolates from sporadic cases of infection are very diverse. Strong associations between common phagetypes (PG52, PG121 and PG55) and the three most common serotypes (HS1, HS2 and HS4) found in the study were evident. CONCLUSIONS: Strains of C. jejuni causing human infections in the United Kingdom are very phenotypically diverse. Individual strains characterized by serotype, phagetype and biotype were detected throughout the 7-month study period and from geographically distinct sources, indicating an unrecognized outbreak or other epidemiologically significant source of human infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The low frequency incidence of most C. jejuni strains should enable easy recognition of outbreaks by strain type surveillance at local, regional and national level in the United Kingdom. The characterization of common strain profiles in this study by simple phenotypic methods could provide the basis for strain specific epidemiological studies for reservoirs of infection and transmission routes for human infection.

Bacterial Typing Techniques↗
PubMed2002

Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus I. The influence of environmental salinity and external ammonia.

At a field site in Belize, mangrove killifish Rivulus marmoratus inhabit hypersaline waters (up to 48 per thousand containing approximately 1 mmol l(-1) ammonia. We tested the hypotheses that R. marmoratus modify their nitrogen metabolism and excretion (i) by accumulating free amino acids (FAAs) and urea in the tissues during hyperosmotic stress and (ii) by shifting to ureotelism and accumulating FAAs during hyperammonia stress. Urea excretion (J(Urea)) (but not ammonia excretion, J(Amm)) displayed a diurnal pattern, with significantly less (75 %) urea excreted at night than during the day in both laboratory-reared clones and wild-caught killifish. When fish were exposed to hypersaline conditions (45 per thousand sea water), J(Urea) was significantly reduced and tissue urea and FAA levels were elevated compared with those of control fish (15 per thousand sea water). When R. marmoratus were exposed to 0, 1, 2, 5 and 10 mmol l(-1) NH(4)Cl (pH 8) for 48 h, no differences were found in J(Urea). Remarkably, prolonged exposure (10 days) to 5 mmol l(-1) NH(4)Cl (pH 8) did not result in an elevation of tissue ammonia levels. In addition, tissue urea and total FAA levels did not differ between control and ammonia-exposed fish after > or =4 days. We propose that the euryhaline R. marmoratus retain urea and FAAs within their tissues in response to extreme osmotic stress. In contrast to many ammonia-tolerant fishes, R. marmoratus do not shift to ureotelism during prolonged hyperammonia stress, nor do they convert nitrogenous wastes into FAAs. The data suggest that killifish continue to eliminate ammonia despite an unfavourable blood-to-water gradient, thereby avoiding accumulation of ammonia.

Amino Acids↗
PubMed2002

Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus II. Significant ammonia volatilization in a teleost during air-exposure.

The mangrove killifish Rivulus marmoratus can tolerate prolonged periods of air-exposure (>1 month). During these periods of emersion, we hypothesized that R. marmoratus would convert potentially toxic ammonia into urea and free amino acids (FAAs). In air-exposed fish, both ammonia (J(Amm)) and urea (J(Urea)) excretion continued at approximately 57 % and 39 %, respectively, of submerged rates. Remarkably, approximately 42 % of the total ammonia excreted during air-exposure was through NH(3) volatilization. Ammonia did not accumulate in whole-body tissues of air-exposed fish, but levels of both urea and some FAAs (primarily alanine and glutamine) were up to twofold higher after 10 days. The activities of the ornithine-urea cycle enzymes carbamoyl phosphate synthetase III and ornithine transcarbamylase increased (by approximately 30 % and 36 %, respectively) in whole-body tissues of air-exposed fish, while levels of arginase remained unchanged. The activities of enzymes involved in amino acid and oxidative metabolism were not significantly different between control and air-exposed fish. Partitioning of the anterior and posterior ends of immersed fish revealed that just over half (57 %) of the total nitrogen (ammonia+urea) was excreted through the anterior end of the fish, presumably via the branchial tissues, while emersed fish increased excretion via the posterior end (kidney+skin). R. marmoratus do not undergo a shift towards ureotelism during air-exposure. Rather, we propose that R. marmoratus are able to survive on land for extended periods without significant ammonia accumulation because they continuously release ammonia, partially by NH(3) volatilization.

Air↗
PubMed2001

Solid-state transformations of zinc 1,4-benzenedicarboxylates mediated by hydrogen-bond-forming molecules.

The zinc 1,4-benzenedicarboxylates [Zn3(bdc)3(H2O)3] . 4 DMF (1; bdc = 1,4-benzenedicarboxylate), [Zn(bdc)(H2O)] . DMF (2), and [Zn(bdc)] DMF (3) crystallise at room temperature from mixtures of toluene/ dimethylformamide (DMF) under concentrated, dilute and dry conditions, respectively. The structure of phase 1 (monoclinic: P2(1)/c, a 13.065(1), b = 9.661(1), c = 18.456(1) A, beta = 106.868(2) degrees) consists of layers containing stacks of three zinc cations linked by mono- and bidentate bdc groups. Structure 1 converts to the known phase 2 by an irreversible, reconstructive phase transformation, whereas 2 and 3 interconvert reversibly upon the loss or addition of water. Removal of all solvent molecules included during crystallisation gives poorly crystalline [Zn(bdc)] (4), which is readily converted to highly crystalline solids upon contact with hydrogen-bond-forming molecules such as water, DMF and small alcohols. The crystal structures of the mono- and dihydrates [Zn(bdc)(H2O)] (6) and [Zn(bdc)-(H2O)2] (7) have been determined ab initio from powder X-ray diffraction data (compound 6, monoclinic: C2/c, a = 17.979(1), b = 6.352(1), c = 7.257(1) A, beta=91.477(1) compound 7, monoclinic: C2/c, a = 14.992(1), b = 5.0303(2), c = 12.098(1) A, beta = 103.82(1) degrees). The methanol adduct [Zn3(bdc)3] . 6CH3OH (5) is the same as that prepared previously by direct crystallisation. Comparison of these adduct structures with those prepared directly reveal that they are formed by in situ recrystallisations. Subsequent removal of included molecules gives amorphous [Zn(bdc)], which can be recrystallised again when placed in contact with hydrogen-bond-forming molecules.

Journal Article↗
PubMed2001

Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus.

During the denning period, black bears (Ursus americanus) are capable of enduring several months without food. At the same time, female bears that are pregnant or lactating have an added metabolic stress. Based on laboratory studies, much of the energy required to support metabolism and lactation during denning in black bears comes from lipid reserves. These lipid reserves are mobilized and the most metabolically active lipid fraction in the blood are nonesterified fatty acids (NEFA). Therefore, we hypothesized that plasma NEFAs would be higher in denning relative to active bears and in lactating relative to non-lactating female bears. We further hypothesized that in bears with elevated plasma NEFA levels, other lipid-related parameters (e.g., ketone bodies, albumin, cholesterol, lipase) would also be elevated in the plasma. Denning bears had significantly increased NEFA levels in all classes (saturates, monoenes, and polyenes). A doubling of plasma NEFA levels and a 33% increase in albumin, the plasma fatty acid binding protein, in denning bears, resulted in NEFA/albumin ratios that were higher in denning bears (4:1) compared to those of active bears (3:1). Bears became relatively ketonemic with a 17-fold increase in D-beta-hydroxybutyrate levels during the denning period. Plasma cholesterol approximately doubled and lipase was ten-fold lower in denning relative to active bears. These findings indicate a strong correlation between plasma lipid metabolites and the denning period in a wild population of black bears.

Animals↗
PubMed2001

Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (Oncorhynchus mykiss).

The purpose of this study was to determine if carbamoyl phosphate synthetase III (CPSase III) and related urea cycle enzyme activities in skeletal muscle tissue of juvenile rainbow trout (Oncorhynchus mykiss) increase during short- or long-term exercise, in parallel with changes in whole-body urea excretion rates. Urea excretion was elevated by 65% in fish that swam at high-speed (50 cm/s) vs. low-speed (20 cm/s) over a 2-h period, with no significant changes in CPSase III, ornithine transcarbamoylase or glutamine synthetase activities in muscle tissue. Fish that swam for 4 days at high-speed had higher rates of ammonia excretion and GSase activity in muscle and liver tissue relative to low-speed swimmers. Calculations showed that 47-53% of excreted urea, theoretically could be accounted for by total muscle CPSase III activity in juvenile and adult trout. The data indicate that increases in the rate of urea excretion during short-term high intensity exercise are not linked to higher activities of urea cycle enzymes in muscle tissue, but this does not rule out the possibility of increased flux through muscle CPSase III and related enzymes. Furthermore, these results indicate that urea cycle enzyme activities in skeletal muscle tissue can account for a significant portion of total urea excretion in juvenile and adult trout.

Animals↗
PubMed2001

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.

Acylation↗
PubMed2001

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.

Animals↗
PubMed2001

Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch.

In elasmobranch fishes, urea occurs at high concentrations (350-600 mM) in the body fluids and tissues, where it plays an important role in osmoregulation. Retention of urea by the gill against this huge blood-to-water diffusion gradient requires specialized adaptations to the epithelial cell membranes. Experiments were performed to determine the mechanisms and structural features that facilitate urea retention by the gill of the spiny dogfish Squalus acanthias. Analysis of urea uptake by gill basolateral membrane vesicles revealed the presence of a phloretin-sensitive (half inhibition 0.09 mM), sodium-coupled, secondary active urea transporter (Michaelis constant = 10.1 mM, maximal velocity = 0.34 micromol. h(-1). mg protein(-1)). We propose that this system actively transports urea out of the gill epithelial cells back into the blood against the urea concentration gradient. Lipid analyses of the basolateral membrane revealed high levels of cholesterol contributing to the highest reported cholesterol-to-phospholipid molar ratio (3.68). This unique combination of active urea transport and modification of the phospholipid bilayer membrane is responsible for decreasing the gill permeability to urea and facilitating urea retention by the gill of Squalus acanthias.

Acetamides↗
PubMed2001

Ammonia detoxification and localization of urea cycle enzyme activity in embryos of the rainbow trout (Oncorhynchus mykiss) in relation to early tolerance to high environmental ammonia levels.

The present study investigated the role of ammonia as a trigger for hatching, mechanisms of ammonia detoxification and the localization of urea cycle enzymes in the early life stages of freshwater rainbow trout (Oncorhynchus mykiss). The key urea cycle enzyme carbamoyl phosphate synthetase III was found exclusively in the embryonic body (non-hepatic tissues); related enzymes were distributed between the liver and embryonic body. 'Eyed-up' trout embryos were exposed either acutely (2h) to 10 mmol l(-1) NH(4)Cl or chronically (4 days) to 0.2 mmol l(-1) NH(4)Cl. Time to hatching was not affected by either acute or chronic NH(4)Cl exposure. Urea levels, but not ammonia levels in the embryonic tissues, were significantly higher than in controls after both acute and chronic NH(4)Cl exposure, whereas there were no significant changes in urea cycle enzyme activities. Total amino acid levels in the embryonic tissues were unaltered by chronic ammonia exposure, but levels of most individual amino acids and total amino acid levels in the yolk were significantly lower (by 34-58%) than in non-exposed controls. The data indicate that trout embryos have an efficient system to prevent ammonia accumulation in embryonic tissue, by conversion of ammonia to urea in embryonic tissues and through elevation of ammonia levels in the yolk.

Ammonia↗
PubMed2001

Pathways for urea production during early life of an air-breathing teleost, the African catfish Clarias gariepinus Burchell.

Embryos and larvae of the African catfish Clarias gariepinus excrete significant quantities of urea. The present study focused on the potential urea-generating pathways during early development of this teleost; uricolysis, argininolysis and the ornithine-urea cycle (OUC). Uricase, allantoinase, allantoicase and ureidoglycollate lyase of the uricolytic pathway were expressed in all early life stages and in adult liver of C. gariepinus. Uricase activity increased in starved larvae compared with yolk-sac larvae. The key regulatory enzyme of the teleost OUC, carbamoyl phosphate synthetase III (CPSase III), was expressed predominantly in muscle of developing C. gariepinus larvae and showed negligible activity in the absence of its allosteric effector N-acetyl-L-glutamate. CPSase III and ornithine carbamoyl transferase activities increased in fed larvae compared with starved larvae. In contrast to the early developmental stages, adult C. gariepinus expressed only low and variable levels of CPSase III, suggesting that, under the experimental conditions employed, OUC expression is influenced by developmental stage in this species. The data indicate that early C. gariepinus life stages express the enzymes necessary for urea production by uricolysis, argininolysis and the OUC, and this may explain why urea tissue levels and urea excretion rates are substantial during the early development of this air-breathing teleost.

Air↗
PubMed2000

'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.

beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.

Animals↗