Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a ß-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. ß-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

Obesity and kidney disease in type 1 and 2 diabetes: an analysis of the National Diabetes Audit

Background: Obesity is increasingly prevalent in many countries. Obesity is a major risk factor for the development of type 2 diabetes but its relationship with diabetic kidney disease (DKD) remains unclear. Some studies have suggested that the metabolic syndrome (including obesity) may be associated with DKD in type 1 diabetes. Aim: To investigate the association between obesity and DKD. Design: Retrospective cross-sectional study. Methods: National Diabetes Audit data were available for the 2007–08 cycle. Type 1 and 2 diabetes patients with both a valid serum creatinine and urinary albumin:creatinine ratio were included. DKD was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2, albuminuria or both. Logistic regression was used to analyse associations of obesity (body mass index ≥30 kg/m2) and other variables including year of birth, year of diagnosis, ethnicity and stage of kidney disease. Results: A total of 58 791 type 1 and 733 769 type 2 diabetes patients were included in the analysis. After adjustment, when compared with type 1 diabetes patients with normal renal function those with DKD were up to twice as likely to be obese. Type 2 DKD patients were also more likely to be obese. For example, type 2 diabetes patients with an eGFR <15 ml/min/1.73 m2 and normoalbuminuria, microalbuminuria or macroalbuminuria were all more likely to be obese; odds ratios (95% CI) 1.65 (1.3–2.1), 1.56 (1.28–1.92) and 1.27 (1.05–1.54), respectively. Conclusions: This study has highlighted a strong association between obesity and kidney disease in type 1 diabetes and confirmed their association in type 2 diabetes.

Tracking down the cause of proteinuria in primary care

Proteinuria originates from the kidney and occurs as a result of injury to either the glomerulus or the renal tubule or both. It is relatively common in the general population with reported point prevalence of up to 8% but the prevalence falls to around 2% on repeated testing. Chronic glomerular injury resulting in proteinuria may be secondary to prolonged duration of diabetes or hypertension. A tubular origin of proteinuria may be associated with inflammation of renal tubules triggered by prescribed drugs or ingested toxins. In the absence of obvious clues to the cause of persistent proteinuria on history or clinical examination it is worthwhile reviewing the patient's prescribed drugs to identify any potentially nephrotoxic agents e.g. NSAIDs. NICE guidelines recommend screening for proteinuria in individuals at higher risk for chronic kidney disease (CKD). These include patients with diabetes, hypertension, cardiovascular disease, connective tissue disorders, a family history of renal disease and those prescribed potentially nephrotoxic drugs. Patients with sudden onset of lower limb oedema and associated proteinuria should have a serum albumin level measured to exclude the nephrotic syndrome. Renal tract ultrasound will measure kidney size, and detect scarring associated with chronic pyelonephritis or prior renal stone disease which can cause proteinuria.

Tolvaptan in patients with autosomal dominant polycystic kidney disease

The course of autosomal dominant polycystic kidney disease (ADPKD) is often associated with pain, hypertension, and kidney failure. Preclinical studies indicated that vasopressin V(2)-receptor antagonists inhibit cyst growth and slow the decline of kidney function.

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD(+) or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

Narrow-band ultraviolet B exposure increases serum vitamin D levels in haemodialysis patients

Chronic kidney disease (CKD) patients are especially prone to vitamin D insufficiency. Narrow-band ultraviolet B (NB-UVB) treatment increases serum 25-hydroxyvitamin D [25(OH)D] in dermatological patients, and we studied whether it also improves vitamin D balance in CKD patients on haemodialysis.

Could occupational physical activity mitigate the link between moderate kidney dysfunction and coronary heart disease?

Background

Chronic kidney disease is now regarded as a risk factor for cardiovascular disease. The impact of occupational or non-occupational physical activity (PA) on moderate decreases of renal function is uncertain.

We aimed to identify the potential association of PA (occupational and leisure-time) on early decline of estimated glomerular filtration rate (eGFR) and to determine the potential mediating effect of PA on the relationship between eGFR and heart disease.

From the PRIME study analyses were conducted in 1058 employed men. Energy expended during leisure, work and commuting was calculated. Linear regression analyses were used to determine the link between types of PA and moderate decrements of eGFR determined with the KDIGO guideline at the baseline assessment. Cox proportional hazards analyses were used to explore the potential effect of PA on the relationship between eGFR and heart disease, ascertained during follow-up over 10 years.

For these employed men, and after adjustment for known confounders of GFR change, more time spent sitting at work was associated with increased risk of moderate decline in kidney function, while carrying objects or being active at work was associated with decreased risk. In contrast, no significant link with leisure PA was apparent. No potential mediating effect of occupational PA was found for the relationship between eGFR and coronary heart disease.

Occupational PA (potential modifiable factors) could provide a dual role on early impairment of renal function, without influence on the relationship between early decrease of e-GFR and CHD risk.

Factors influencing survival after kidney transplant failure

BACKGROUND: The failure of a kidney transplant is now a common reason for initiation of dialysis therapy. Kidney transplant recipients commencing dialysis have greater morbidity and mortality than transplant-naïve, incident dialysis patients. This study aimed to identify variables associated with survival after graft failure.

METHODS: All recipients of first, deceased donor kidney transplants performed in Northern Ireland between 1986 and 2005 who had a functioning graft at 12 months were included (n = 585). Clinical and blood-derived variables (age, gender, primary renal disease, diabetic status, smoking status, human leukocyte antigen (HLA) mismatch, acute rejection episodes, immunosuppression, cardiovascular disease, graft survival, haemoglobin, albumin, phosphate, C reactive protein, estimated glomerular filtration rate (eGFR), rate of eGFR decline, dialysis modality, and access) were collected prospectively and investigated for association with re-transplantation and survival. The association between re-transplantation and survival was explored by modelling re-transplantation as a time-dependent covariate.

RESULTS: Median follow-up time was 12.1 years. Recipients with a failing graft (158/585) demonstrated rapid loss of eGFR prior to graft failure, reducing the time available to plan for alternative renal replacement therapy. Median survival after graft failure was 3.0 years. In multivariate analysis, age and re-transplantation were associated with survival after graft failure. Re-transplantation was associated with an 88% reduction in mortality.

CONCLUSIONS: Optimal management of kidney transplant recipients with failing grafts requires early recognition of declining function and proactive preparation for re-transplantation given the substantial survival benefit this confers. The survival benefit associated with re-transplantation persists after prolonged exposure to immunosuppressive therapy.

A Recyclable Acidic Ionic Liquid Gel Catalyst for Dehydration: Comparison with an Analogous SILP Catalyst.

An acid-functionalized ionic liquid was entrapped within a silica gel to yield a recyclable liquid phase catalyst for the dehydration of rac-1-phenyl ethanol. Hot filtration tests showed that the activity was within the gel. Comparison with an analogous SILP system revealed fundamental differences in the properties and behavior of the materials.

Development of a slurry continuous flow reactor for photocatalytic treatment of industrial waste water

The water treatment capability of a novel photocatalytic slurry reactor was investigated using methylene blue (MB) as a model pollutant in an aqueous suspension. A pellet TiO ₂ catalyst was employed and this freed the system from the need of filtration of catalyst following photocatalysis. This configuration combines the high surface area contact of catalyst with pollutant of the slurry reactor and also offers a high illumination of catalyst by its unique array of weir-like baffles. In this work, the batch adsorption of MB from aqueous solution (10μM) onto the TiO ₂ catalyst was studied, adsorption isotherms and kinetics were determined from the experimental data. Complete degradation of MB was achieved within 60 min illumination with various loadings of catalyst (30-200 g L ^-1). A modest catalyst loading (30 g L ^-1) achieved 98% degradation within 60 min of irradiation. Experimental results indicate that this novel reactor configuration has a high effective mass transfer rate and UV light penetration characteristics. 

Planning Urban Food Production into Today's Cities

If cities are to become more sustainable and resilient to change it is likely that they will have to engage with food at increasingly localised levels, in order to reduce their dependancy on global systems. With 87 percent of developed regions estimated to be living in cities by 2050 it can be assumed that the majority of this localised production will occur in and around cities.
As part of a 12 month engagement, Queen’s University Belfast designed and implemented an elevated aquaponic food system spanning the top floor and exterior roof space of a disused mill in Manchester, England. The experimental aquaponic system was developed to explore the possibilities and difficulties associated with containing fish tanks, filtration units, vertical growing systems and roof top growing systems within and upon existing buildings, including the structural considerations needed when undertaking such transformations. Although capable of producing 4000 crops at any one time, the elevated aquaponic system utilised space within the existing building, which could otherwise be used as lettable area, and also located some crop growth within the building where light levels are reduced.
The following paper takes the research collected from the elevated aquaponic system and extrapolates the findings across a whole city. The resulting research enables the agricultural productive capacity of todays cities to be determined and a frame work of implementation to be developed for city wide food production. The research focuses specifically on facade and roof based systems, thus elevating the need to utilise lettable area within cities in addition to locating crops where light levels are highest.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

Biosensor based toxicity dissection of tannery and associated environmental samples

Kenyan tannery and associated environmental samples were selected for ecotoxicological assessment. A tool-kit of techniques was developed, including whole-cell biosensor and chemical assays. A luminescence based bacterial biosensor (Escherichia coli HB101 pUCD607) (via a multi-copy plasmid) was used for toxicity assessment. Samples were manipulated prior to biosensor interrogation to identify the nature of the toxic contaminants. Untreated samples (before any manipulations) showed a strong toxic effect at the discharge point in comparison to other sampling points. Sparging was used to identify toxicity associated with volatile organics. The toxicity of contaminants, removed by treatment with activated charcoal was identified for all the sampling points except for those upstream of effluent discharges. Filtration identified toxicity associated with suspended solids. Changes in availability of toxic contaminants due to pH adjustment of most samples from the tannery effluent treatment pits were also associated with the extreme pH values (4.0 and 8.0). The approach used has highlighted the complexicity of toxic pollutants in effluent from the tanning industry and the dissection of toxicity points to possible remediation strategies for effluents from the tanning industry.