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Biomedical subjects

W R Robertson

Publications and source records attributed to W R Robertson.

At least 127 records · Page 7Linked to original sources
PubMed1983

Negative feedback effects on ACTH secretion by cortisol in Cushing's disease.

Bioassayable plasma ACTH and corticosteroid levels were measured during constant infusions of low doses of cortisol (3-15 mg/h) into normal subjects and into two patients with Cushing's disease. Plasma ACTH levels decreased significantly in all subjects within 60 min from the start of the infusion. The rate of rise of plasma corticosteroid levels concomitant with this feedback suppression were considerably higher in the patients with Cushing's disease (greater than 13 nmol/l/min) than in normal subjects (3-6 nmol/l/min). In one of the Cushing's patients, ACTH secretion resumed in spite of the corticosteroid levels rising at a rate of greater than 20 nmol/l/min. It is concluded that cortisol-induced suppression of ACTH secretion may be operational in Cushing's disease but that the mechanism is less sensitive than in normal subjects.

Adrenal Cortex↗
PubMed1982

20 alpha-Hydroxysteroid dehydrogenase activity in the rat corpus luteum; a quantitative cytochemical study.

A quantitative cytochemical method for the demonstration of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-HSD) in the regressing corpora lutea of the adult rat ovary is described. The method employs unfixed tissue sections and relies upon the oxidation of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) with nitro blue tetrazolium as the hydrogen acceptor. The enzyme was dependent upon NADP+ for its activity and was inactive when 20 beta-hydroxy-4-pregnen-3-one (20 beta-OH-P) was used as a substrate. The apparent Km values for 20 alpha-OH-P and NADP+ were 3 x 10(-4) M and 2.5 x 10(-5) M respectively. Inhibition of 20 alpha-HSD activity by steroids was demonstrable at pH 8. Androstenedione was by far the most potent inhibitor, followed by progesterone (the product of the enzyme activity) 17 alpha-hydroxyprogesterone, Compound S and 20 beta-OH-P. At pH 6.8, a pH more favourable to the progesterone leads to 20 alpha-OH-P reaction, only progesterone and 17 alpha-hydroxyprogesterone were inhibitory. Testosterone was without demonstrable effect at either pH.

20-Hydroxysteroid Dehydrogenases↗
PubMed1982

Characteristics of the response of dispersed guinea-pig adrenal cells to low doses (1-50 pg/ml) of alpha 1-24 adrenocorticotrophin.

Isolated adrenal cells prepared by tryptic digestion of the guinea-pig adrenal gland are sensitive to low concentrations (less than 25 pg/ml) of adrenocorticotrophin (ACTH). Cell which have been pre-incubated for 2 h. centrifuged and resuspended in fresh culture medium prior to the introduction of 10 pg/ml ACTH for 60 min show a marked increase (328 +/- 109 nmol/l; mean +/- SD) in cortisol secretion over the control compared to freshly dispersed cells (75 +/- 45 nmol/l). Further potentiation of the ACTH effect was seen with the pre-incubated cells by suplementing the medium with calcium (8 mM) and ascorbate (2 mM) but not with theophylline (1 mM). Basal cortisol secretion was not affected by any of the additives. In the presence of 8 mM calcium and after 60 min incubation 10 pg/ml ACTH stimulated cortisol secretion from 328 nmol/l over the control to 839 +/- 382 nmol/l. The effect of ascorbate (2 mM) was to further increase the effect of ACTH at all dose levels tested (1-25 pg/ml). The concentration of ACTH required to provoke half maximal cortisol secretion decreased from 95 pg/ml with normal medium to 12 pg/ml with calcium -ascorbate supplemented medium. Using this supplemented medium the cells were sensitive to 1 pg/ml and cortisol secretion was stimulated 10-fold over the control with 50 pg/ml, a dose which saturated the system.

Adrenal Glands↗
PubMed1982

Negative rate-sensitive feedback effects on adrenocorticotrophin secretion by cortisol in normal subjects.

Plasma ACTH and corticosteroid levels were measured in normal subjects during constant infusion of either 0.9% (W/V) NaCl solution or cortisol, and during insulin-induced hypoglycaemia. During infusions of 0.9% NACl solution the secretion of ACTH and corticosteroids was episodic. Fast, rate-sensitive, negative feedback inhibition of ACTH secretin was observed during cortisol infusions, when the corticosteroid levels were within the physiological range (200-750 nmol/l) and were rising at a rate of between 5 and 10 nmol/l per min for 30 min or longer. When plasma corticosteroid levels were in a steady state, the initial fast feedback effects were abolished and ACTH secretion resumed. However, this recovery of ACTH secretion was not seen when the corticosteroid levels were persistently above 800 nmol/l. It appears that corticosteroid-induced negative feedback in man may be both rate- and level-sensitive. During insulin stress test ACTH secretion fell at time when the plasma corticosteroid level was rising rapidly (greater than 5 nmol/1 per min) despite persistent hypoglycaemia.

Adrenal Cortex Hormones↗
PubMed1982

Effect of human plasma on the bioactivity of adrenocorticotrophin.

The effect of plasma on the bioactivity of ACTH was investigated with the cytochemical section bioassay. Levels of ACTH in normal human plasma were underestimated if the plasma concentration in the incubation medium exceeded 2% and if the plasma ACTH level was less than 2 ng/l. A retrospective analysis of 250 plasma ACTH assays revealed that the ACTH concentration was consistently underestimated in the 1:100 dilution compared with the 1:1000 dilution, although not to the extent that any assay would be rejected for non-parallelism. The extent of the underestimation increased as the ACTH concentration increased. Guinea-pig adrenal sections responded faster to 1:10 or 1:50 dilutions of human plasma (containing 2 ng ACTH/l) than to either 1:100 or 1:1000 dilutions of that same plasma, or to ACTH (over the range 0.005-5 ng/l)in the absence of plasma. This faster response could also be seen in the absence of plasma by increasing the ACTH concentration above 5 ng/l. It seems that plasma contains factors which potentiate the biological effect of ACTH in the cytochemical assay system.U

Adrenal Glands↗
PubMed1981

The influence on enzyme activity of storage of tissue blocks at -70 degrees C.

Blocks of tissue from various organs of the rat have been chilled by precipitate immersion in n-hexane cooled to -70 degrees C, and then stored at -70 degrees C. At various intervals (up to 14 days) after chilling, cryostat sections were prepared from these blocks and assayed for the activity of a variety of enzymes. Enzyme activity was measured by scanning and integration microdensitometry. With the exception of acid phosphatase and cytochrome oxidase, all enzymes assayed were stable for at least 7 days after storage at -70 degrees C and most were stable for 14 days, Storage of fresh-frozen sections at -30 degrees C in the cabinet of the cryostat, for up to 24 h, had little effect on enzyme activity.

Animals↗
PubMed1980

The use of an inexpensive, general purpose microcomputer in quantitative cytochemistry.

A 32K Commodore Pet microcomputer has been interfaced with two Vickers microdensitometers. This system allows for the simultaneous logging of data from two densitometers being operated independently of each other. Software for the statistical analysis of data generated by the densitometers, and for use with the cytochemical bioassay of hormones, is described. The densitometer/Pet system is relatively cheap, reliable, and readily adaptable to other applications.

Computers↗
PubMed1980

A quantitative study of N-acetyl-beta-glucosaminidase activity in unfixed tissue sections of the guinea-pig thyroid gland.

A post-coupling procedure for the quantitative measurement of N-acetyl-beta-glucosaminidase activity in unfixed tissue sections of guinea-pig thyroid is described. The method depends on the cleaving of a naphthol AS-BI substrate and the insoluble reaction product is post-coupled with Fast Garnet GBC salt (in acetate buffer, pH 6.2) at 4 degrees C. Even though this enzyme is localized in the lysosomes, an inert colloid stabiliser, polyvinyl alcohol (G18/140) is included in the reaction medium to allow the use of the optimal substrate concentration (0.5 mg/ml) whilst employing a low concentration (5%) of ethylene glycol monomethyl ether. The high molecular weight (90 000) grade of polyvinyl alcohol used did not stabilize the lysosomal membrane, although a lower molecular weight (15 000) grade of polyvinyl alcohol (G04/140) may do. The enzyme activity was not affected by the metal ions Ca2+ and Zn2+ and was totally abolished by the specific inhibitor 2-acetamido-2-deoxy-D-gluconolactone.

Acetylglucosaminidase↗
PubMed1980

A cytochemical section-bioassay for thyrotropin.

A cytochemical section-bioassay of thyrotropin has been developed which retains the sensitivity of the earlier segment-assay (10(-4) microunits/ml) and allows a considerable increase in the rate at which these within-animal bioassays can be done. The index of precision (n=23) was 0.11 +/- 0.06; intra-assay variation was 1.3%; inter-assay variation was 10.9 +/- 6%. Fiducial limits (p = 0.95) ranged from 65-150% to 78-127%. The system is also capable of detecting thyroid-stimulating immunoglobulins.

Aminopeptidases↗
PubMed1979

A quantitative cytochemical method for the demonstration of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary.

A method for the quantitative measurement of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD+ or NADP+ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP+ dependence is not abolished by inhibiting nonspecific alkaline phomonoesterase. The activity of delta5,3beta-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10(-4) M.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Comparison of radioimmunoassay with homogeneous enzyme immunoassay for the determination of theophylline concentration in serum.

The homogeneous enzyme immunoassay (EMIT) and radioimmunoassay (RIA) procedures for the quantitation of theophylline concentrations in serum were evaluated and compared. Both methods exhibited curves linear over the therapeutic range (range of concentration, 2.0-33.0 microgram/ml). Precision was acceptable for both methods with the coefficient of variation being less than 8.3%. The RIA procedure was found to be slightly more precise. The correlations between the EMIT and RIA procedures was found to be sufficient to allow the procedures to be used interchangeably. This would facilitate the test availability on a 24 hr, 7 day basis.

Humans↗
PubMed1978

A quantitative study of the effects of different grades of polyvinyl alcohol on the activities of certain enzymes in unfixed tissue sections.

Different grades of the colloid stabilizer, polyvinyl alcohol, used for protecting unfixed cryostat sections during cytochemical reactions, may have different effects on enzymatic activity. The influence of three grades of polyvinyl alcohol on the activities of "soluble", membrane-bound and membrane-enclosed enzymes has been investigated in unfixed sections; the activities were measured microdensitometrically. The largest molecular weight polyvinyl alcohol (G18/140, mol. wt. about 90 000) did not retain glucose-6-phosphate dehydrogenase activity in sections of rat liver even when used at the maximum convenient concentration (12%); G04/140 and M05/140 (molecular weights of 15 000 and 25 000 respectively) retained this soluble enzyme if used at concentrations of 30 and 20% respectively. At these concentrations, lactate dehydrogenase activity was apparently decreased when G04/140 and M05/140 were used; this diminished activity has been shown to be due to the need to establish optimal concentrations of reactants for each grade of polyvinyl alcohol and for each reaction. When optimal concentrations of reactants were used, the activities of this enzyme in the presence of each grade of polyvinyl alcohol were identical. The presence of any type of polyvinyl alcohol did not influence the activities of mitochondrial succinate dehydrogenase or of the smooth endoplasmic reticulum enzyme, delta5,3beta-hydroxysteroid dehydrogenase. However, the presence of polyvinyl alcohol improved the state of the section.

3-Hydroxysteroid Dehydrogenases↗
PubMed1978

The synthesis of DNA by DNA-membrane complexes from irradiated E. coli B/r and Bs-1: the role of the membrane.

DNA-membrane complexes were isolated from lysed E. coli B/r and Bs-1, either by low g forces from a low salt solution, or by high g forces through a discontinuous sucrose gradient. The latter method was more gentle. Irradiation of the intact bacteria had no effect on the membrane macromolecules or on RNA components of these complexes. DNA loss was not significant after irradiation under anoxic conditions but complexes isolated from from Bs-1 irradiated in air showed an appreciable decrease in DNA content. In the presence of the appropriate nucleotide mixture, both 'free' DNA, found in the supernatant fractions, and rapidly sedimented membrane-associated DNA were able to synthesize DNA in the absence of added polymerase. DNA synthesis associated with 'free' DNA was more sensitive to radiation than that associated with DNA bound to the membrane, which appeared to moderate the effects of radiation on new DNA synthesis. It is concluded that the depression of DNA synthesis is primarily a result of irradiation-induced changes on genome-DNA. The interpretation of earlier work from our laboratories that DNA-membrane complexes contained the macromolecular structure which responded to radiation with a high o.e.r. is not supported by the evidence in this work.

Cell Membrane↗
PubMed1977

Exchange of ribosomal proteins among the ribosomes of Escherichia coli.

The exchange of ribosomal proteins among ribosomes of E. coli has been measured, using a density label technique. As expected most of the proteins do not exchange appreciably. However a substantial fraction of each of proteins S1, S2, S21, L7/L12, L9, L10, L11, L26 and L33 is found to exchange, but exchange of S1, S2, L7/L12, L10, L11 and L26 is found to occur in vitro after lysis of the cells, and therefore it is not possible to say whether or not these proteins also exchange in vivo. tin contrast S21, l9 and L33 do not exchange after lysis of the cells and we therefore conclude that these proteins exchange in vivo. The maximum level of exchange of S21, L9 and L33 is attained so rapidly that we were unable to show whether or not it was dependent on protein synthesis.

Bacterial Proteins↗