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Biomedical subjects

R G Price

Publications and source records attributed to R G Price.

At least 55 records · Page 3Linked to original sources
PubMed1990

The effect of polycythemia and hypoxia on urinary N-acetyl-beta-D-glucosaminidase activity in newborns.

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-acetyl-beta-D-glucosaminidase excretion on the first day of life was significantly raised in polycythemic newborns (P less than 0.01). Fourteen days after partial plasma exchange the enzyme activity returned to normal. N-acetyl-beta-D-glucosaminidase activities in preterm babies with respiratory distress syndrome were significantly (P less than 0.01) raised on the 1st, 2nd, 4th days and fell sharply to the 14th day. N-acetyl-beta-D-glucosaminidase isoenzyme studies revealed that urine samples taken from preterm babies with respiratory distress syndrome in the first week after birth contained increased amounts of intermediate and B isoenzyme forms while there was a concomitant reduction in the amount of the A form present.

Acetylglucosaminidase↗
PubMed1990

"VRA-GlcNAc": novel substrate for N-acetyl-beta-D-glucosaminidase applied to assay of this enzyme in urine.

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 condensation of vanillyl N-acetyl-beta-D-glucosaminide and rhodanine-3-ethanoic acid. The phenol released by enzyme action has an intense absorption peak at 492 nm (epsilon = 37,000 L.mol-1.cm-1). The Km for NAG in urine was 0.5 mmol/L at pH 4.5. The substrate solution may be stored at 4-7 degrees C for one week without any increase in the substrate blank. For optimal assay conditions, we used a substrate concentration of 3 mmol/L and a 15-fold sample dilution at pH 4.5-5.0, with measurement at 505 nm, not significantly different from that at 492 nm. The sensitivity of the assay with VRA-GlcNAc as substrate is twice that of the MNP-GlcNAc [2-methoxy-4-(2'-nitrovinyl)-phenyl GlcNAc] procedure (Clin Chim Acta 1982;124:195-204) and can detect low amounts of minor NAG isoenzymes. This assay is an improvement on previously available colorimetric methods and could be easily incorporated into kits based on either an enzyme calibrant or the molar absorptivity of the phenol.

Acetylglucosamine↗
PubMed1989

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster).

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

Amino Acids↗
PubMed1989

The glomerular basement membrane defect in Alport-type hereditary nephritis: absence of cationic antigenic components.

Alport-type hereditary nephritis is a familial disorder which results in progressive renal insufficiency and sensorineural hearing loss. It is thought to result from a biochemical defect affecting basement membranes. To study this further, non-collagenous components of type IV collagen were prepared from the glomerular basement membrane (GBM) by collagenase digestion from three male patients with hereditary nephritis. The normal Goodpasture antigenicity of the 28 and 26 kD monomers and 54 and 50 kD dimers which may be isolated from the GBM was absent on one-dimensional immunoblots. Two-dimensional electrophoresis and immunoblotting studies showed absence of Goodpasture antigenicity of these molecular weight components as well as all cationic monomeric and dimeric spots. It is concluded that the expression of the Goodpasture antigen is altered in basement membranes of hereditary nephritis patients. The altered antigenicity thus acts as a marker for the underlying abnormality.

Anti-Glomerular Basement Membrane Disease↗
PubMed1987

Isoenzymes of urinary N-acetyl-beta-D-glucosaminidase (NAG) in patients with renal transplants.

Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography. Although raised NAG activity was found in stable transplant patients compared to controls, the level of activity was constant and no change in the isoenzyme profile was found. In reversible rejection there was a marked increase in the intermediate forms, particularly I2 and a concomitant fall in the relative amount of the A-form present but the profile became normal when the patient stabilised. Much more complex patterns were observed in patients who did not respond to treatment. Both the B and I forms were elevated with a fall in the A-form and in one case excretion of the serum As form was observed. The intermediate forms were always increased in rejection.

Acetylglucosaminidase↗
PubMed1986

Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins.

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 the N-hydroxysuccinimide ester which reacts with lysine residues under mild conditions to form photostable amide links. The Stokes shift of 100 nm compared to 30 nm for Fluorescein isothiocyanate (FITC) allows easy filter discrimination of exciting and emitting radiation. The agent has been demonstrated in use for fluorohistochemical examination of human kidney glomeruli, using the sandwich technique and compared with the same procedure using FITC-labelled antibodies. The good quantum yield coupled with convenient emission lines in the mercury spectrum allows photographic exposure time of fluorescent labelled sections to be reduced to a quarter of that required for a corresponding FITC conjugate. AMCA-immunoglobulin conjugates were not susceptible to photobleaching and have a storage life at -20 degrees C of more than two years.

Antibodies, Monoclonal↗
PubMed1986

Renal toxicity of propyleneimine: assessment by non-invasive techniques in the rat.

The nephrotoxicity of three different dose levels of propyleneimine (10, 20 and 30 microliter/kg body wt) administered intraperitoneally to rats was studied and 20 microliters/kg body weight was found to be the most appropriate sublethal dose. Injection of propyleneimine (10 microliters/kg body wt) produced a small rise in N-acetyl-beta-D-glucosaminidase (NAG) activity, minor histological damage but no change in urine volume. Six rats were injected with 20 microliters/kg body weight, and urine was collected over the following 16 days. An immediate increase in urine volume, osmolality together with a concomitant decrease in specific gravity, was accompanied by a small increase in creatinine excretion and a more marked increase in the sodium and potassium content of urine after the administration of the nephrotoxin. NAG activity increased immediately and peaked on day 3, the activity remained elevated until day 12 when it fell to near normal levels. The activity of both beta-D-galactosidase and beta-D-glucosidase increased 9 days after administration of the nephrotoxin. In contrast, no consistent change was found in the excretion of the brush border marker enzymes, leucine aminopeptidase (LAP), alanine aminopeptidase (AAP) or alkaline phosphatase (ALP). Proteinuria increased sharply the day after injection and remained abnormal. Increased urinary albumin excretion and the predominance of low molecular weight proteins was demonstrated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Evidence is presented that propyleneimine exerts its early toxic effect on the renal papilla.

Acetylglucosaminidase↗
PubMed1986

Increased prolylhydroxylase activity in chondrocytes but not synovial cells from adjuvant arthritic rat knees.

Prolylhydroxylase and galactosylhydroxylsyl glucosyl (GGH) transferase were assayed in cells cultured from cartilage and synovium (adipose type) removed from normal and adjuvant arthritic rat knees. There was a significant increase in prolylhydroxylase in chondrocytes from arthritic knees compared to the normals but no change in GGH transferase activity was found. In the cells derived from the synovium no variation in either enzyme was observed. No significant changes in DNA or protein content was found in either type of cells from the arthritic joints. Comparison of the prolylhydroxylase activity in the supernatant and pellet fractions prepared from chondrocytes from normal and arthritic joints, demonstrated that the majority of the activity was recovered in the supernatant fraction rather than the pellet in arthritic chondrocytes. The data presented indicate that the degree of hydroxylation of cartilage collagen alters in arthritis.

Animals↗
PubMed1986

Penicillamine-induced elastosis perforans serpiginosa. Tip of the iceberg?

Elastosis perforans serpiginosa (EPS) is now a well-recognized potential complication of long-term penicillamine therapy. By itself, EPS appears to be a relatively innocuous cutaneous side effect of penicillamine. However, suspicion has been raised in recent literature that EPS may represent only a superficial manifestation of more serious penicillamine-induced systemic elastic tissue damage, particularly involving blood vessels. This is a report of a patient with Wilson's disease who was treated with penicillamine for 14 years. She developed EPS, and histologic examination of the skin revealed the characteristic penicillamine-induced "lumpy-bumpy" elastic fibers in the dermis. More important, nonlesional skin showed the same elastic fiber changes. Of greatest significance was the finding of identical elastic fiber alterations in an artery.

Adult↗
PubMed1985

Fluorescent and radiolabelling of pepsin-digested human glomerular basement membrane with a newly developed hydroxy-coumarin derivative (CASE).

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 apparent binding. When CASE was conjugated to pHGBM prior to iodination 11% binding was achieved. CASE acting as an iodine acceptor may be useful for proteins containing few or inaccessible tyrosine residues or which are destroyed by introduction of 125I. Since CASE is fluorescent, small amounts of material can be detected during isolation prior to iodination.

Basement Membrane↗
PubMed1985

Age-related changes in rat glomerular basement membrane components solubilised with pepsin.

The proportion of glomerular basement membrane (GBM) solubilised following pepsin treatment increased with age when the digestion was carried out at 4 degrees C, but remained constant at 50% of the membrane when the digestion was carried out at 10 degrees C irrespective of rat age. Amino-acid analysis of the solubilised material indicated that it became more collagen-like with age, as judged by the increase in glycine, hydroxyproline and hydroxylysine content, if the digestion was carried out at 10 degrees C. Polyacrylamide gel electrophoresis of pepsin-solubilised material demonstrated that components with apparent molecular weights of 180,000, 160,000 and 130,000 increased in intensity with age. Components with apparent molecular weights less than 85,000 were only present in pepsin-soluble material prepared from GBM isolated from young rats. The intensity of the bands in the pro-alpha-region varied with age and some procollagen-like material was not solubilised by pepsin treatment. These studies confirm that aging of rat GBM is characterised by an increase in the proportion of the collagenous components present in rat GBM.

Aging↗
PubMed1984

Differential solubility of subcomponents of rat glomerular basement membrane.

Marked differences were found in the electrophoretic profiles and amino-acid compositions of components prepared from rat glomerular basement membrane (GBM) by a number of different solubilization procedures. Treatment with reducing agent resulted in a simplified electrophoretic pattern which was characterized by the presence of a major collagenous component with a mol.wt. of 150 000. In contrast, detergent solubilized mainly lower-mol.-wt. material which had a more polar amino-acid composition. When both reagents were used together the majority of the basement-membrane material was solubilized within 2 h and components with mol.wts. of 170 000 and 135 000 were predominant in the pro-alpha region of the gel. Treatment for a further 16 h was required to solubilize higher-mol.-wt. material and to achieve maximum solubility of components in the pro-alpha region with mol.wts. of 185 000 and 150 000. These methods provide a means of separating subcomponents of rat GBM while avoiding the problems of degradation inherent in enzymatic procedures.

Animals↗
PubMed1984

Colorimetric assay for N-acetyl-beta-D-glucosaminidase (NAG) in pathological urine using the omega-nitrostyryl substrate: the development of a kit and the comparison of manual procedure with the automated fluorimetric method.

This paper describes a comparison of the recently developed substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl-2-acetamido-2-deoxy-beta-D-gluc opyranoside (MNP-GlcNAc) and the corresponding 4-methylumbelliferyl substrate (4-MU-GlcNAc) for the determination of urinary NAG. A good correlation (r = 0.977) was found between NAG activities in 366 urine samples from renal transplant patients determined by either the fluorimetric method or the colorimetric procedure. The colorimetric method may be used with confidence as an alternative to the fluorimetric assay and does have the advantage that colorimetry is widely used in clinical chemistry laboratories. Sample blanks are not normally required unless there is blood contamination in the urine which can easily be identified visually. The incorporation of the MNP-GlcNAc substrate into a kit suitable for the assay of urinary NAG using a simple battery-operated miniphotometer is also described. The presence of elevated NAG levels in urine can be detected rapidly and this procedure is suitable for routine monitoring of renal patients in the laboratory and may prove to be suitable for use by non-laboratory personnel in clinics or on the ward.

Acetylglucosamine↗
PubMed1984

Detection of Goodpasture antigen in fractions prepared from collagenase digests of human glomerular basement membrane.

Preparations of human glomerular basement membrane (GBM) were digested with collagenase, and a Goodpasture (GP) antigen rich pool from gel filtration column runs was identified by antibody inhibition radioimmunoassay. The components of the GP antigen pool were separated on polyacrylamide gels, and transferred to nitrocellulose sheets by the 'western' blotting technique. The blots were separately reacted with thirteen GP sera as primary antibody, followed by peroxidase labelled goat anti-human IgG and revealed 45-50K (two bands) and 25-28K (one-three bands) components. No corresponding reactivity was observed using convalescent GP sera or other control sera (normal human serum, rapidly progressive glomerulonephritis with or without pulmonary haemorrhage, and lupus erythematosus) as primary antibody.

Amino Acids↗
PubMed1983

The diagnostic value of urinary enzyme measurements in hypertension.

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.

Acetylglucosaminidase↗
PubMed1983

Comparison of urinary N-acetyl-beta-D-glucosaminidase and urinary fibrin degradation products for diagnosis of rejection after renal transplantation.

A comparison was made of two established tests to see which was the better indicator of rejection after renal transplantation. Daily urine samples were assayed for excretion of N-acetyl-beta-D-glucosaminidase (NAG) and fibrin degradation products (FDP). Twenty-five rejection episodes were studied in 19 patients. Both indicators tended to move in parallel. NAG was more often elevated in rejection, namely in 96% of rejection episodes as against 76% for FDP, FDP had the advantage of fewer false positive results and when it correctly indicated rejection its increase above the baseline was relatively greater than for NAG, NAG and FDP can both provide early warning of rejection. NAG is preferred for its ease of assay. Assays are performed daily and are of practical value in clinical management. The results of assays must be interpreted in conjunction with all relevant information.

Acetylglucosaminidase↗