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P F Hall

Publications and source records attributed to P F Hall.

At least 37 records · Page 2Linked to original sources
PubMed1993

Structure-function studies of human aromatase.

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.e. C299A, E302L, P308F, D309N, D309A, T310S, T310C, and S312C, have been generated using a mammalian cell stable-expression system. The results from site-directed mutagenesis studies indicate that the region containing Gln-298 to Val-313 is indeed a very important part of the active site of aromatase. The catalytic properties of P308F, D309N, and D309A have been examined in detail and are discussed. Active site-directed labeling is also an important approach to investigate the structure-function relationship of aromatase. HPLC-linked electrospray mass spectrometry is indicated as a useful technique for the characterization of active site-directed probe-modified enzyme. The mass spectral analysis of aromatase suggests that aromatase is glycosylated.

Amino Acid Sequence↗
PubMed1993

Binding of lipid droplets and mitochondria to intermediate filaments in rat Leydig cells.

We have examined the distribution of lipid droplets and mitochondria in relation to the cytoskeletons of Leydig cells in primary culture by using light and electron microscopy on living, intact and detergent-extracted cells. After mild extraction with Triton X-100 lipid droplets and mitochondria retained their original distribution within the cell. Double immunofluorescent microscopy showed that both structures co-localise with intermediate filaments. Transmission electron microscopy of intact (unextracted) and mildly extracted Leydig cells showed that intermediate filaments are closely associated with mitochondria and lipid droplets. By examination of stereo pairs, intermediate filaments were shown to establish direct contact with mitochondria and lipid droplets. The association of droplets and mitochondria with intermediate filaments suggests possible mechanisms by which the transport of cholesterol takes place from droplets to mitochondria where this substrate enters the steroidogenic pathway.

Animals↗
PubMed1992

Cyclic AMP regulates expression of the rat gene for steroid 17 alpha-hydroxylase/C17-20 lyase P-450 (CYP17) in rat Leydig cells.

The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5' upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.

Animals↗
PubMed1992

A serious issue.

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Canada↗
PubMed1992

Gene for 17 alpha-hydroxylase/C (17-20) lyase P-450: complete nucleotide sequence of the porcine gene and 5' upstream sequence of the rat gene.

We describe the isolation and characterization of a full-length clone for the porcine 17 alpha-hydroxylase/C(17-20) lyase (CYP17) gene. The complete exon and partial intron sequences are presented including approx. 1000 bp of the 5' upstream sequence. In addition we describe the isolation and characterization of the 5' upstream region of the rat CYP17 gene. The sequences of the first exon, part of the first intron, and approx. 3.5 kb of the 5' upstream region are presented.

Amino Acid Sequence↗
PubMed1992

Attachment of mitochondria to intermediate filaments in adrenal cells: relevance to the regulation of steroid synthesis.

The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis.

Adrenal Glands↗
PubMed1992

Endozepine/diazepam binding inhibitor in adrenocortical and Leydig cell lines: absence of hormonal regulation.

One of the many effects which have been attributed to the peptide endozepine/diazepam binding inhibitor (Ep/DBI) is the stimulation of adrenocortical and testicular Leydig cell mitochondrial steroidogenesis. We have used two cell lines (Y-1 mouse adrenal cell tumour and MA-10 mouse Leydig cell tumour), both of which exhibit hormone stimulated steroid production, to investigate the role of Ep/DBI in acute hormone stimulated steroidogenesis. The time course of incorporation of 35S-translabel into Ep/DBI and its turnover rate when the isotope was removed were examined. Cell samples were extracted and separated on Sep-Pak C18 columns and analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis followed by fluorography as well as by direct scintillation counting. This allowed us to estimate the in vivo half-life of Ep/DBI and also to investigate the hormonal dependence of the peptide. Data presented here suggest that (i) Ep/DBI levels are not regulated by trophic hormones in these steroidogenic cell lines, and (ii) that the peptide has a relatively long half-life (greater than 3 h), a finding incompatible with suggestions of it having a rapid turnover. Therefore, it seems unlikely that control of Ep/DBI steroidogenic effects is via hormonal modulation of the peptide levels.

Adrenal Cortex↗
PubMed1992

Angiotensin II regulates cytochrome P-450 steroid hydroxylase enzyme expression in human adrenal glomerulosa cells.

We have examined the regulation of cytochrome P-450 side chain cleavage enzyme (P-450SCC) and P-45011 beta (18) hydroxylase (P-450(11) beta) enzyme expression by angiotensin II (AII), the major regulator of aldosterone biosynthesis, in cultured human adrenal glomerulosa cells. We also examined the effect of lipoxygenase products of arachidonic acid on the expression of these enzymes since it has been previously shown that the 12-lipoxygenase pathway plays a key role in AII-induced aldosterone synthesis in rat and human adrenal glomerulosa cells. AII (10(-7) mol/L) induced a 3-fold stimulation of P-450SCC and over a 2-fold increase in P-450(11) beta protein expression. The 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) caused a 2-fold increase in P-450(11) beta levels without altering P-450SCC levels. These results show for the first time that AII can directly increase the levels of P-450SCC and P-450(11) beta enzymes in glomerulosa cells. The results also suggest that 12-HETE may mediate long term effects of AII action by stimulating P-450(11) beta levels.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells.

Light microscopy of living and extracted adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures. Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5 nm wide. The droplets in Y-1 cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.

Adrenal Glands↗
PubMed1991

Stimulation by endozepine of the side-chain cleavage of cholesterol in a reconstituted enzyme system.

Addition of endozepine in nanomolar concentrations to a system for side-chain cleavage reconstituted from highly purified P-450scc and electron carriers (adrenodoxin reductase and adrenodoxin) stimulates the conversion of cholesterol to pregnenolone (side-chain cleavage). This response is concentration and time-dependent and specific to the extent that a second steroidogenic P-450 located in the inner mitochondrial membrane (ie 11 beta-hydroxylase) was not stimulated by endozepine. Homogeneous endozepine prepared from bovine brain, the corresponding genetically engineered peptide and des(glu-ilu)-endozepine isolated from bovine adrenal cortex are all approximately equipotent in this system. Moreover, endozepine accelerates the rate of reduction of P-450scc by NADPH and the electron carriers. The results suggest that endozepine acts directly on P-450 and hence the rate of side-chain cleavage.

Adrenal Cortex↗
PubMed1991

Cancer risks after medical radiation.

Radiation-induced tumors cannot be distinguished from tumors in general by means other than a statistical excess. Epidemiological studies are the only means by which answers can be given as regards the carcinogenic effect of ionizing radiation. Age at exposure is perhaps the most important host factor influencing cancer risk and it is generally believed that cancer risk decreases with increasing age at exposure. For most cancers the temporal pattern follows the natural incidence, i.e. the cancers do not occur before ages normally associated with increased incidence. The induction period for solid tumors is at least 10 years while the corresponding figure for leukemia is 2 years. The breast, thyroid, lung and bone marrow seem to be the most radiosensitive tissues, while the risk of chronic lymphatic leukemia and possibly Hodgkin's disease and prostatic cancer does not seem to increase after exposure to ionizing radiation.

Female↗
PubMed1991

Cytochrome P-450 C21scc: one enzyme with two actions: hydroxylase and lyase.

Testis, adrenal, ovary and placenta contain a microsomal cytochrome P-450 that is capable of converting progesterone to androstenedione and pregnenolone to dehydroepiandrosterone. This conversion requires 17 alpha-hydroxylation followed by C17,20-lyase activity which are both catalyzed by this one protein. Gene cloning and Northern blotting reveal that, at least in man, the same gene is responsible for both testicular and adrenal enzymes. The enzyme was first purified from neonatal pig testis. Both the testicular and adrenal enzymes show a marked preference for the 5-ene substrate (pregnenolone) in keeping with the extensive use of the 5-ene pathway in that species. Affinity alkylation with 17 alpha-bromoacetoxyprogesterone reveals a conserved cysteine at the active site of the enzyme and confirms the conclusion that a single enzyme catalyzes both reactions. Under some circumstances the enzyme catalyzes only 17 alpha-hydroxylation to permit the formation of the C21 steroid cortisol. The regulation of lyase activity, i.e. the determination of the extent to which the second activity is expressed, results from the availability of P-450 reductase. No doubt the greater concentration of this protein in testicular as opposed to adrenal microsomes (x 3.5) is responsible for the production of androgens in the testis and cortisol in the adrenal. Testicular cytochrome b5 also specifically stimulates lyase activity and also causes the porcine enzyme to catalyze a new reaction, i.e. delta 16-synthetase, resulting in synthesis of the important pheromone androsta-4,16-dien-3-one from progesterone.

Adrenal Glands↗
PubMed1991

The role of diazepam binding inhibitor in the regulation of steroidogenesis.

It has been well established that one factor influencing the rate of side-chain cleavage of cholesterol is the rate of delivery of the substrate, cholesterol, from depots in the cytoplasm to the inner mitochondrial membrane within the organelle. This process of intracellular transport consists of two steps: transport to the mitochondria and transport from outer to inner membrane. Transport to mitochondria requires the cytoskeleton and Ca(2+)-calmodulin but not newly synthesized protein. Transport within the mitochondria requires newly synthesized protein. Both steps are stimulated by the trophic hormones ACTH and LH, in their respective target organs. Since the steroidogenic responses to these hormones are inhibited by cycloheximide, it is proposed that the new protein(s) required for these responses are synthesized in the cytoplasm. Using an assay based on the production of pregnenolone by mitochondria from bovine adrenal cortex, a protein of mol. wt approximately 8200 (temporarily referred to as 8.2 K) was isolated. The protein was purified to homogeneity and found to possess the following properties: (i) 8.2 K accelerated the synthesis of pregnenolone by isolated mitochondria (ii) it promoted entry of cholesterol from the incubation medium into mitochondria (iii) 8.2 K accelerated the transport of cholesterol from outer to inner membrane (iv) it also promoted loading of P-450scc with cholesterol, when either mitoplasts of the inner mitochondrial membranes were incubated with 8.2 K in vitro. Moreover, (v) the synthesis of 8.2 K was increased by ACTH and (vi) the half-life of the protein was less than 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1991

Synthesis of aldosterone by mitochondria and homogeneous 11 beta-hydroxylase from beef and pig.

It was found that homogeneous 11 beta-hydroxylase from bovine and porcine adrenals catalyzes the conversion of DOC to aldosterone. Mitochondria from both glomerulosa and fasciculata also convert DOC to aldosterone but glomerulosa is much more active than fasciculata. Cholate extracts of mitochondria from the two zones were equally active in converting DOC to aldosterone. Moreover all the enzyme activities of 11 beta-hydroxylase (including 18-hydroxylation and aldehyde synthetase) were precipitated by a polyclonal antibody raised in rabbit against the pure 11 beta-hydroxylase. It is concluded that in beef and pig a single adrenocortical 11 beta-hydroxylase is responsible for the synthesis of aldosterone. To determine the influence of the mitochondrial membrane from glomerulosa and fasciculata on the activities of 11 beta-hydroxylase we examined the activities of rotenone-insensitive reductase enzymes in mitochondria from the two zones. Semidehydroxyascorbate reductase and NADH-cytochrome C reductase activities are considerably more active in glomerulosa than in fasciculata mitochondria. Moreover ascorbate plus NADH (but not ascorbate alone) greatly increases the ability of malate and NADPH to support synthesis of aldosterone without affecting 11 beta- or 18-hydroxylations in mitochondria. It is proposed that maximal synthesis of aldosterone by adrenocortical mitochondria requires in addition to the usual electron transport system (NADPH- greater than ADR- greater than ADX- 11 beta-OHase) an auxilliary system in the outer mitochondrial membrane: NADH- greater than Fp- greater than cyt b- greater than semidehydroascorbate reductase.

Adrenal Glands↗
PubMed1990

Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells.

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.

Adrenal Gland Neoplasms↗
PubMed1990

Ascorbate as a source of reducing equivalents for the synthesis of aldosterone.

The three steps in the synthesis of aldosterone (11 beta/18-hydroxylations and aldehyde synthetase) were examined in mitochondria from bovine glomerulosa and fasciculata to study the regulation of aldehyde synthetase. Ascorbate plus NADH shows synergism with malate in stimulating aldehyde synthetase without affecting 11 beta/-18-hydroxylations. The concentration of semidehydroascorbate reductase in mitochondria from glomerulosa is more than twice that from fasciculata. We propose that in glomerulosa, ascorbate provides a source of reducing equivalents that specifically support the last step in the synthesis of aldosterone.

Adrenal Glands↗