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P J Stoward

Publications and source records attributed to P J Stoward.

At least 19 recordsLinked to original sources
PubMed1995

The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases.

The kinetics of two enzyme systems in situ that have been studied with real-time image analysis systems are reviewed in detail. The enzymes are a structurally-bound mitochondrial enzyme, succinate dehydrogenase (SDH) and a soluble cytoplasmic enzyme, lactate dehydrogenase (LDH). The image analysis system is used to capture successive images of a tissue section at constant time intervals whilst it is being incubated on a substrate-containing gel film. The increasing absorbances of the final reaction products in each cell are measured in the successive images as a function of incubation time. The absorbances of the formazan reaction products formed by SDH, for example, in sections of liver determined by such means increase linearly during the first minute of incubation, but non-linearly afterwards. The initial velocities of SDH in single hepatocytes in sections incubated on gel substrate films are calculated from the activities during the first 20 s of incubation. In contrast, the activities of LDH measured in various cell types, including hepatocytes, with the gel film technique increase nonlinearly during the first minute of incubation, but linearly for incubation times between 1 and 3 min. The initial velocities (vi) of LDH in single cells can be, calculated, however from the activities during the first interval, 10 s, of the image capturing sequence. Unfortunately, the experimental errors of the initial velocities of LDH determined in this way are relatively high. To overcome this problem, we have found empirically that the equations vi = a1oA and vi = v + a2oA enable reliable initial velocities (vi) of the LDH reaction in single cells of various types to be calculated using the data of the linear activities for incubation times between 1 and 3 min. Dependence of the initial velocities of the SDH and LDH reactions on substrate concentrations gave the Michaelis constants (Km) and maximum velocities (Vmax). The Km values determined in situ for SDH in hepatocytes and for LDH in various cell types with the gel film technique are in the same order of magnitude as the corresponding values determined biochemically. The constants a1, a2 and Km of LDH are characteristic for each cell type and seem to be related to the intracellular localization of the enzyme and to its ligand-binding rather than to the different isozyme compositions in various cell types.

Animals↗
PubMed1992

Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system.

The initial reaction kinetics of succinate dehydrogenase in situ were investigated in sections of mouse unfixed liver using an ARGUS-100 image analyser system. The sections were incubated on substrate-containing agarose gel films. Images of a section, illuminated with monochromatic light (584 nm), were captured with the image analyser in real time at intervals of 10 s during the incubation. The absorbances of selected hepatocytes in the successive images were determined as a function of time. In every cell, the absorbance increased nonlinearly after the first minute of incubation. The initial velocity of the dehydrogenase was calculated from the linear activities during the first 20 s of incubation. Hanes plots of the initial velocities and succinate concentration yielded the following mean kinetic constants. For periportal hepatocytes, the apparent Km = 1.2 +/- 0.8 mM and Vmax = 29 +/- 2 mumol hydrogen equivalents formed/cm3 hepatocyte cytoplasm per min. For pericentral hepatocytes, Km = 1.4 +/- 1.0 mM and Vmax = 21 +/- 2 mumol hydrogen equivalents/cm3 per min. The Km values are very similar to those determined previously from biochemical assays. These results, and the observed dependence of the initial velocity on the enzyme concentration, suggest that the technique reported here is valid for the histochemical assay of succinate dehydrogenase.

Animals↗
PubMed1991

Secretory pathway of vitellogenesis in the liver of the cockerel as revealed by immuno-gold and computer-assisted digitization techniques.

The protein A-gold immunocytochemical technique was used to localize the secretory pathway of oestradiol-induced vitellogenin in hepatic parenchymal cells of the cockerel. Liver was removed from experimental birds on the 1st, 4th and 8th day following oestradiol-treatment, and embedded in Lowicryl K4M resin cured at -20 degrees C. In selected electron micrographs the fractional surface area of each of the intracellular compartments was measured by the computer-assisted digitization technique. Labelling was detected over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the immature secretory vacuoles (ISV) including condensing vacuoles and the mature secretory vacuoles (MSV). Counts of the gold particles demonstrated an increasing concentration which progressed in the order RER less than Golgi less than ISV less than MSV and identified the secretory pathway of the protein. The highest density of labelling was obtained on the 4th day, when vitellogenin reaches its peak activity. Autophagic activity (or crinophagy) was also found in lysosomes and its labelling intensity increased daily. A hypothesis concerning the secretory pathway of non-stored proteins by the liver is discussed further.

Animals↗
PubMed1991

Immunoelectron microscopical demonstration of egg-yolk plasma precursor vitellogenin in the hepatocyte of oestradiol-treated cockerels.

Vitellogenin has been localized at the electron microscopical level in the liver of the cockerel using a colloidal gold technique. White leghorn cockerels were treated with 17 beta-oestradiol to induce vitellogenesis. Pieces of liver were removed from control and experimental birds on the 4th and 8th days following hormone treatment, and embedded in Lowicryl K4M. Vitellogenin was isolated from the plasma of oestradiol-treated cockerels, and the antibody to it elicited in rabbits and made vitellogenin-specific by affinity chromatography on lipovitellin-Sepharose columns. At the light microscopical level, the intensity of immunohistochemical staining was considerably above background levels in oestradiol-treated cockerels. At the electron microscopical level, gold particles indicating antigenic sites of vitellogenin were largely confined to the rough endoplasmic reticulum, Golgi apparatus, immature and lysosomes and phagosomes within hepatocytes and sinusoidal cells respectively. These observations strongly suggest that the intracellular pathway of vitellogenin secretion in chicken hepatocytes under the experimental conditions studied involves external stimuli and secretory vacuoles. The labelling of lysosomes may reflect catabolic turnover (crinophagy).

Animals↗
PubMed1989

Effects of therapeutic percutaneous electrical stimulation of atrophic human quadriceps on muscle composition, protein synthesis and contractile properties.

The effects of percutaneous electrical stimulation (70 V, 300 microseconds pulses at 30 Hz) on muscle composition and rate of protein synthesis were studied in seven patients with quadriceps atrophy secondary to unilateral osteoarthritis of the knee (stimulated group). Quadriceps were stimulated on the affected side for 1 h per day. The results were compared to those from seven patients who did not use a muscle stimulator (control group), in whom muscle biopsy at surgery provided evidence of wasting of tissue protein on the side of osteoarthritis (normal leg 608 +/- 266 micrograms protein micrograms-1 DNA, affected leg 256 +/- 100 micrograms protein micrograms-1 DNA, means +/- SD, P less than 0.05; type I fibre diameters: normal 53.2 +/- 6.7 microns, affected 43.8 +/- 4.0 microns, P less than 0.05). In patients who had received stimulation there was no residual difference between the legs in either muscle protein concentration (normal 411 +/- 168 micrograms protein micrograms-1 DNA, affected 373 +/- 112 micrograms protein micrograms-1 DNA) or fibre diameter (type I diameters: normal 56.1 +/- 7.8 microns, affected 58.0 +/- 10.7 microns). Stimulation did not influence the ratios of muscle force elicited by acute stimulation at 20 and 50 Hz (normal 75 +/- 15%, affected 79 +/- 15%), or rates of muscle relaxation (percentage losses of tetanic force 10 ms-1: normal 7.66 +/- 1.2%, affected 8.67 +/- 2.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1988

Dipeptidyl peptidases in the soleus muscle of the rat before and after treatment with 5-hydroxytryptamine.

A moderate peptidase activity against L-lysyl-L-proline-4-methoxy-beta-napththylamide was detected histochemically in unfixed sections of soleus muscle fibres of inbred male Wistar rats using two variants of the semipermeable membrane technique. One variant involved simultaneous coupling with tetrazotised 3,3'-dimethoxybenzidine, the other post-coupling. The activity at pH 6 increased approximately three-fold in many fibres showing signs of insult in rats that had been given a single low dose of 5-hydroxytryptamine (10 mg/kg body weight) 48-72 h earlier. The hydroxytryptamine treatment was found to induce a selective myopathy. Some of the increased peptidase activity within insulted muscle fibres appeared to arise from invading mononuclear cells, but the majority seemed endogenous to muscle fibres. The peptidase activity persisted in some fibres 21-28 days after 5-hydroxytryptamine administration, by which time the whole muscle appeared histologically normal. The variation of the activity of the peptidase with pH in the presence of various inhibitors was investigated in both control and insulted muscle fibres. From its sensitivity and behaviour towards Zn2+, Hg2+, Cu2+, puromycin, benzethonium chloride and phenylmethylsulphonyl fluoride and its indifference towards Co2+, Cd2+, Mn2+ and o-phenanthroline, it is concluded that the activity can be attributed to a mixture of at least two peptidases, dipeptidyl peptidase II and an unidentified neutral dipeptidyl peptidase. The possible role of the peptidase(s) in muscle regeneration in discussed.

Animals↗
PubMed1988

Limitations of the quantitative cytochemical assay of catechol oxidase in melanoma cells.

The cytochemical quantification of catechol oxidase activity in fixed B16 melanoma cells was investigated using dopa as the substrate. Inhibitors showed that peroxidases do not significantly interfere. The kinetics of melanin formation were studied initially in solution with purified catechol oxidase. Two key parameters were identified: lag-time and the rate of melanin formation. The lag-time was taken as the time required by intermediates to reach a critical concentration at which the polymerization process starts and melanin production becomes measurable (at 640 nm). In solution, the lag-time decreases as the enzyme activity increases, particularly when the activity is very low. The rate at which melanin is formed by pure enzyme in solution is independent of dopa concentration when its activity is low but increases linearly with dopa concentration when the activity is comparatively high. In fixed melanoma cells, the lag-time decreases linearly with increases of dopa concentrations up to 20 mM; at concentrations higher than this, the lag decreases more slowly. In contrast, the rate of melanin production is unaffected by changes in dopa concentration. The lag-times of different cells lines incubated at the same substrate concentration decrease as the enzyme activity of the cells increases. The rate of melanin production seems to be affected by factors other than catechol oxidase activity, such as the intracellular organization and distribution of the enzyme.

Catechol Oxidase↗
PubMed1988

Simultaneous histochemical assay of two dehydrogenases in the same cell.

A new approach has been developed for the simultaneous assay of the activities of two enzymes (lactate and succinate dehydrogenases) in the same cell in sections of unfixed liver. The sections, mounted on coverslips, were placed on top of 0.6-mm thick 0.8% low gelling-temperature agarose films containing the substrates of both enzymes (70 mM lactate and 50 mM succinate, respectively) plus 80 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 10 mM NaN3, 1.5 mM NAD+, 1.2 mM Nitro BT and 0.26 mM phenazine methosulphate. The integrated absorbance (A) at 585 nm of the final reaction product formazans deposited by the two enzymes in a selected hepatocyte was measured continuously at 37 degrees C as a function of incubation time, using a Vickers M85 microdensitometer. The intercept A0 on the A-axis of the linear regression line of A on time was determined. After a known incubation time t, the absorbance A1, was noted and the section placed on another gel film lacking the substrates in order to estimate the final reaction product either formed in the gel film or lost from the cell. The absorbance A2 of the hepatocyte was remeasured. The reaction velocities (activities) vL and vS of lactate and succinate dehydrogenases, respectively, were calculated from the following equations: vL = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t and vS = (A2-alpha LA1)/(1-alpha L)t where alpha L = A2/A1 for hepatocytes incubated on gel films containing only lactate as the substrate. This parameter was found to be virtually constant (0.44) over a wide range of vL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Rates of muscle protein synthesis in paraspinal muscles: lateral disparity in children with idiopathic scoliosis.

1. The rate of paraspinal (multifidus) muscle protein synthesis was measured bilaterally at the top, apex and bottom of the thoracic curve in nine children with an idiopathic scoliosis, using the stable-isotope-labelled amino acid L-[1-13C]leucine. 2. No significant difference was observed in rates of muscle protein synthesis between the two sides of the spine, at the levels of the first vertebrae in neutral alignment at the top and bottom of the curve. However, in every patient, at the apex of the spinal curve, synthesis was higher on the convexity than on the concavity (0.077 +/- 0.04 %/h convex, 0.052 +/- 0.02 %/h concave, mean +/- SD, P less than 0.01). 3. Muscle RNA activity (microgram of protein synthesized h-1 microgram-1 of RNA) was lower at the curve apices on the concave than the convex side (0.019 +/- 0.09 microgram h-1 microgram-1 convex apex, 0.016 +/- 0.06 microgram h-1 microgram-1 concave apex, P less than 0.05). Activities were similar on the two sides at the top and bottom of the curve. 4. Differences in muscle histology between the two sides were also observed only at the apex, with a lower type I fibre diameter (50.9 +/- 8.5 micron convex, 38.3 +/- 2.4 microns concave, P less than 0.05) and a lesser proportion of type I fibres (63 +/- 12% convex, 49 +/- 9% concave, P less than 0.05) on the concavity. 5. The results are consistent with effects on muscle protein turnover secondary to an increased muscle contractile activity on the curve convexity and functional immobilization of the muscle on the curve concavity.

Adolescent↗
PubMed1987

Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization.

Quadriceps muscle protein turnover was assessed in the post-absorptive state in six men immediately after the end of unilateral leg immobilization (37 +/- 4 days) in a plaster cast after tibial fracture. A primed-constant intravenous infusion of L-[1-13C]leucine was administered over 7 h. Quadriceps needle biopsies, taken bilaterally at the end of the infusion, were analysed for muscle protein leucine enrichment with 13C. Quadriceps muscle protein synthetic rate, calculated from the fractional incorporation of [13C]leucine into protein compared with the average enrichment of blood alpha-ketoisocaproate, was 0.046 +/- 0.012%/h in the uninjured leg, but was only 0.034 +/- 0.007%/h in the quadriceps of the previously fractured leg (P less than 0.05, means +/- SD). Muscle RNA activity (i.e. protein synthetic rate per RNA) fell from 0.27 +/- 0.08 microgram of protein synthesized h-1 microgram-1 of RNA in the control leg to 0.14 +/- 0.03 microgram of protein synthesized h-1 microgram-1 of RNA in the immobilized leg (P less than 0.02). Immobilization was associated with a significant atrophy of type I muscle fibres (mean diameter 69.5 +/- 21 microns immobilized, 81.1 +/- 18 microns control, P less than 0.05), but no significant change occurred in type II fibre diameter. Mean quadriceps fibre volume calculated from the values for fibre diameter and percentage of each fibre type, was smaller in the injured leg by 10.6%; this value was near to the calculated difference in muscle thigh volume (calculated from thigh circumference and skin-fold thickness) which was less by 8.3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters. A quantitative histochemical study.

The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Although the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.

Acid Phosphatase↗
PubMed1985

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey.

Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and gamma-glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for gamma-glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low gamma-glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermyosial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.

Adolescent↗
PubMed1984

Implications of the immunohistochemical localization of the carbonic anhydrase isozymes for their function in normal and pathologic cells.

Histochemical knowledge of the distribution of CA and the two isozymes CA I and CA II has been reviewed here. An abundance of CA occurs most commonly in epithelial cells specializing in transport of ions and water. A mechanism is favored whereby the polarity of efflux of CA-generated protons and bicarbonate across the apical versus the basolateral plasmalemma depends not on the location of CA, which is probably in the cytosol in most sites, but rather on the transport properties of the luminal compared with the serosal region of the plasma membrane in each epithelial cell type. CA exists also in some protein-secreting, merocrine cells including serous cells of salivary and tracheobronchial glands. Available evidence supports the possibility that CA stored as a secretory product in the cytoplasmic granules is released from these cells and, thus, implies extracellular biologic activity for CA in these sites. CA exists also in abundance in various nonepithelial cells performing different and not fully defined biologic functions in these cells. Prevalence of one isozyme over another varies in different cell types. A question remains whether the significance of this variability depends on work load or other undetermined factors.

Animals↗
PubMed1982

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle. IV. A post-coupling technique.

A post-coupling semipermeable membrane technique for determining the activity of acid phosphatase in sections of skeletal muscle has been developed and investigated for its reproducibility and validity. Cryostat sections of unfixed muscle mounted on dry dialysis membranes are first incubated for 1-4 at 37 degrees C on a gelled medium containing 4 mM naphthol AS-BI phosphate buffered at pH 5. They are next transferred to another gel containing only hexazotised Pararosanaline and incubated for a further 30 min. Finally, they are treated with 70% ethanol, dried in air, and mounted. The final reaction product (FRP) deposited within muscle fibres is mostly distributed as a fine reticular network, tentatively identified as sarcoplasmic reticulum. Large FRP "granules' of the kind observed with Meijer's simultaneous coupling membrane technique are not formed. The method is reproducible and valid in terms of several working criteria. For example, the mean absorbance of the FRP at its absorption maximum increases linearly with incubation time; and in model sections containing various amounts of a subcellular fraction rich in acid phosphatase, the mean absorbance after a constant incubation time is proportional to the enzyme concentration. FRP is formed at approximately twice the rate it is deposited in the simultaneous coupling method. The most important advantage of the post-coupling method over the simultaneous coupling method is that the inhibition of the enzyme by coupling reagents is avoided.

Acid Phosphatase↗
PubMed1982

The histochemical basis of quantitative histology.

The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed. In principle, the morphological parameters of cells can be measured in suitably-stained sections using image analysers. In practice, however, the individual cells often cannot be distinguished from each other in such systems. This problem might be overcome, at least in muscle, by visualizing types III and IV collagen in the connective tissue and basement membranes surrounding the cells. The functional capacities of cells within tissues can be inferred by measuring the activities of their key enzymes with quantitative histochemical techniques. At present, the wrong enzymes are frequently chosen for this purpose. Flux-generating and physiologically significant enzymes are preferable, but this requires new histochemical techniques to be devised and validated quantitatively for use with the computer-assisted analytical microscopes that are now becoming available commercially.

Animals↗
PubMed1981

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle. II. Non-specific reactions in control sections.

The absorbances of skeletal muscle fibres at 530 nm in control sections of either fresh or boiled tissue, incubated according to Meijer's semipermeable membrane technique for acid phosphatase, increase linearly with incubation time. Moreover, the absorbances continue to increase even after the incubation has been terminated by immersion of the sections in formalin. The comparatively weakly absorbing yellow reaction product originally formed is transformed with 24 h of mounting the sections into a strongly absorbing purple material with an absorption maximum at 570 nm. The original yellow product seems to be hexazotised Pararosanaline adsorbed onto tissue proteins. The formation of the purple product can be prevented by treating control sections immediately after incubation with 70% ethanol for 30 min at room temperature in place of formalin. Ethanol stabilises the yellow non-specific reaction product but does not extract it from tissue sections. The ethanol treatment thus seems useful for improving the validity of Meijer's technique.

Acid Phosphatase↗