Robert G Price

• King's College Hospital NHS Foundation Trust

Cadmium is a non-essential metal which because of its extensive use in industry and agriculture presents a worldwide health hazard. Long-term exposure to cadmium results in nephropathy characterised by interstitial nephritis affecting the renal tubules. In addition to occupational exposure, cadmium can contaminate food and water increasing the numb...

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics-a process associated with promoting antibiotic resistan...

To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFO) on Panton-Valentine leukocidin-positive community acquired methicillin-susceptible Staphylococcus aureus (PVL +ve CA-MSSA) biofilm formation. Biofilms from PVL +ve CA-MSSA (clinical isolate) were subjected to pH, temperature,...

Elucidation of the regulation of ChiB production in Aspergillus nidulans. Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental...

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies...

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-aceta...

Polycystin-1 is a putative 460 kDa membrane protein with a unique structure and is possibly representative of a new family of proteins. Its structure suggests an involvement in cell signalling and cell-matrix interactions. The amino acid sequence of polycystin-1 has to date been predicted from its gene sequence. This, to our knowledge, is the first...

Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expre...

A large number of compounds, which are in common usage in industry and medicine, are potentially nephrotoxic. Renal damage and disease resulting from toxic exposure is progressive and will, if unarrested, culminate in irreversible renal disease. There is, therefore, a need to develop a battery of tests with which to monitor and characterise the nep...

The current investigation is the largest to date concerned with the assessment of the value of different urinary biomarkers to detect nephrotoxic effects in children exposed to cadmium and lead. A battery of tests which had proved valuable in previous studies on men and women where used, together with a number of more recently developed biomarkers....

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]- quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-oc...

Children have been considered a risk group for lead (Pb) toxicity, mainly because of neurophysiological or neuro-cognitive deficits following Pb exposure. Blood Pb levels (b-Pb) of 100 microg/l currently have been defined as the lowest adverse effect level. The aim of this study was to compare, with the help of urinary markers, the kidney function...

Damage to the kidneys is one of the primary toxic actions of metals. Nephrotoxic substances not only cause renal disease directly, but they can also destroy renal reserve capacity, potentially placing those people with additional risk factors, such as diabetes, hypertension, cardiovascular disease, and genetic predispositions, at greater risk. To d...

Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme...

Cyst fluid samples obtained from eight patients with autosomal dominant polycystic kidney disease (ADPKD) were analysed for the presence of the basement membrane component laminin and its breakdown products, using ELISA and immunoblotting techniques. Whole laminin was not detected, whereas laminin fragments of 270, 155, 87, 56, and 14 kDa were dete...

Normal renal tissue, ranging from 8 weeks' gestation to full term to adult, was probed with polyclonal antibodies raised to peptide epitopes within the translated PKD1 gene sequence. Three antibodies were studied, all of which gave similar results. Renal tissue from patients with autosomal dominant polycystic kidney disease (ADPKD) and samples from...

Groups of industrial workers exposed to heavy metals (cadmium, mercury, and lead) or solvents were studied together with corresponding control groups. The cohorts were collected from several European centers (countries). Eighty-one measurements were carried out on urine, blood, and serum samples and the results of these analyses together with quest...

The categories of tests that were discussed in the opening session included the following, with examples: 1. Clinical indicators of renal disease: GFR, serum creatinine, dipstick-positive proteinuria, cystatin C 2. Early indicators of glomerular damage: elevated albumin or transferrin excretion with normal LMW protein excretion, extracellular matri...

Two enhanced enzyme-linked-immunofilter assay (ELIFA) methods for the rapid and quantitative detection of whole bacterial cells are described. In the first method, specific antibody bound to bacterial cells was amplified using a secondary antibody and detected by the conjugated enzyme activity (peroxidase) of a third antibody in a chemiluminescent...

An improved method is described for the detection of Escherichia coli by an enzyme linked immunofilter assay (ELIFA) using nitrocellulose membrane sandwiched between two 96-well plates. The incorporation of a pumping system permits a continuous flow of reagents and/or wash fluids through the membrane and provides an assay procedure capable of detec...

Within the framework of an European Commission-funded project, groups of industrial workers exposed to heavy metals (cadmium, mercury and lead) or solvents were studied together with corresponding control groups. Eighty-one measurements were carried out on urine and serum samples and the scientific results together with individual questionnaire inf...

Laminin isolated from human placenta was subjected to prolonged pepsin digestion. Seven peptide fragments (designated N1 to N7) were separated by ion-exchange chromatography and gel filtration and characterised by SDS-polyacrylamide gel electrophoresis and immunoblotting. The molecular size of the laminin fragments varied from approx. 900,000 (N1)...

An ELISA procedure for the assay of laminin fragments in serum and urine is described. Samples from solvent-exposed workers and diabetic patients were studied. In cohorts exposed to perchloroethylene serum and urine laminin fragments were elevated but the urinary N-acetyl-beta-D-glucosaminidase (NAG) was unaffected. The data indicate that the urina...

A number of chemicals may adversely affect one or more of the anatomical structures of the kidney, such as the glomerulus, the tubular apparatus, the medullary, or interstitial cells. To recognize subclinical renal dysfunction, a battery of new, non-invasive tests was applied in comparison to established ones. The study on cadmium exposed subjects,...

Urinary N-acetyl-beta-D-glucosaminidase activity was measured in 123 healthy children aged between 1-14 years by two newly developed colorimetric procedures using MNP-GlcNAc and VRA-GlcNAc substrates and was compared to the well established PNP-GlcNAc assay. The enzyme activity was factored with the urinary creatinine concentration and expressed as...

A study was conducted to explore the effects of different sources of dietary carbohydrate on the altered metabolism of basement membrane proteins (type IV collagen and laminin) that occurs in the streptozotocin-diabetic rat. The substitution of barley for sucrose reduced the elevated plasma concentration of 7-S collagen and of laminin P2, peptide f...

The application of enzyme-linked immunofilter assay (ELIFA) techniques to the detection of whole bacterial cells in aqueous samples is described. In a qualitative assay, Escherichia coli cells were collected on 25 mm diameter cellulose ester membranes in filter holders and treated in situ with rabbit anti-E. coli IgG, phosphatase-conjugated anti-ra...

The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate in...

The kinetic characteristics of β-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhib...

Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acid N-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining pr...

The NAG activity present in urine from newborn babies was assayed using two colorimetric procedures with either MNP-GlcNAc or VRA-GlcNAc as substrate and compared with data obtained with the well established PNP-GlcNAc procedure. Both new assays were easy to perform and reproducible. The MNP-GlcNAc method has the advantage that it is now available...

High field 1H-NMR spectra of fluid collected from the cysts of six renal transplant recipients with autosomal dominant polycystic kidney disease (ADPKD) have been measured and the major metabolite signals assigned. Quantitative NMR measurements have revealed a combination of unusual biochemical features of the cystic fluids that shows them to be di...

Anti-fluorescein antibodies were found to prevent the fading of emitted fluorescence from fibroblasts stained with fluorescein-labelled fibronectin antibodies. The prevention of fading is the result of specific binding of the fluorochromes present on the stained cells by the anti-fluorescein antibodies. The sheep anti-FITC antibody used in this stu...

The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline++ +-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for po...

The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a...

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cell...

Urinary N-acetyl-beta-D-glucosaminidase activity was assayed in fullterm and preterm polycythemic neonates, in preterm infants with hypoxia, and in healthy newborns. There were no significant differences between fullterm and preterm babies or between appropriate for gestational age and small for gestational age neonates in the normal group. N-acety...

We describe a new chromogenic substrate and assay for determining N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) activity in normal and pathological human urine. The novel substrate, ammonium 5-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3- methoxyphenylmethylene]-2-thioxothiazolidin-4-one-3-ethan oate (VRA-GlcNAc), is prepared by condens...

The condensation of vanillyl 2-acetamido-2-deoxy-β-D-glucopyranoside (1b) and vanillyl β-D-galactopyranoside (1c) with various heterocycles having either an active methyl or methylene group has now been studied in order to enhance the colour of the released phenol and the solubility of the substrate. The resulting substrates are suitable for the as...

Immunological techniques, especially fluorescent staining, aid the diagnosis of immune-based glomerulonephritis. MAb’s* enable the isolation and characterization of BM antigens that may be involved in disease. Two MAb’s directed against conformational and sequential epitopes in type IV collagen have been investigated using rotary shadowing and immu...

A new fluorescent protein labelling agent, 7-amino-4-methyl coumarin-3-acetic acid (AMCA), emits in the blue region (440–460 nm) on activation with UV light (350 nm). The active reagent is theN-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links. The Stokes shift of 100 nm compared to 30...

The labelling of pepsin-digested human glomerular basement membrane (pHGBM) with a newly developed fluorescent iodine acceptor 7-hydroxy-coumarin-3-acetic acid N-hydroxysucciniimydyl ester (CASE) is described. The binding of a monoclonal antibody to pHGBM was assessed by radiobinding assays, and when directly iodinated pHGBM was used there was no a...