Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus.
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Amino Acid Sequence↗
Biomedical subjects
W L Miller
Publications and source records attributed to W L Miller.
Regulation of human cytochrome P450scc and adrenodoxin messenger ribonucleic acids in JEG-3 cytotrophoblast cells.
Cycloheximide generally inhibits steroidogenesis, but has different effects on the accumulation of the mRNAs for various steroidogenic enzymes in different species, tissues, and cell lines. In bovine adrenocortical cells, cycloheximide prevents ACTH- or cAMP-induced accumulation of the mRNAs for cytochrome P450scc and adrenodoxin, but in human cells, cycloheximide induces the accumulation of adrenodoxin mRNA. To study the potential role of the 3'-untranslated regions, and especially the AU-rich regions, of adrenodoxin and P450scc mRNAs in cycloheximide-sensitive regulation of mRNA accumulation, we constructed a series of vectors expressing P450scc or adrenodoxin mRNA with its own or each other's 3'-untranslated sequences and transfected them into human JEG-3 cytotrophoblast cells. Removal of the AU-rich 3'-untranslated sequences of adrenodoxin mRNA and replacing them with the 3'-untranslated region of P450scc did not alter the abundance or apparent stability of this mRNA, or its inducibility by cycloheximide or cAMP. Substituting the AU-rich 3'-untranslated region of adrenodoxin mRNA (which contains three copies of the AUUUA sequence) for the 3'-untranslated region of P450scc did not alter the inducibility of P450scc mRNA with forskolin. Inhibition of transcription with actinomycin-D elicited no difference in the adrenodoxin mRNA half-life in JEG-3 cells treated with forskolin, cycloheximide, or both. RNA polymerase run-on assays show little effect of forskolin on adrenodoxin gene transcription, while P450scc gene transcription was induced. These data suggest that the principal means for regulating P450scc mRNA is transcriptional, while the principal regulation of adrenodoxin is posttranscriptional. This posttranscriptional regulation of adrenodoxin mRNA is not mediated by the AUUUA sequences or other segments of the 3'-untranslated region.
Adrenodoxin↗
Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc.
The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.
8-Bromo Cyclic Adenosine Monophosphate↗
Categorical data analysis in primary care research: log-linear models.
Primary care researchers often wish to perform multiple variable analyses using variables measured at a nominal or ordinal level. This paper provides a step-by-step description of log-linear modeling, an approach uniquely well suited to explore and describe interactions among three or more nominal or ordinal variables. The method of log-linear analysis is illustrated with the use of an example from a primary care research project in which the relationships among hypertension, diet, and sodium were examined. The advantages and disadvantages of log-linear models and logistic regression are compared and available computer software programs discussed.
Humans↗
Routine, ceremony, or drama: an exploratory field study of the primary care clinical encounter.
BACKGROUND: Practicing family physicians often find family systems theory and the biopsychosocial model attractive, but have difficulties applying them in a busy office practice. METHODS: Two family physicians in a four-person group practice were identified as "exemplars" at managing daily office practice. A collaborative, qualitative methodology was used to explore their strategy. A series of semistructured key informant interviews with the two physicians and the head nurse identified themes later clarified using ethnoscience interviewing techniques. The resultant clinical encounter typology and decision-making taxonomy were evaluated using participant observation and key informant review. The final results were compared with the literature on physician-patient relationships. RESULTS: Three clinical encounter types were identified. "Routines" were simple, single, and brief visits in which a contractual style and the biomedical model were used. "Ceremonies" were linked rituals that invoked a covenantal style. "Dramas" were a series of visits concerning situations of conflict and emotion and included psychosocial problems. The family was often convened for dramas. Determination of the presenting concern, the trigger for coming, the patient request, the illness prototype, and the type of communication allowed recognition of the clinical encounter type. CONCLUSIONS: Identifying a clinical encounter as a routine, ceremony, or drama may help family physicians integrate family systems concepts into their busy office practices. These findings have numerous implications for future research, clinical practice, and teaching.
Adult↗
Chronic venous insufficiency.
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Bandages↗
Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency.
Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities. We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam. We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast. Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities. Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities. An HhaI restriction site created by the mutation should permit screening of large populations.
Adolescent↗
Clinical and molecular genetics of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
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17-alpha-Hydroxyprogesterone↗
The role of transcriptional regulation in steroid hormone biosynthesis.
The regulated expression of the genes encoding the various steroidogenic enzymes is a crucial component in the control of steroid hormone biosynthesis. Tissue-specific transcription of each of the steroidogenic enzyme genes determines the array of enzymes present within a steroidogenic tissue, and therefore the types of steroid hormones the tissue produces. Transcriptional regulation also determines developmental changes in the steroid hormones synthesized by steroidogenic tissues and for the quantitative regulation of steroid hormones necessary for reproduction and for maintaining physiological homeostasis. The molecular mechanisms governing transcriptional regulation of steroidogenic enzyme genes is now being studied. The results so far indicate that, like most other genes, transcription of steroidogenic enzyme genes is regulated by cis-elements in the 5' flanking DNA of the genes that bind trans-acting proteins found in the nucleus. Several types of cis-elements have been identified: elements responsible for basal transcription, for induction by cAMP, and for both basal and cAMP induction. Some of the basal cis-elements identified may have a role in tissue-specific transcription of certain steroidogenic enzyme genes in steroidogenic tissues. We have also identified regions in both the human P450scc and human P450c17 promoters that repress transcription when activated by the Ca2+/protein kinase C intracellular second messenger system used by angiotensin II. This review summarizes our current understanding of transcriptional regulation of the steroidogenic enzyme genes.
Animals↗
Pyopneumopericardium attributed to an esophagopericardial fistula: report of a survivor and review of the literature.
Herein we describe a case of pyopneumopericardium that resulted from formation of an acquired esophagopericardial fistula in a patient with silent, benign esophageal ulcer disease. Atypical features on initial examination suggested congestive heart failure or a pneumonic process (or both). The delayed development of pneumopericardium disclosed on a chest roentgenogram led to the clinical recognition of the esophagopericardial fistula. Subsequent emergent pericardiocentesis relieved cardiac tamponade and enabled us to diagnose pyopneumopericardium. A radiographic contrast study with use of meglumine diatrizoate revealed the site of the fistula in the midesophagus. The esophagopericardial fistula was surgically closed, and our patient had a good final result. Formation of an esophagopericardial fistula is a relatively uncommon finding; of the 60 previously reported cases, only 10 patients have survived. As illustrated in the current case, early diagnosis and treatment, including pericardial drainage and intense antibiotic therapy followed by a well-planned operative closure of the fistula, are paramount for the successful management of esophagopericardial fistulas.
Aged↗
Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23-q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24-q25; and P450c17 to 10q24-q25.
Steroid hormones are synthesized by a complex array of 10 enzymes. The genes for each of these have now been cloned, and previous work has determined the regional chromosomal assignments of six of these. We used in situ hybridization to determine the regional chromosomal assignments of the four remaining enzymes. The CYP11A1 gene encodes mitochondrial P450scc, which converts cholesterol to pregnenolone, and is located on 15q23-q24. The gene for adrenodoxin, which receives electrons from adrenodoxin reductase and transfers them to P450scc, is on 11q22 while its pseudogenes are on 20q11-q12. The gene for adrenodoxin reductase is on 17q24-q25. The CYP17 gene encodes P450c17, which has both 17 alpha-hydroxylase and 17,20-lyase activities, and is located on 10q24-q25. None of the 10 genes involved in human steroidogenesis is closely linked to another gene for a steroidogenic enzyme.
Adrenodoxin↗
The gene encoding ovine follicle-stimulating hormone beta: isolation, characterization, and comparison to a related ovine genomic sequence.
Follicle-stimulating hormone (FSH), the primary stimulus for egg and sperm maturation in mammals, is an alpha/beta heterodimer. Each subunit is encoded by a single-copy gene in the human, bovine, and rat genomes. Transcription of both subunits is inhibited by estradiol and progesterone in ovine pituitary cultures. We report the sequence of one ovine FSH-beta gene (-1,527 to +3,664) that is expressed in vivo and the identification of a novel, second ovine FSH-beta-like sequence. Digestion of ovine genomic DNA with Bgl II yielded two fragments of 10 kb and 15 kb that hybridized to a bovine FSH-beta cDNA. The 10-kb fragment contained 6 kb of 5'-flanking region and all but about 200 bp of the 3' terminus of the ovine FSH-beta gene. This FSH-beta gene encodes a protein that differs from the published ovine protein sequence only at the carboxy terminus (Arg-109Glu-110[STOP codon] instead of Glu-109Arg-110[Glx-111]) and at positions 49 (Ala instead of Thr) and 88 (Arg instead of Ser). This gene is organized similarly to the human, bovine, porcine, and rat FSH-beta genes, and its coding sequence is nearly identical (99.5%) to a reported ovine FSH-beta cDNA. Expression of the FSH-beta gene on the 10-kb fragment in vivo was determined by analysis of wether mRNA using the polymerase chain reaction. A 95-bp sequence of the 15-kb fragment was 87% homologous to the corresponding coding region of the 10-kb fragment. This comparison suggested that the 15-kb fragment contains either an FSH-beta-like sequence or a pseudogene. Several potential steroid response elements were found by sequence analysis of the 5'-flanking region of the FSH-beta gene on the 10-kb fragment. A mechanism by which these elements may act is suggested.
Amino Acid Sequence↗
Progesterone shortens poly(A) tails of the mRNAs for alpha and beta subunits of ovine luteinizing hormone.
Progesterone (P) and 17 beta-estradiol (E) inhibit transcription of alpha mRNA (alpha mRNA) by about 70% in ovine pituitary cultures. In addition, P, but not E, shortens alpha mRNA during the 3-24-h period following treatment. Data presented here focus on this post-transcriptional effect of P, which appears totally independent from transcriptional changes and may alter the translational efficiency of the affected mRNA or its half-life. Results indicate this shortening occurs at the poly(A) tail. The normal length of this tail was estimated to be 70-100 nucleotides in the case of alpha mRNA, and it was shortened by 30-40 nucleotides during P treatment. Shortening was normally observed after 3-10 h of P treatment and was complete by 10-24 h. Significant shortening occurred at 7-10 nM P in ovine pituitary culture; the 10-15-nM concentration of P in the serum of luteal phase ewes also appears sufficient to shorten alpha mRNA in vivo since alpha mRNA from the pituitaries of these ewes was found to be consistently shorter than alpha mRNA from ovariectomized ewes. Finally, P also shortened the poly(A) tail of mRNA for the beta-subunit of LH. This indicates that P-induced poly(A) shortening not only affects alpha mRNA but may affect several or many mRNAs found in ovine gonadotrophs. The significance of poly(A) shortening is not yet understood, but it could involve post-transcriptional inhibition, which, combined with transcriptional effects, may make P a more potent inhibitor of gonadotropin synthesis than E.
Animals↗
Normal genes for the cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal hyperplasia.
Congenital lipoid adrenal hyperplasia is the most severe form of congenital adrenal hyperplasia. Affected individuals can synthesize no steroid hormones, and hence are all phenotypic females with a severe salt-losing syndrome that is fatal if not treated in early infancy. All previous studies have suggested that the disorder is in the cholesterol side chain cleavage enzyme (P450scc), which converts cholesterol to pregnenolone. A newborn patient was diagnosed by the lack of significant concentrations of adrenal or gonadal steroids either before or after stimulation with corticotropin (ACTH) or gonadotropin (hCG). The P450scc gene in this patient and in a previously described patient were grossly intact, as evidenced by Southern blotting patterns. Enzymatic (polymerase chain reaction) amplification and sequencing of the coding regions of their P450scc genes showed these were identical to the previously cloned human P450scc cDNA and gene sequences. Undetected compound heterozygosity was ruled out in the new patient by sequencing P450scc cDNA enzymatically amplified from gonadal RNA. Northern blots of gonadal RNA from this patient contained normal sized mRNAs for P450scc and also for adrenodoxin reductase, adrenodoxin, sterol carrier protein 2, endozepine, and GRP-78 (the precursor to steroidogenesis activator peptide). These studies show that lipoid CAH is not caused by lesions in the P450scc gene, and suggest that another unidentified factor is required for the conversion of cholesterol to pregnenolone, and is disordered in congenital lipoid adrenal hyperplasia.
Adrenal Hyperplasia, Congenital↗
Human proopiomelanocortin-(79-96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells.
Whether the production of human adrenal androgens is stimulated by a hormone other than ACTH or angiotensin-II has long been controversial. A candidate for such a cortical androgen stimulatory hormone (CASH) is the proximal 18-amino acid hinge region of proopiomelanocortin (POMC). This region of POMC lies to the amino-terminal side of the ACTH region, displays considerable amino acid sequence variation among mammals, and has been reported to stimulate the secretion of dehydroepiandrosterone (DHEA) from cultured adult adrenal cells. We cultured human fetal adrenal cells with 10(-8) M CASH-18, 10(-13) M ACTH, 10(-8) M CASH-18 plus 10(-13) M ACTH, 10(-8) M ACTH, or vehicle alone. Culture media were assayed for cortisol, DHEA, and DHEA sulfate, and cellular RNA was assayed for P450scc and P450c17 mRNAs. ACTH (10(-8 M) increased steroid secretion and the mRNAs for P450scc and P450c17, but the other treatments were indistinguishable from the control. Thus, CASH-18 does not affect steroidogenesis in primary cultures of human fetal adrenal cells. These data could be consistent with a model in which adrenarche, the onset of adrenal androgen synthesis at 8-10 yr of age, is mediated by developmentally programmed synthesis of a receptor for CASH. Alternatively, they are also consistent with this region of POMC having no CASH activity.
Adrenal Glands↗
Congenital adrenal hyperplasias.
The congenital adrenal hyperplasias (CAH) are a very common group of genetic disorders of steroid hormone synthesis. The genes encoding each of the steroid biosynthetic enzymes have now been cloned, and the mutations in these genes that cause the various forms of CAH are being determined. The emerging picture is substantially different from traditional views of CAH. Examination of the clinical findings in each form of CAH from a molecular genetic perspective simplifies understanding of these complex and closely related syndromes. Because mild forms of CAH are being recognized with increasing frequency, and severely affected patients now survive and reproduce, these disorders are of interest to endocrinologists dealing with all age groups.
Adrenal Hyperplasia, Congenital↗
Expanding the boundaries of family medicine research.
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Family Practice↗
A qualitative approach to primary care research: the long interview.
Qualitative research methods offer useful complements to the traditional epidemiologic approaches most commonly used by primary care researchers. Narrative texts generated through open-ended interviews are one form of qualitative data that may provide useful insights into primary care issues that are otherwise overlooked by more structured designs. This paper presents a step-by-step process for conducting a long interview, a qualitative approach with distinct potential for primary care research. Advantages and limitations of the long interview and its relationship to other qualitative methods are also discussed.
Connecticut↗