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T Kealey

Publications and source records attributed to T Kealey.

At least 55 records · Page 3Linked to original sources
PubMed1992

Rat hair follicle growth in vitro.

Pelage hair follicles were isolated by gentle microdissection from 8-12-day-old rats, and maintained in supplemented Williams E medium. Length measurements made on freshly isolated hair follicles, and at 24-h intervals, showed a significant increase in hair follicle length over 48 h, after which time no further significant increase in length was observed. Photomicrographs of maintained follicles showed that this increase in hair follicle length could be attributed to the production of a keratinized hair shaft. Histology and [methyl-3H] thymidine autoradiography of freshly isolated hair follicles showed the dermal papilla to be elongated, with thymidine uptake located predominantly in the matrix cells of the hair follicle bulb adjacent to the dermal papilla. This pattern remained unaltered for the first 48 h of maintenance, but after 72 h the dermal papilla had rounded into a tight ball of cells, with very little thymidine uptake occurring in the adjacent matrix cells. On maintenance, fetal calf serum (FCS), epidermal growth factor (EGF) and 12-o-tetradecanoyl phorbol 13-acetate (TPA) all significantly stimulated [methyl-3H] thymidine and [U-14C] leucine uptake, but inhibited hair follicle elongation. Insulin-like growth factor-1 (IGF-1) had no significant effect on rates of hair follicle elongation and [methyl-3H] thymidine uptake, but significantly stimulated rates of [U-14C] leucine uptake. Transforming growth factor-beta 1 (TGF-beta 1) significantly inhibited both the rate of [methyl-3H] thymidine uptake and hair follicle elongation.

Animals↗
PubMed1991

An in vitro model for the study of human hair growth.

Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermosubcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watch makers' forceps. Isolated hair follicles maintained free floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinized hair shaft, and was not associated with the loss of hair follicle morphology. [Methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labeling of keratins showed that their patterns of synthesis did not change with maintenance. Serum was found to inhibit hair follicle growth in vitro; and when follicles were maintained in serum-free medium, they grew for up to 10 days, suggesting that in vitro the hair follicles are able to regulate their own growth, possibly by the production of relevant growth factors. This may prove useful in identifying the autocrine/paracrine mechanisms that operate in the hair follicle. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth regulatory effect on hair follicles in vitro and that EGF and its other receptor ligand TGF-alpha mimic the in vivo depilatory effects of EGF that have been reported for sheep and mice.

Adenosine Triphosphate↗
PubMed1991

cDNA cloning of a human androgen-induced mRNA exhibiting an early and protein synthesis-independent induction.

The detailed mechanism of action of androgens remains unknown. We have used an androgen-dependent human prostate cancer cell line and a subtractive cDNA hybridisation strategy to enrich for androgen-dependent sequences. This yielded a cDNA clone which exhibits the characteristics of a primary trans-activated target for androgens. This androgen-regulated gene encodes a polyadenylated 4.5 kb mRNA which is induced 30-50-fold within 3 h of treatment with 10 nM dihydrotestosterone. The induction does not require continued protein synthesis as it is maintained in the presence of protein synthesis inhibitors.

Androgens↗
PubMed1991

Metabolic studies on isolated hair follicles: hair follicles engage in aerobic glycolysis and do not demonstrate the glucose fatty acid cycle.

The matrix cells of the hair follicle have one of the highest rates of cell division in the mammalian body, but their fuel metabolism is poorly understood, due mainly to the difficulty in obtaining viable intact follicles from the skin. We have previously shown that viable and intact rat hair follicles can be isolated by shearing, and in this study we now report on their fuel metabolism. In this study we have shown that the hair follicle exhibits aerobic glycolysis, in that of the total glucose utilized by the hair follicle, only 10% is oxidized to CO2. We have also shown that, in the absence of glucose, the hair follicle is capable of utilizing other fuels such as palmitate and beta-hydroxybutyrate. However, neither palmitate or beta-hydroxybutyrate had any effect on the rate of glucose utilization or on [U-14C] glucose oxidation, showing that glucose sparing via the glucose fatty acid cycle does not operate in the hair follicle. Measurements of glucose flux through the pentose phosphate pathway accounted for only 3% of the total glucose utilized by the hair follicle, although this value represented 32% of the total glucose oxidized. Both palmitate and beta-hydroxybutyrate inhibited glucose flux through the pentose phosphate pathway.

3-Hydroxybutyric Acid↗
PubMed1991

Androgen metabolism by isolated human axillary apocrine glands in hidradenitis suppurativa.

Androgen metabolism was investigated in normal human apocrine glands and in those isolated from age-matched patients with hidradenitis suppurativa. Axillary glands were isolated by shearing and androgen interconverting enzyme activities were measured in cell-free homogenates by incubation with [3H] dehydroepiandrosterone, [3H] androstenedione and [3H] testosterone. The activities (pmol/mg protein/min: mean + SEM) of 3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (10.0 +/- 1.2 vs. 5.3 +/- 0.5: n = 5) and 17 beta-hydroxysteroid dehydrogenase (58.1 +/- 4.5 vs. 35.7 +/- 5.2: n = 5) were significantly lower (P less than 0.005) in hidradenitis suppurativa, whereas 5 alpha-reductase activity (12.5 +/- 2.3 vs. 12.5 +/- 2.0: n = 5) was similar. This report suggests that hidradenitis suppurativa cannot be attributed to exaggerated activities of end-organ androgen interconverting enzymes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Organ maintenance of human sebaceous glands: in vitro effects of 13-cis retinoic acid and testosterone.

Human sebaceous glands were isolated by shearing, and maintained for 7 days either on defined medium, on medium supplemented with 3 microM-testosterone or on medium supplemented with both 3 microM-testosterone and 1 microM-13-cis retinoic acid. Freshly isolated glands retained their in vivo morphology. On maintenance, the glands retained their freshly isolated rates of cell division, but the sebocytes showed increased keratinization and there was multilayering of the peripheral undifferentiated cells. However, glands maintained in the presence of 1 microM-13-cis retinoic acid showed very little luminal keratinization and only a small degree of multilayering. On autoradiography, freshly isolated glands retained their in vivo pattern of [methyl-3H]thymidine incorporation. Similar patterns were seen when glands were maintained for 7 days with or without testosterone. However, in the presence of both testosterone and 13-cis retinoic acid there was only slight graining. Following 7 days maintenance the rate of lipogenesis fell significantly. This was partially reversed by testosterone, but further inhibited by 13-cis retinoic acid. The patterns of lipids that are synthesised after a week's maintenance are very similar to those seen in freshly isolated glands, except that the squalene:cholesterol ratio is reversibly regulated by 3 microM-testosterone and 1 microM-retinoic acid. Protein synthesis was maintained at the same rates as for freshly isolated glands under all conditions of maintenance. Whereas DNA synthetic rates were maintained in the presence of testosterone, they were significantly inhibited by 13-cis retinoic acid. Glandular wet weights were retained under all conditions of maintenance, except that they were significantly reduced by 13-cis retinoic acid. This study shows that human sebocytes continue to divide on organ maintenance, but that they do not differentiate fully. However, this provides the first demonstration that 13-cis retinoic acid acts on human sebaceous glands directly, reducing the rate of cell division and the rate of lipogenesis, which shows that the maintained human sebaceous gland might provide a useful model for studying the effect of 13-cis retinoic acid on human sebocytes.

Adult↗
PubMed1990

Human hair growth in vitro.

We report for the first time the successful maintenance and growth of human hair follicles in vitro. Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers' forceps. Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with the loss of hair follicle morphology. [methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labelling of keratins showed that their patterns of synthesis did not change with maintenance. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth-regulatory effect on hair follicles in vitro and that EGF mimics the in vivo depilatory effects that have been reported in sheep and mice.

Adenosine Triphosphate↗
PubMed1989

An NADH-linked luciferase assay for glycogen: the preparation of glycogen-free, viable human eccrine sweat glands.

A glycogen assay based on bacterial NADH luciferase is described. It is free of tissue interference. The detection limit is 0.12 nmol glycogen, and the coefficient of variation is 5.5%. A method of depleting human eccrine sweat glands while retaining their viability is described. This depends on their incubation in 10(-5) M acetylcholine and 1 mM pyruvate. This method may be applicable to other tissues. The evidence for the viability of glycogen-depleted human eccrine sweat glands is reported and includes tissue contents of ATP and the rates of oxidation of glucose, pyruvate, beta-hydroxybutyrate, and palmitate.

Adenosine Triphosphate↗
PubMed1989

Lipogenesis by isolated human apocrine sweat glands: testosterone has no effect during long-term organ maintenance.

Lipid synthesis by freshly isolated human apocrine glands has been measured by the incorporation of [U-14C] acetate. Incorporation is linear over 6 h at 1010 +/- 282 pmol/mg wet weight/h (n = 11; mean +/- sem). The lipid classes, as percentages of the total lipid synthesized, were found by TLC to be cholesterol 12.3 +/- 2.0, mono-glycerides 7.5 +/- 1.5, 1,2 di-glycerides 3.0 +/- 0.9, 1,3 di-glycerides 3.5 +/- 0.5, tri-glycerides 28.4 +/- 1.8, free fatty acids 2.0 +/- 0.4, lysolecithin 15.4 +/- 3.9, sphingomyelin 9.9 +/- 4.3, phosphatidyl-choline 8.4 +/- 0.4, phosphatidyl-ethanolamine -inositol and -serine 1.8 +/- 0.1, phosphatidic acid and cardiolipin 3.3 +/- 0.5, and unidentified 3.3 +/- 0.5 (mean +/- sem, n = 5). Glands were maintained on permeable supports. After 10 d maintenance, electron microscopy showed that the cellular architecture had been preserved, that the ATP contents were the same as in freshly isolated glands, and that [U-14C] acetate incorporation was not significantly altered at 851 +/- 237 pmol/mg/h (n = 18). The addition of 3 microM testosterone had no effect on acetate incorporation at 844 +/- 231 pmol/mg/h (n = 18). The lipid classes and their proportions were similar to the values for fresh glands after 10 d maintenance both with and without testosterone.

Acetates↗
PubMed1989

Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles.

We have already shown that shearing can be used to yield large numbers of viable intact hair follicles. We now show that these follicles can be viably maintained on permeable supports for 7 days in vitro as determined by their adenine nucleotide contents, rates of [methyl-3H]thymidine and [U-14C]leucine uptake, [methyl-3H]thymidine autoradiography, patterns of keratin synthesis and light and electron microscopy. These studies, however, show that after 7 days maintenance the morphology of maintained follicles shows a closer resemblance to the telogen rather than the anagen follicle. We therefore conclude that the failure of previous attempts at maintaining hair growth in culture is due to hair follicles prematurely entering the resting stage of their hair growth cycle, possibly as a response to isolation.

Adenosine Diphosphate↗
PubMed1987

Electrophysiological and dye-coupling studies on secretory, myoepithelial and duct cells in human eccrine sweat glands.

1. Electrophysiological properties and dye-coupling status of secretory, myoepithelial and coiled duct cells in isolated human eccrine sweat glands have been assessed by single-micro-electrode studies and intracellular micro-iontophoresis of the fluorescent naphthalimide dye Lucifer Yellow CH (molecular weight 457). Treated glands were embedded in LKB HistoResin and examined by transmission fluorescence microscopy, first as wholemounts and then as 5 microns serial sections. Sections positive for Lucifer Yellow were photographed and then stained with Toluidine Blue for observation by conventional microscopy to confirm the site of penetration. 2. Out of forty-five successful micro-iontophoreses, three were confirmed in secretory cells, twelve in myoepithelial cells and thirty in cells of the coiled duct wall. The latter were identified as the most penetrable in the coiled part of the isolated human eccrine sweat gland. 3. Of the three secretory cells labelled (resting potentials -40, -52 and -63 mV), all demonstrated dye coupling to neighbouring secretory cells although in one case this was found to be selective. Not every secretory cell was involved in coupling. No fluorescent label spread to the myoepithelial cells which form a network on the basal surface of the secretory tubule. 4. When myoepithelial cells were penetrated, they demonstrated dye coupling to neighbouring myoepithelial cells but not to secretory cells with which they also made contact. Basal resting potentials of -35 to -65 mV were recorded (mean = -52 mV, S.E. of mean = +/- 2.4 mV, n = 12) and, in eight out of the twelve cells penetrated and labelled, spontaneous depolarizing transients were also observed whose amplitude but not frequency increased with increasing membrane polarization. Administration of acetylcholine to produce a final concentration of 10(-6) to 10(-7) M produced either depolarization or micro-electrode dislodgement. 5. Of the thirty cells labelled in the coiled duct, twenty-six showed obvious dye spread to neighbouring cells in both layers of the wall. There was no relation between dye-coupling status and basal resting potential which lay in the range -40 to -82 mV (mean = -60 mV, S.E. of mean = +/- 2.4 mV, n = 30). Repeated doses of acetylcholine produced either no response from cells in this range or depolarization in cells with resting potentials more negative than -70 mV and hyperpolarization in cells with resting potentials more positive than -70 mV. In addition, there was a biphasic response, depolarization followed by hyperpolarization in a cell of resting potential -67 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1986

Lipogenesis in isolated human sebaceous glands.

Lipogenesis in isolated human sebaceous glands from [U-14C]glucose, [U-14C]leucine, [U-14C]isoleucine, and [U-14C]valine has been determined by thin-layer chromatography. Total lipogenesis from 2 mmol/l [U-14C]glucose was 114.8 +/- 22.3 pmol/gland per h (mean +/- SE), with 53.8% being incorporated into triglycerides, 20.2% into squalene, 12.8% into phospholipids, 2.1% into cholesterol and 7.1% into wax monoester and cholesterol ester and 5% into di- and monoglycerides and free fatty acids. Total lipogenesis from 2 mmol/l [U-14C]leucine, 2 mmol/l [U-14C]isoleucine, and 2 mmol/l [U-14C]valine in the presence of 2 mmol/l glucose was 26, 29 and 9%, respectively, of that seen with 2 mmol/l glucose alone. The pattern of 14C distribution in the various lipid classes from the three U-14C-labelled branched-chain amino acids was not significantly different from that seen with [U-14C]glucose.

Carbon Radioisotopes↗
PubMed1986

Rapid isolation in large numbers of intact, viable, individual hair follicles from skin: biochemical and ultrastructural characterization.

A rapid, novel method is described by which large numbers of intact, viable, individual hair follicles may be isolated from rat skin. Follicles are freed from the surrounding connective tissue by shearing, which is effected by repeated cutting with a loosely fitting pair of scissors, and collected individually under liquid using gentle aspiration. Ultrastructural analysis indicates that the follicles are sheared away from the surrounding dermis in the region of the connective tissue capsule which encircles the hair. The follicles appear viable by light and electron microscopy and, within 2 h of isolation, retain the capacity to incorporate [3H]thymidine into DNA and [35S]methionine into proteins as judged by autoradiography. A histologic comparison indicates that the structural integrity of follicles isolated by this new method is significantly superior to those plucked from the animal at the same time. The method affords the isolation of large numbers of hair follicles, without resort to enzyme treatments, suitable for biologic studies in the absence of other skin appendages and dermis.

Animals↗