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P F Hall

Publications and source records attributed to P F Hall.

At least 19 recordsLinked to original sources
PubMed1996

Expression of the gene for cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (CYP17) in porcine Leydig cells: identification of a DNA sequence that mediates cAMP response.

Regulation of CYP17 gene expression in porcine Leydig cells was investigated in primary culture. We previously reported the sequence of the 5' upstream and much of the pig gene. (Zhang et al. (1992) Biochim. Biophys. Acta. 1131, 345-348). DNase I footprinting assays identified a region between -193 and -174 that was bound by nuclear proteins. Examination of the DNA sequence in this region revealed putative Sp1 and AP-2 binding sites, but gel retardation assays using an oligonucleotide from -198 to -168 as a probe revealed two specific DNA-protein complexes that were not Sp1 or AP-2. The oligonucleotide was cloned into a reporter gene containing a minimal porcine CYP17 promoter and the resultant construct was transiently transfected into porcine Leydig cells. This chimeric construct had both basal and cAMP-induced transcriptional activities. Southwestern blot identified a prominent binding of a nuclear protein around 68 kDa and a weaker binding of a nuclear protein around 110 kDa. Sequences between -250/+1 are highly homologous to those sequences from human, bovine and rodent CYP17 gene, but the -193/-174 region has no homology to those genes. Other regions of the porcine CYP17 were also important for the basal and cAMP-mediated regulation. Luciferase expression vectors were prepared with 5' flanking DNA from the porcine CYP17 gene and were expressed in primary culture of porcine Leydig cells. The region between -587/-325 was important for basal transcription, and a region of DNA between -325 and -140 was important for cAMP regulation.

Adrenal Glands↗
PubMed1995

GTP-binding proteins in adrenocortical mitochondria.

We have identified two GTP-binding proteins in mitochondria from bovine adrenal cortex (fasciculata). Sub-mitochondrial particles were fractionated into inner membrane, contact point and outer membrane vesicles on sucrose density gradients. These sub-mitochondrial fractions were identified by the presence of enzyme markers and electron microscopy. Photoaffinity labelling with [gamma-32P]GTP identified a 45 kDa GTP-binding protein in outer mitochondrial membranes and a 19 kDa protein in the contact points. The molecular weight of 45 kDa and requirement for Mg2+ ions raise the possibility that this protein is an alpha subunit of a heterotrimeric GTP-binding protein or a novel GTP-binding protein. The specificity of nucleotide binding, the requirement for low concentrations of Mg2+ (0.1 mM) and molecular weight of 19 kDa suggest that this protein is a typical member of the so-called small GTP-binding protein family. The location of 45 kDa in the outer membrane and that of 19 kDa in the contact points suggest roles for these proteins in the interaction with the extramitochondrial environment and in the regulation of mitochondrial membranes, respectively.

Adrenal Cortex↗
PubMed1995

The role of endozepine in the regulation of steroid synthesis.

Endozepine has recently been isolated from various steroid-forming organs. The following article explores the role of endozepine in the regulation of steroid synthesis. Steroid hormone synthesis from cholesterol begins in the inner mitochondrial membrane, where cytochrome P450 converts cholesterol to pregnenolone. Scientists thought that ACTH would stimulate this conversion, but experiments showed no such stimulation. However, addition of aminoglutethimide to block side-chain cleavage caused the expected reaction of ACTH to take place. Next the role of protein synthesis on the actions of ACTH was explored. Then endozepine was isolated from bovine fasciculata based on stimulation of pregnenolone production by freshly prepared mitochondria. After further experimentation it was concluded that endozepine is a peptide with at least two groups of actions: It binds GABAA receptors in the central nervous system, and it increases the mitochondrial synthesis of pregnenolone.

Adrenocorticotropic Hormone↗
PubMed1995

Impact of a nurses' strike on the cesarean birth rate.

OBJECTIVE: Our purpose is to describe the impact of a 31-day nurses' strike on the cesarean birth rate in the province of Manitoba, Canada. STUDY DESIGN: Computerized hospital records, obtained for all births over a 24-month period, were used to identify complications of labor indicating cesarean section, method of delivery, and adverse maternal and newborn outcomes. The strike interval was compared with a 16-month prestrike period. RESULTS: The cesarean section rate in the strike interval, 12.5 per 100 deliveries, was significantly lower than the prestrike rate of 14.6 per 100 deliveries (p < 0.05). Reductions occurred primarily among breech deliveries and among women with a previous cesarean section. No differences were observed in the rates of individual adverse maternal or newborn outcomes. However, the pooled incidence of adverse newborn outcomes was significantly higher during the strike than during the prestrike period (10.2 vs 8.1/100 deliveries, odds ratio 1.27, 95% confidence interval 1.07 to 1.52). CONCLUSION: In response to constraints imposed by a reduced nursing complement, physicians increased the frequency of vaginal birth in breech presentation and among women with previous cesarean section.

Breech Presentation↗
PubMed1995

The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis.

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol--the substrate for the first enzyme, namely P450scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca2+/calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca2+/calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cyclic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca2+/calmodulin possibly by redistributing cellular Ca2+. Ca2+/calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca2+/calmodulin) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.

Actin Cytoskeleton↗
PubMed1995

Transmission of Ureaplasma urealyticum from mothers to full and preterm infants.

This study assessed maternal genital colonization and subsequent neonatal transmission rate of Ureaplasma urealyticum in pregnant women in an average socioeconomic population. In addition very low birth weight infants were assessed to determine whether the presence of U. urealyticum correlated with increased risk of developing respiratory problems. The study group consisted of 108 sequential full term mothers and 104 preterm mothers delivering in a tertiary care hospital in central Canada. The genital carriage rates (assessed using placental sampling) of ureaplasmas in term and preterm mothers were 25.9 and 19.2%, respectively (P = 0.3185). Acquisition of ureaplasmas in the neonatal respiratory tract of neonates occurred significantly (P = 0.0182) more often in preterm neonates (11 of 130; 8.5%) than in term neonates (2 of 110; 0.9%). Very low birth weight (VLBW) infants (< or = 1500 g) were at greater risk (P = 0.042) of acquiring ureaplasmas in their respiratory tracts (5 of 26; 19%) than larger preterm neonates (6 of 104; 5.8%). All VLBW infants with respiratory colonization by ureaplasmas (5 of 5) developed bronchopulmonary dysplasia compared with 33% (7 of 21) of VLBW neonates without ureaplasmas (P = 0.028). This difference in bronchopulmonary dysplasia development among VLBW infants was independent of further stratification by birth weight. These VLBW neonates with ureaplasmas also stayed significantly (P = 0.037) longer in the neonatal intensive care unit (43.6 +/- 10.4 days) than did other preterm neonates (22.1 +/- 20.8 days). Our results demonstrate that VLBW preterm neonates have increased risk of acquiring U. urealyticum.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes.

Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.

Acyl-CoA Oxidase↗
PubMed1994

Calcium/calmodulin induces phosphorylation of vimentin and myosin light chain and cell rounding in cultured adrenal cells.

In previous reports on adrenal cells we have shown that calcium/calmodulin regulates cholesterol transport to mitochondria and induces phosphorylation of cytoskeleton homogenates. In this study, we have used bovine fasciculata cells permeabilized in situ to identify the phosphorylated proteins and to investigate the manner in which the cytoskeleton components may act together in any subsequent reorganization of the cell. The main cytoskeletal proteins namely vimentin, tubulin, actin, and the associated protein myosin light chain were identified on polyacrylamide gel electrophoresis by their molecular weights and by Western blotting using affinity-purified monoclonal antibodies. In permeabilized cells, calcium/calmodulin promoted phosphorylation of vimentin and myosin light chain within the first 10 min. When incubation time was extended in the presence of 1 mM non-radiolabeled ATP, cell contraction was seen after 15 min. Immunofluorescent microscopy showed that actin microfilaments and myosin light chain displayed a similar pattern of distribution which indicates the actomyosin. Electron microscopy revealed the actomyosin as a dense ring around the cell beneath the plasma membrane. Intermediate filaments (10 nm) were seen within this ring which gave rise to a mixed network in which microfilaments appeared to interconnect intermediate filaments. Immunogold electron microscopy revealed that the 10-nm filaments, found within the actomyosin ring, are vimentin intermediate filaments. It is proposed that calcium/calmodulin causes phosphorylation of the myosin light chain which triggers contraction, and this process involves the intermediate filament protein vimentin. The redistribution of the cytoskeleton and hence the cell rounding is due, in part to the interconnection between vimentin intermediate filaments and actin microfilaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Rethinking risk.

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Canada↗
PubMed1993

Indirect immunofluorescence modified to display two antigens with one light filter.

We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter. This, in turn, allows more efficient comparison of the distribution of the two antigens in a single field or photograph than is possible by comparing two fields or photographs by conventional double staining. The method is especially useful for determining possible co-localization of two cellular structures. We illustrate the method in adrenal cells in which mitochondria and intermediate filaments are seen to be co-localized.

Adrenal Gland Neoplasms↗
PubMed1993

Rat Sertoli cell aromatase cytochrome P450: regulation by cell culture conditions and relationship to the state of cell differentiation.

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗