Biomedical subjects

T Kealey

Publications and source records attributed to T Kealey.

61 records · Page 4Linked to original sources

PubMed1986

The isolation of human sebaceous glands and apocrine sweat glands by shearing.

A new method of isolating human sebaceous and apocrine sweat glands by the repeated dissection of skin biopsies with scissors is described. The success of the technique is attributed to a line of weakness between the investing capsule and the surrounding connective tissue which parts under shear forces. The glands are judged to be viable by: (i) light and electron microscopy; (ii) ATP, ADP and AMP contents of 148.8 +/- 30.3, 30.6 +/- 4.7 and 14.9 +/- 4.7 pmol (mean +/- s.e.m.) for sebaceous glands and 310.2 +/- 34.1, 90.35 +/- 16.3 and 40.1 +/- 11.8 pmol (mean +/- s.e.m.) for apocrine sweat glands, which gave energy charges of 0.84 and 0.81, respectively; and (iii) a rate of sebaceous gland lipogenesis of 39.7 +/- 3.7 pmol glucose incorporated into lipid/gland/h (mean +/- s.e.m.).

Adenine Nucleotides↗

PubMed1986

Electrophysiological studies on isolated human eccrine sweat glands.

Human eccrine sweat glands were isolated by shearing and the potential differences across the basolateral membranes determined using bevelled micro-electrodes filled with 4 M potassium acetate. Stable resting potentials of up to -81 mV were recorded. Alterations in external potassium concentration from 1.2 to 100 mM caused the membrane potential to change over a 70 mV range in cells of high resting potential, indicating that the basolateral membrane is largely potassium permeable. Input impedance was determined by constant current injection and found to be in the range 4-80 M omega. On giving a bolus injection of acetylcholine to produce a final concentration of 10(-6)-10(-7) M, four types of response were observed: depolarization, in a proportion of cells with resting potentials of -66 to -80 mV (n = 19), hyperpolarization, in a group of cells with resting potentials of -47 to -70 mV (n = 22), no change, in some cells of -40 to -81 mV resting potential (n = 22) and micro-electrode dislodgement (n = 8). In cells depolarizing to acetylcholine, the depolarization was short-lived and in thirteen cases was followed by a 'rebound' hyperpolarization. Input impedance decreased during depolarization in one-third (n = 5) of the cells in which satisfactory measurement could be made and increased during the final phase of depolarization or during rebound hyperpolarization. In cells hyperpolarizing to acetylcholine, the hyperpolarization was usually accompanied by an increase in input impedance. In ten of the twenty-two cells which showed no change to a first dose of acetylcholine, the agonist was administered at least two more times. In two cells (resting potentials -62 mV, -64 mV) a hyperpolarization was observed whereas in three others (resting potentials -66 mV, -70 mV, -81 mV) depolarization occurred. The effects of acetylcholine, whether depolarizing or hyperpolarizing, were reversibly inhibited by atropine and irreversibly reduced by ouabain. Experiments performed on glands maintained for up to 30 h in supplemented RPMI 1640 tissue culture medium yielded essentially similar results to those performed on freshly isolated glands.

Acetylcholine↗

PubMed1986

The primary culture of epithelia from the secretory coil and collecting duct of normal human and cystic fibrotic eccrine sweat glands.

Isolated human eccrine sweat glands have been microdissected into their secretory and reabsorptive components. Complete separation of these epithelia was confirmed by differential uptake of Neutral Red stain by an intermediate section of gland containing the junction between the secretory coil and the collecting duct. Primary cultures were obtained from explants of both tissues in medium RPMI-1640 or Williams E supplemented with foetal calf serum, insulin, transferrin, epidermal growth factor and hydrocortisone. The cells in the initial coil cultures had an elongated morphology while those of ductal origin were polyhedral. After 10 days both cultures were composed of polyhedral cells of varying diameter. All these morphological types were of epithelial lineage, as demonstrated by the binding of a monoclonal antibody to cytokeratin, the intermediate filament specific for epithelial cells. Outgrowth from both secretory and reabsorptive epithelia were multilayered, with plentiful desmosomal connections and an underlying basal lamina. Ultrastructural features typical of the epithelial cell types present in intact eccrine sweat glands were absent in a high proportion of the proliferating cells but domes, indicative of transepithelial active ion transport, were present in dense cultures from the reabsorptive duct. Outgrowth was also obtained from the secretory and reabsorptive epithelia of sweat glands from two cystic fibrotic patients. Since the most characteristic malfunction of cystic fibrosis is the impaired ion transport in the eccrine sweat gland, the availability of cultured epithelia should provide a useful model for study of the disease.

Cells, Cultured↗

PubMed1985

The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay.

An enzyme/bioluminescent assay for the determination of oxalate in plasma is described in which NADH, a reaction product of the enzymic degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, is determined using a commercially available bioluminescent system. In contrast to most previously documented methods, this sensitive and specific assay requires minimal sample preparation allowing oxalate concentrations to be determined within 2 h of sample collection. The limit of detection for plasma samples is 0.8 mumol/l. The recovery of oxalate added to plasma averaged 99%. The inter-batch coefficient of variation, calculated by analysis of a plasma sample from a uraemic patient (oxalate concentration = 45.8 mumol/l) on 8 occasions, over a period of 5 wk, was 3.2%. Plasma oxalate concentrations in 35 normal subjects ranged from less than 0.8-1.5 mumol/l, which is in excellent agreement with values obtained by in vivo isotope dilution studies. Plasma oxalate was found to be strikingly elevated in a group of uraemic patients maintained on regular haemodialysis.

Adult↗

PubMed1984

Studies on rat parotid-cell actomyosin.

Actomyosin was partially purified from rat parotid cells dispersed by collagenase digestion and found to possess different solubility characteristics from that from (undispersed) rat parotid tissue. This is attributed to the decrease in vascular contamination effected by the isolation of parotid cells, yielding a non-muscle actomyosin [Adelstein, Conti, Johnson, Pastan & Pollard (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3693-3697]. Myosin light-chain kinase was partially purified from dispersed rat parotid cells by calmodulin affinity chromatography and shown to be activated by Ca2+-calmodulin. The calmodulin content of dispersed rat parotid cells was shown to be 6.50 +/- 0.59 ng of calmodulin/micrograms of rat parotid-cell protein (mean +/- S.E.M.), as determined by the activation of purified bovine brain phosphodiesterase by heat-treated extracts of dispersed rat parotid cells.

Actomyosin↗

PubMed1984

Biochemical and ultrastructural studies of human eccrine sweat glands isolated by shearing and maintained for seven days.

A new method of isolating human eccrine sweat glands by the repeated dissection of skin biopsies with scissors is described. The success of the technique is attributed to a potential line of weakness between the investing capsule and the surrounding connective tissue, which parts under shear forces. The yield is 20-50 glands per biopsy (5 cm X 0.5 cm). The glands are judged to be viable by: (i) light and electron microscopy; (ii) ATP, ADP and AMP contents of 81.0 +/- 12.7, 13.8 +/- 3.3 and 3.8 +/- 1.0 pmol/gland, respectively (mean +/- S.E.M.), which gave an energy charge of 0.90; (iii) the 28-fold rise in cyclic GMP content and the sevenfold rise in cyclic AMP content effected by treatment for 2 min with 10(-5) M-acetylcholine and for 10 min with 10(-5) M-isoprenaline, respectively; (iv) the rate of [3H]leucine uptake into protein; and (v) the concentration of Neutral Red by the collecting duct. Glands were maintained for 7 days on polycarbonate filters floating on RPMI 1640 tissue-culture medium. After this time the ATP, ADP and AMP contents were 63.2 +/- 7.3, 8.5 +/- 2.2 and 3.5 +/- 0.8 pmol/gland, respectively (mean +/- S.E.M.), which gave an energy charge of 0.90. During maintenance a dilatation of the intercellular spaces developed in both secretory coil and collecting duct. Following maintenance there was a significant rise in the rate of [3H]leucine uptake into protein. Maintained glands demonstrated a fivefold greater accumulation of cyclic AMP in response to isoprenaline than did freshly isolated glands, but there was no comparable maintenance hypersensitivity of cyclic GMP to acetylcholine. This pattern of adrenergic, but not cholinergic, maintenance hypersensitivity matches the known lack of denervation hypersensitivity of human eccrine sweat glands to acetylcholine in vivo.

Adenosine Diphosphate↗

PubMed1983

The metabolism and hormonal responses of human eccrine sweat glands isolated by collagenase digestion.

1. Collagenase digestion of biopsies of human skin yields eccrine sweat glands that can be picked out under binocular light microscopy. The glands are viable as determined by the exclusion of Trypan Blue, the uptake of Methylene Blue, electron microscopy, the rate of lactate dehydrogenase release, ATP content and the rates of glucose oxidation and lactate release. 2. It is proposed that eccrine sweat glands engage in aerobic glycolysis, which accounts for the high content of lactate in sweat (15--60 mM) and the high lactate/pyruvate ratio (100: 1) [Emrich & Zwiebel (1966) Pfluegers Arch. 290, 315--319]. 3. Acetylcholine causes a 4-fold increase in cyclic GMP content, dilatation of the intercellular canaliculi and a reversible, atropine-sensitive, 2-fold increase in the rates of glucose oxidation and lactate release. 4. Isoprenaline causes a 2.5-fold increase in cyclic AMP content. Phenylephrine does not significantly alter cyclic nucleotide metabolism.

Acetylcholine↗