Biomedical subjects

K Docherty

Publications and source records attributed to K Docherty.

At least 109 records · Page 6Linked to original sources

PubMed1988

Gonadal regulation of pituitary hormone mRNA levels in male rats.

The control of anterior pituitary hormone gene expression by testosterone in male rat pituitaries in vivo was investigated using dot-blot mRNA-cDNA hybridization assays. Common alpha subunit mRNA levels doubled by 2 days after orchidectomy and rose progressively to reach plateau levels three to four times intact control values by 2 weeks. LH-beta mRNA increased significantly (congruent to 50%) within 12 h, and thereafter progressively to seven times intact control values by 3 weeks after orchidectomy. The changes in alpha mRNA were likely to have occurred in gonadotrophs and not thyrotrophs, since TSH-beta mRNA levels were unaltered by orchidectomy. LH subunit mRNA changes were accompanied by an initial (1-4 days) decrease in pituitary LH content; thereafter, pituitary LH increased in parallel with and by a similar magnitude to the LH-beta mRNA. Serum LH rises occurred before significant increases in LH subunit mRNA after orchidectomy. The lack of temporal correlation between mRNA levels and serum and pituitary LH in the early stages after removal of testosterone feedback contrasts with the good correlation when a new steady state was achieved after 3-4 weeks, and indicates differing kinetics for changes in these aspects of gonadotroph function. An inhibitory effect of testosterone on LH subunit gene expression was confirmed by prevention of the rise in alpha and LH-beta mRNAs when treatment commenced immediately after castration. However, pituitary LH content and serum LH levels were reduced relative to control values, suggesting additional inhibitory actions of testosterone on translational and post-translational events in gonadotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

DNA and RNA analysis of cytochrome P-450 21-hydroxylase: transcriptional activity in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a family of inherited disorders of adrenal steroidogenesis, most commonly due to deficiency of P-450 21-hydroxylase (21-OH). There are two genes for 21-OH on the short arm of chromosome 6, the A gene which is thought to be inactive, and the B gene. These genes appear as 3.2 and 3.7 kb TaqI fragments on Southern blots. In a study of DNA from 60 normal controls with TaqI and a 21-OH cDNA probe, 12% exhibited a homozygous deletion of the A gene, and 22 and 8% heterozygous deletions of A and B genes respectively. TaqI analysis of eight patients with CAH revealed four without A or B gene deletions, three with heterozygous deletions of the B gene and one with a homozygous deletion of the B gene. On further analysis with KpnI, EcoRI, PvuII and BglII, however, these genotypes were amended to two with heterozygous deletions of the B gene and two with possible B to A gene conversions. The genotypes of the four patients without deletions remained unchanged. RNA from CAH and Cushing's adrenal tissue was also analysed using A and B gene-specific oligodeoxynucleotide probes. B gene transcripts were detected in both CAH and Cushing's adrenals, while no A gene transcripts could be detected in either tissue. The level of B gene-derived mRNA was greater in the Cushing's adrenal than in the CAH adrenal, which in turn was greater than that in the adrenal from a normal individual.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1988

Changes in insulin receptor expression in HL60 cells induced to differentiate towards neutrophils or monocytes.

HL60 is a human promyeloid cell line capable of differentiating towards monocytes or granulocytes when treated with appropriate agents. Changes in insulin receptor number, affinity and mRNA levels were observed when HL60 cells were induced to differentiate with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulphoxide (DMSO). Total and high-affinity insulin receptor numbers decreased following treatment of HL60 cells with DMSO, whereas total insulin receptor number increased and high-affinity receptor number decreased in cells treated with TPA. Three distinct insulin receptor mRNA species of 9.1, 6.3 and 2.8 kb were identified in HL60 cells. The larger 9.1 and 6.3 kb species were increased in both TPA- and DMSO-treated HL60 cells, and the 2.8 kb mRNA was reduced in differentiated cells. Thus HL60 cells differentiated towards monocytes or granulocytes showed similar changes in the levels of individual insulin receptor mRNAs, but displayed contrasting alterations in low-affinity insulin binding. Three HL60 variant lines, which have different capacities to respond to inducers of monocyte and neutrophil differentiation, showed similar levels of total insulin receptors, but differed in their expression of high-affinity receptors. The data provide evidence for the existence of two distinct insulin receptors.

Blotting, Northern↗

PubMed1987

A comparison of the effects of bromocriptine and somatostatin on growth hormone gene expression in the rat anterior pituitary gland in vitro.

The effects of the dopamine agonist bromocriptine (BCR) have been compared with those of somatostatin (SS) on growth hormone (GH) synthesis and secretion by rat anterior pituitary cells in vitro. Both BCR and SS produced a dose-related reduction in GH release. Cytoplasmic GH mRNA levels were unchanged by BCR treatment and this finding was associated with an increase in total intracellular GH content. The reduction in GH release seen following SS treatment was accompanied by a fall in cytoplasmic GH mRNA levels and no significant change in intracellular GH content. These results suggest that the effects of BCR are predominantly on GH release mechanisms, whereas SS appears not only to regulate hormone release but also to regulate GH gene expression at a pre-translational level.

Animals↗

PubMed1987

Effect of hypothyroidism and thyroid hormone replacement in vivo on pituitary cytoplasmic concentrations of thyrotropin-beta and alpha-subunit messenger ribonucleic acids.

We have studied the influence of hypothyroidism and thyroid hormone replacement in vivo on rat pituitary cytoplasmic concentrations of TSH beta and alpha-subunit mRNA, measured by cytoplasmic dot hybridization, as well as circulating TSH and pituitary TSH content. Cytoplasmic concentrations of GH, PRL, and LH beta-subunit mRNA were measured in parallel. Marked increases in serum TSH and TSH beta and alpha mRNA were found in hypothyroidism; these changes reversed 72 h after beginning T3 replacement. A more marked effect of hypothyroidism on TSH beta mRNA than on alpha mRNA was evident, suggesting differential control of regulation of the TSH genes. In contrast to a rapid fall in serum TSH after T3 administration, an increase in TSH beta and alpha mRNA was evident at 1 and 6 h, followed by a later fall in TSH mRNA to euthyroid values. Pituitary cytoplasmic concentrations of GH and PRL mRNA were reduced in hypothyroidism and increased after administration of T3; LH beta mRNA was unaffected by hypothyroidism or T3 replacement. The differential effects of thyroid status on TSH, GH, PRL, and LH mRNA indicate specificity of regulation of these anterior pituitary hormone genes.

Animals↗

PubMed1987

The effect of thyroid hormones on growth hormone gene expression in vivo in rats.

Thyroid hormones are important regulators of GH synthesis and secretion. In this study we have made a detailed examination of the time-course of the effects of hypothyroidism and tri-iodothyronine (T3) replacement in the intact rat on GH gene expression in the anterior pituitary gland. Changes in pituitary cytoplasmic GH messenger (m)RNA levels were compared with total pituitary GH content and serum GH concentration during the development of hypothyroidism and following short-term T3 replacement in vivo. Hypothyroidism was associated with a fall in pituitary GH mRNA levels. Treatment of hypothyroid animals with T3 rapidly stimulated GH mRNA levels to values above those seen in euthyroid controls. The reduction in GH mRNA levels seen during the development of hypothyroidism was accompanied by a fall in serum GH and pituitary GH content, both of which were partially restored by T3 replacement. Thus thyroid hormone replacement in hypothyroidism rapidly stimulates GH mRNA synthesis, which is followed by the gradual restoration of pituitary GH stores and serum GH concentration.

Animals↗

PubMed1987

Modulation by oestrogen of thyroid hormone effects on thyrotrophin gene expression.

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Studies of the parathyroid hormone gene in normal subjects, and in subjects with primary hyperparathyroidism and familial benign hypercalcaemia.

Familial benign hypercalcaemia (FBH) closely resembles primary hyperparathyroidism (PHPT) both clinically and biochemically. Using a cDNA probe for the parathyroid hormone (PTH) gene we have studied restriction fragment length polymorphisms in normal British subjects and have shown them to be similar to those found in previous studies in a German population. The pattern of inheritance of these restriction fragment length polymorphisms in a family with FBH shows that the PTH gene is not involved in the pathogenesis of the condition. Limited studies in PHPT indicate that it is unlikely that a major structural defect or rearrangement is responsible for the sporadic form of the disease.

Alleles↗

PubMed1987

Thyroid status affects the regulation of prolactin mRNA accumulation by tri-iodothyronine and thyrotrophin-releasing hormone in cultured rat anterior pituitary cells.

The effects of tri-iodothyronine (T3) and TRH on prolactin mRNA accumulation in monolayer pituitary cell cultures prepared from both euthyroid and hypothyroid rats were investigated. Basal prolactin mRNA concentrations and prolactin release into culture medium were increased in hypothyroid cultures, the increase being related to the duration of hypothyroidism in vivo. The inhibitory effects of T3 seen in euthyroid cells were preserved in cells derived from hypothyroid animals, and the degree of inhibition was greater in cells from the most severely hypothyroid rats. However, the stimulation of prolactin synthesis and secretion induced by TRH in euthyroid cultures was not found in the hypothyroid cells. Hypothalamic and anterior pituitary TRH content were measured in similarly hypothyroid and euthyroid rats. A large hypothalamic pool of TRH was found, which was unchanged in hypothyroidism, whereas anterior pituitary TRH content was increased in the hypothyroid rats. The consequent down-regulation of anterior pituitary TRH receptors may explain the poor response of prolactin to TRH seen in vitro.

Animals↗

PubMed1987

Hormonal regulation of thyrotrophin synthesis and secretion.

We have examined the effects of triiodothyronine treatment on serum TSH and pituitary cytoplasmic TSH alpha and beta mRNA levels in the hypothyroid rat. Serum TSH and the cytoplasmic levels of the TSH alpha and beta subunit mRNAs rose concomitantly in hypothyroidism. Treatment of hypothyroid animals with triiodothyronine resulted in a fall in serum TSH but an initial increase in subunit mRNAs, followed by a reduction to euthyroid levels. We have also studied the modulation of these responses by oestrogen and testosterone administration. Treatment with oestradiol during the development of hypothyroidism gave rise to lower levels in alpha and beta subunit mRNAs compared with hypothyroid animals not receiving the oestrogen. Oestrogen also abolished the early rise in subunit mRNA levels seen following triiodothyronine replacement. Testosterone administration during the development of hypothyroidism had a similar effect to that of oestrogen in that subunit mRNA levels were lower in the androgen-treated hypothyroid animals than in hypothyroid animals receiving no exogenous androgen. These data suggest that gonadal steroids render the pituitary thyrotroph cell more sensitive to thyroid hormone.

Animals↗

PubMed1986

Effect of hypothyroidism on pituitary cytoplasmic concentrations of messenger RNA encoding thyrotrophin beta and alpha subunits, prolactin and growth hormone.

Thyroid hormones may directly regulate gene expression in the anterior pituitary. In order to examine this possibility we have studied the effect of hypothyroidism in the rat on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding thyrotrophin (TSH) beta and alpha subunits, prolactin and GH. We demonstrated a marked increase in TSH beta and alpha subunit mRNA, accompanied by a decrease in GH mRNA, in the hypothyroid state, changes largely reversed by thyroid hormone replacement. We have thus shown a direct influence of thyroid status on the pretranslational events occurring in pituitary hormone synthesis. The simultaneous rise in cytoplasmic TSH beta and alpha mRNA levels and fall in GH mRNA in hypothyroidism suggests that thyroid status exerts a differential effect on the expression of these genes.

Animals↗

PubMed1986

Tri-iodothyronine and phenytoin reduce prolactin messenger RNA levels in cultured rat pituitary cells.

Thyroid hormones may regulate prolactin gene transcription. We have previously found that phenytoin inhibits tri-iodothyronine (T3) nuclear binding, and have suggested that phenytoin may act as a partial T3 agonist. We have therefore investigated the effects of phenytoin and T3 on prolactin release and gene transcription, using the technique of cytoplasmic dot hybridization with complementary DNA probes to estimate prolactin messenger (m) RNA concentrations in cytoplasm from cultured rat pituitary cells. Tri-iodothyronine treatment led to a small but significant fall in prolactin release by 72 h, but caused marked dose- and time-dependent reductions in prolactin mRNA levels at 48-72 h. Phenytoin, however, caused more rapid falls in both prolactin release and mRNA concentrations. Neither T3 nor phenytoin significantly altered GH mRNA levels. These studies suggest effects of phenytoin similar, but not identical, to those of T3 in the lactotroph.

Animals↗

PubMed1986

Cyclic adenosine nucleotides and growth hormone-releasing factor increase cytosolic growth hormone messenger RNA levels in cultured rat pituitary cells.

The cellular mechanisms involved in GH biosynthesis have been investigated by the measurement of steady-state levels of cytosolic GH messenger RNA (mRNA) in primary cultures of rat pituitary cells using an RNA-complementary DNA (cDNA) hybridization assay. Growth hormone mRNA-cDNA hybridization increased in a linear manner with increasing cytosol concentration. Cellular GH mRNA levels rose by an average of 2.4-fold (range, 1.6-3.3; n = five experiments) after exposure to GH-releasing factor (GRF(1-40); 10 nmol/l) for 3 days. Treatment with GRF increased the release of GH into the culture medium, and depleted the cellular GH content by 40%. Total GH (in the medium plus cells) after GRF treatment increased by between 1.5- and 3.8-fold, a magnitude similar to the increase in GH mRNA levels. Treatment of cells with dibutyryl adenosine 3':5'-cyclic monophosphate (1 mmol/l) or forskolin (5 mumol/l) increased the levels of cytosolic GH mRNA by between 1.6- and 4.7-fold. These agents increased GH release into the medium, depleted cellular GH content and increased total GH in the system to the same extent as GRF (10 nmol/l). These data demonstrate that cyclic adenosine nucleotides may mediate the GRF induction of GH gene transcription. In addition, we have shown that increases in the levels of cellular GH mRNA are reflected by increased GH biosynthesis, suggesting that the regulation of hormone gene transcription is one cellular site for the control of hormone biosynthesis and, ultimately, hormone available for release.

Animals↗

PubMed1986

Demonstration of thyrotrophin beta-subunit messenger RNA in rat pituitary cells in primary culture--evidence for regulation by thyrotrophin-releasing hormone and forskolin.

We have reported previously the effect of thyroid status in vivo on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding the thyrotrophin (TSH) beta-subunit (Franklyn, Lynam, Docherty et al, 1985). Studies in vitro of the regulation of TSH beta gene transcription have been confined to thyrotrophic tumour cells. We now report the demonstration of TSH beta-subunit mRNA in non-tumorous rat pituitary cells in primary culture. Treatment of cells with thyrotrophin-releasing hormone (TRH) and with forskolin resulted in a marked increase in cellular concentration of TSH beta-mRNA. These results suggest that TRH exerts a direct effect on the pretranslational events involved in TSH synthesis and further that the adenylate cyclase system may be involved in the regulation of synthesis. We have thus described a novel system for the study of TSH beta-subunit gene expression in normal rat pituitary cells in vitro.

Animals↗

PubMed1985

Castration increases luteinizing hormone subunit messenger RNA levels in male rat pituitaries.

The effect of castration on pituitary common alpha and LH-beta subunit mRNA levels was examined in adult male rats in a dot-blot hybridization assay using cytosolic mRNA and 32P labelled plasmids containing cDNA encoding sequences of the subunits. Orchidectomy increased alpha subunit mRNA levels threefold by day 4 with no further rise at 2 months. The LH-beta subunit mRNA levels increased by threefold at 2 months. Serum LH levels increased by up to fortyfold (2 months). Pituitary LH content increased by fourfold at 2 months, though was reduced at 4 days after castration. These results suggest that transcription of the alpha and LH-beta genes are similarly regulated after castration. However the greater magnitude of serum LH increase after castration than that of both LH subunit mRNA levels and the bidirectional changes in pituitary LH content implies additional translational/post-translational control of gonadotrophin biosynthesis by gonadal hormones.

Animals↗

PubMed1984

Cathepsin B-related proteases in the insulin secretory granule.

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.

Adenoma, Islet Cell↗

PubMed1984

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview.

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Proinsulin in human serum: problems in measurement and interpretation.

The immunoreactivity of an extracted pancreatic human proinsulin standard in an indirect immunoradiometric assay was found to be at least one hundred times higher than that of biosynthetic human proinsulin. Limited tryptic digestion of the biosynthetic proinsulin increased its immunoreactivity in the assay and this was attributed to the production of partially cleaved proinsulin molecules which still retained the C-peptide moeity. This inference was confirmed by the finding that pure samples of 65/A1 and 32/33 split proinsulins reacted in the assay very similarly to the pancreatic proinsulin standard. The implications of these results are that the immunoassays of proinsulin using antisera to C-peptide may recognise the intact proinsulin molecule very poorly, if at all, and that the 'proinsulin' measured by such assays of human serum may be largely if not entirely intermediates of proinsulin cleavage.

C-Peptide↗