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P J Schofield

Publications and source records attributed to P J Schofield.

At least 19 recordsLinked to original sources
PubMed1999

Characterisation and expression of the carbamate kinase gene from Giardia intestinalis.

The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.

Amino Acid Sequence↗
PubMed1998

Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis.

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Amino Acid Sequence↗
PubMed1998

Giardia intestinalis: characterization of a NADP-dependent glutamate dehydrogenase.

Glutamate dehydrogenase from Giardia intestinalis was purified 680-fold to electrophoretic homogeneity with a 42% recovery through a two-step procedure. The most effective step in the purification was the use of CM-Trisacryl that eliminated nearly 99% of the total proteins with 100% recovery. Matrix-assisted laser desorption ionization time-of-flight mass spectrometer was used to analyze the giardial glutamate dehydrogenase after deposition of the purified enzyme on a crystalline layer of 3,5-dimethoxy-4-hydroxy-trans-cinnamic acid. Use of this sample preparation technique allowed the first successful determination of the molecular mass of the enzyme (50,120 +/- 75). Since the molecular weight of the native enzyme was determined to be 270,000 by gel filtration, the enzyme appears to be a hexamer. The enzyme was specific for NADP(H) and functioned more favorably in the direction of glutamate formation than catabolism. The pH optimum was 7.5 for reductive amination of 2-oxoglutarate and 9.3 for oxidative deamination of glutamate. The apparent K(m) values were 0.28 mM for 2-oxoglutarate and 17 microM for NADPH. An unusual biphasic saturation curve characterized the effect of ammonium ion on the activity with a plateau between 40 and 55 mM.

Animals↗
PubMed1998

A novel and simple colorimetric method for screening Giardia intestinalis and anti-giardial activity in vitro.

A new and simple colorimetric method has been developed for determining activity in vitro against Giardia intestinalis. The microtitre plate assay is based upon the nucleoside hydrolase activity released from G. intestinalis by lysis. Action of the nucleoside hydrolase on the substrate analogue, 4-nitrophenyl beta-D-ribofuranoside (NPR), gives rise to a coloured product which may be determined directly by the change in absorbance. A number of other such nucleoside analogues can be similarly used, but NPR is the preferred substrate, since it gives high enzymic activity at a relatively low substrate concentration. The IC50 values determined using this method for the known anti-giardials metronidazole, tinidazole and furazolidone were consistent with previously published values. The method is simple, does not involve radioisotopes or complex instrumentation, and thus provides a convenient method for screening potential anti-giardial agents.

Animals↗
PubMed1998

Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes.

It is increasingly evident that cell swelling is associated with the triggering of many biological processes, including progression of the cell cycle, hormonal response, and gene expression. However, the mechanism by which cell swelling is initially sensed and converted into intracellular signals is still ill-defined. We report here an early event in the detection of cell swelling and initiation of the volume regulatory response in Giardia intestinalis, an ancient representative of the eukaryotic kingdom. Giardial cell swelling, irrespective of the extent, was sensed at a cell volume of 1.06 x isosmotic volume (the threshold volume), at which the transition of the volume regulatory transport system from the 'resting' to the 'open' state occurred. Irreversible modification by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) of reduced thiols affected the threshold volume, but in opposing manners: pCMB increased the threshold volume to 1.14 x and NEM decreased to 0.85 x isosmotic volume. The simple modification of the threshold volume by NEM caused a drastic reduction of giardial cell volume under isosmotic conditions, with a process strikingly similar to the opening of mitochondrial permeability transition pore, a causative event in stress-induced programmed cell death. Substantial evidence supports the hypothesis that modulation of the membrane thiol moieties at the threshold volume, causing the 'all-or-nothing' type of swelling detection, represents the event linking cell swelling to the second messenger systems for volume regulation in present eukaryotes. Pathophysiological implications of alteration of the threshold volume are discussed.

Alanine↗
PubMed1997

Giardia intestinalis: volume recovery in response to cell swelling.

The trophozoite from of the protozoan parasite Giardia intestinalis is subjected to a changing osmotic environment in the small intestine of the host, and consequently effective osmoregulation and control of cell volume are essential to its survival. As a first step toward investigating the mechanism by which hypoosmotically-activated transport is controlled in this organism, we used a light scattering technique to monitor continuously changes in cell volume after osmotic challenge. There was a hyperbolic relationship between A550 and giardial protein concentration, resulting in linear double reciprocal plots and allowing the calculation of relative Giardia cell volumes from A550 values. The initial rate of cell swelling was directly proportional to the hypoosmotic gradient when the hypoosmotic difference was greater than 50 mOsm kg-1. However, a hypoosmotic challenge of < 30 mOsm kg-1 had little effect on cell swelling, suggesting that giardial cell rigidity can resist small changes in medium osmolarity. The use of light scattering as a measure of giardial cell volume changes was validated using a rapidly penetrating solute, ethylene glycol, to induce isoosmotic cell swelling. We have previously shown that trophozoites swelled initially when subjected to a hypoosmotic challenge and that a subsequent regulatory volume decrease was accompanied by rapid alanine efflux and activation of the uptake of an alanine analog, 2-aminoisobutyrate. The ethylene glycol-induced isoosmotic cell swelling was also followed by a regulatory volume decrease, accompanied by a similar rapid release of intracellular alanine and activation of 2-aminoisobutyric acid uptake. This suggests that an increase in cell volume is the primary stimulus for the rapid alanine efflux after hypoosmotic challenge.

Alanine↗
PubMed1995

Amino acid exchange activity of the alanine transporter of Giardia intestinalis.

The influx and efflux of alanine and other amino acids was studied in trophozoites of Giardia intestinalis. Transport of L-[2,3-3H]alanine was used as the index of influx. On the basis of the competition of L-[2,3-3H]alanine uptake by analogues of alanine, the substrate specificity of the alanine transporter was determined. The transporter is an antiport. Influx of alanine or those analogues which inhibited alanine influx caused the efflux of intracellular alanine and a number of amino acids structurally related to alanine. Amino acids unrelated to alanine, such as glutamate, effluxed at a slow rate, and the efflux was not stimulated by extracellular alanine or alanine analogues. However, there was a subset of intracellular amino acids, the alanine subset comprising alanine, serine, glycine, and threonine, the efflux of which was stimulated by external alanine or alanine analogues. Direct measurement by amino acid analysis demonstrated intracellular accumulation of alanine analogues concomitant with the efflux of the alanine subset. These data indicate unequivocal evidence of exchange of intracellular alanine with extracellular alanine analogues, with a 1:1 molar stoichiometry. This is the first demonstration in G. intestinalis of the antiport function of an amino acid transporter.

Alanine↗
PubMed1995

L-arginine transport and metabolism in Giardia intestinalis support its position as a transition between the prokaryotic and eukaryotic kingdoms.

Arginine is metabolized by the arginine dihydrolase pathway in Giardia intestinalis trophozoites and is an important metabolic fuel for this parasite. Radiolabelled arginine was used to characterize the transport of arginine into Giardia intestinalis trophozoites. The transporter had a high affinity for arginine (Km 15 microM) and a high activity [Vmax 76 nmol min-1 (mg protein)-1 at 25 degrees C]. Substrate specificity studies indicated an absolute requirement for the alpha-amino and carboxyl groups, but a tolerance for some substitutions in the guanidino group. The use of non-metabolized arginine analogues in combination with HPLC amino acid analysis of intra- and extracellular pools demonstrated that the arginine transporter is an arginine-ornithine antiport. Investigations of the first step of arginine metabolism, involving arginine deiminase, revealed a relatively high affinity for arginine (Km 0.16 mM) and a large maximal velocity [Vmax 550 nmol min-1 (mg protein)-1 at 37 degrees C]. Substrate specificity studies showed that the arginine deiminase had a characteristically different substrate recognition profile to that of the arginine transporter. Overall, the combination of the transporter and the deiminase result in very low intracellular arginine concentrations and their properties are consistent with the rapid transport of arginine for metabolism via the arginine dihydrolase pathway.

Amino Acid Transport Systems↗
PubMed1995

Physical and linkage mapping of human carbamyl phosphate synthetase I (CPS1) and reassignment from 2p to 2q35.

Carbamyl Phosphate Synthetase I (CPS1) (EC 6.3.4.16) is a highly conserved mitochondrial enzyme catalyzing the first committed step of waste nitrogen metabolism in the urea cycle. Using FISH for physical mapping and CEPH families for linkage analysis, we mapped the CPS1 gene (CPS1) to 2q34-->q35, reassigning it from 2p where it was originally mapped.

Carbamoyl-Phosphate Synthase (Ammonia)↗
PubMed1994

The intracellular amino acid pools of Giardia intestinalis, Trichomonas vaginalis, and Crithidia luciliae.

The total intracellular amino acid profiles of Giardia intestinalis trophozoites, Trichomonas vaginalis, and Crithidia luciliae were determined by sensitive amino acid analysis. The three protozoan parasites exhibited distinctively different amino acid profiles, but all three were dominated by high concentrations of intracellular alanine. This common feature suggests that alanine synthesis is a major aspect of intermediary metabolism in these protozoan parasites. There were also distinctively different aspects, particularly those related to arginine metabolism. Ornithine, citrulline, and ammonia were found in G. intestinalis trophozoites, but no intracellular arginine was detected. This pattern is consistent with the high activity of giardial arginine deiminase and the arginine dihydrolase pathway. However, in contrast, both T. vaginalis and C. luciliae contained considerable intracellular pools of arginine. When the G. intestinalis trophozoites were divided into the two populations existing in in vitro culture--attached and nonattached--there were no significant differences between the amino acid profiles of the two populations, with the exception of citrulline, which was found in lower concentrations in the nonattached cells. The T. vaginalis profile was characterised by high concentrations of valine and leucine, whereas the C. luciliae profile was dominated by high levels of glutamate and proline. Overall, the analysis of the total amino acid pool provides a valuable technique to rapidly highlight those amino acids of potential metabolic significance and to provide a rapid technique for defining the nature of amino acid metabolic interactions in situ.

Amino Acids↗
PubMed1994

Efflux of alanine by Giardia intestinalis.

Giardia intestinalis trophozoites synthesise and then secrete large amounts of alanine into the external medium during growth. This efflux of alanine was studied by preloading cells with L-[2,3-3H]alanine, and determining efflux of radiolabel from intact trophozoites. The efflux of alanine was also determined by measurement of alanine concentration in trophozoites and external medium using high pressure liquid chromatography amino acid analysis. Over the temperature range 4 degrees C to 37 degrees C there was a slow efflux of alanine, but this efflux was greatly stimulated by a number of amino acids structurally similar to alanine, notably glycine, L-serine, L-threonine, L-asparagine and L-glutamine. In contrast, 2-aminoisobutyrate, D-amino acids, and other naturally occurring amino acids had no effect. Those amino acids which stimulated the efflux of intracellular alanine are the same amino acids which inhibited uptake of extracellular alanine. This concordance suggests that an alanine antiport functions for both the influx and efflux of alanine, and acts to maintain a balance between intracellular and extracellular alanine concentrations.

Alanine↗
PubMed1993

The transport and metabolism of alanine by Giardia intestinalis.

The transport and metabolism of L-alanine by Giardia intestinalis trophozoites was characterised. G. intestinalis formed 14CO2 from L-[1-14C]alanine (1 mM) at a rate of 4.8 nmol min-1 (mg protein)-1 at 30 degrees C. The system was saturable, with an apparent Km of 0.29 mM for alanine, and a maximal rate of 6.1 nmol min-1 (mg protein)-1. L-cycloserine inhibited the metabolism, as did a number of amino acids including glycine, serine and threonine. D-alanine and 2-aminoisobutyrate had no effect. G. intestinalis was shown to have a functional transport system for L-alanine. The transporter was saturable with a Km of 1.5 mM and a maximal velocity of 6.1 nmol min-1 (mg protein)-1 at 23 degrees C. It was temperature dependent, with a Q10 of 2.2 and activation energy of 15.9 kcal mol-1. It was not inhibited by potential inhibitors of energy dependent transport. Glycine, L-serine and L-threonine potently inhibited L-alanine transport, whereas D-alanine, beta-alanine and 2-aminoisobutyrate had no effect. L-serine competitively inhibited L-alanine influx. In trophozoites preloaded with [3H]alanine, rapid exchange occurred with external L-alanine and L-serine, but not with D-alanine confirming that L-alanine and L-serine share a common transport site. These observations indicate that G. intestinalis has a functional alanine transporter, which may be an antiport catalysing the exchange of alanine, serine, glycine and threonine.

Alanine↗
PubMed1993

Diabetes care in general practice: an approach to audit of process and outcome.

As a chronic condition in which the major adverse outcomes only occur after many years, diabetes poses special problems for continuing medical audit. The feasibility of continuous audit of process and outcome in diabetes care has been tested in four general practices with organized diabetes care in Newcastle upon Tyne. For all patients with previously diagnosed non-insulin dependent diabetes, the data already collected according to published protocols were assembled into a single database. The time and resource costs of this exercise, together with measures of process, complications, risk factors, and metabolic outcomes were analysed. Data were successfully collected at minimal cost where structured records were completed. Recommended processes had been completed in a high percentage of patients, adverse patient outcomes were limited, and metabolic output measures not unsatisfactory. Nevertheless, attention has been directed to areas where care could be improved. Continuing diabetes audit in primary health care is feasible and helpful, and can use the same measures as in the hospital setting.

Aged↗
PubMed1992

Arginine metabolism during culture of Giardia intestinalis.

The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.

Ammonia↗