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Biomedical subjects

W L Miller

Publications and source records attributed to W L Miller.

At least 127 records · Page 7Linked to original sources
PubMed1995

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Aldosterone↗
PubMed1995

Hybridization of the complementary mRNAs for P450c21 (steroid 21-hydroxylase) and tenascin-X is prevented by sequence-specific binding of nuclear proteins.

The CYP21 gene that encodes the steroid 21-hydroxylase, P450c21, is overlapped on the opposite strand of DNA by the TX-X gene encoding the extracellular matrix protein, tenascin-X. These transcripts contain perfectly complementary segments of 299 bases at their 3'-ends. As these genes are tandemly duplicated and are transcribed in the adrenal cortex, we investigated whether these self-complementary transcripts formed RNA-RNA hybrids in vivo. Formation of heterogeneous nuclear ribonucleoprotein complexes between nascent RNA transcripts and nuclear proteins might modulate such potential RNA-RNA interactions. Using a double RNase protection assay, we found that these RNAs form very small amounts of double-stranded RNA-RNA hybrids in adrenal cells in vivo. To understand why these mRNAs fail to hybridize in vivo, we studied the actions of nuclear proteins on the binding and annealing of their complementary regions in vitro. The nucleation of interstrand annealing was kinetically favored over binding and was efficiently promoted by nuclear extracts. However, RNA-RNA strand zippering was inhibited, suggesting that protein binding and/or stable RNA secondary structures contribute to discontiguous base pairing. Increasing concentrations of nuclear proteins increased the relative proportion of these RNAs in perfect RNase-resistant duplexes but reached only about 20% of the total available RNA strands at saturating concentrations of nuclear proteins. Preincubation of either of the two single-stranded RNAs with nuclear proteins strongly inhibited the nucleation step of annealing, whereas preincubation of both strands abolished the annealing. RNase footprinting of the wild type and mutagenized overlapping transcripts suggested that sequence-specific binding of nuclear proteins is limited to the 5'-half of each RNA strand. These results indicate that the transcription of complementary, opposite-strand RNAs is not a mechanism for the regulation of the abundance of adrenal P450c21 mRNA and suggest that nuclear proteins strongly interfere with interstrand RNA base pairing in vitro as well as in vivo.

Base Composition↗
PubMed1995

Mutation T318M in the CYP11B2 gene encoding P450c11AS (aldosterone synthase) causes corticosterone methyl oxidase II deficiency.

Corticosterone methyl oxidase (CMO) deficiency refers to disorders of aldosterone synthesis due to mutations in the CYP11B2 gene encoding cytochrome P450c11AS, which is the adrenal aldosterone synthase. Type I CMO deficiency is associated with low concentrations of 18OH-corticosterone and aldosterone, due to severe mutations in P450c11AS; while type II CMO deficiency is associated with high concentrations of 18OH-corticosterone and low concentrations of aldosterone, due to less severe mutations of P450c11AS. A single type of mutation, compound homozygosity for R181W and V386A, has been reported as the cause of CMOII deficiency in an inbred population. We now report a patient with a typical clinical and hormonal picture of CMOII deficiency. Direct sequencing of patient and parent DNAs showed that the mother's allele contributed R181W and the deletion/frameshift mutation delta C372, while the father's allele contributed T318M and V386A. These mutants were recreated in cDNA expression vectors singly and in the parental pairs, showing that neither allele contributed any measurable activity. This would suggest the patient should have CMOI deficiency. These studies suggest that other factors besides P450c11AS are involved in the genesis of the distinctive CMOI and CMOII phenotypes.

Base Sequence↗
PubMed1995

Reading and evaluating qualitative research studies.

Qualitative research is now published across the family practice and medical literature. This article is designed to help busy family physicians decide which qualitative studies are worth reading and to provide them with the tools to appreciate and evaluate research design and analysis. By using clinical analogies, the qualitative research process can be better understood.

Family Practice↗
PubMed1994

The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.

Steroidogenesis is initiated by the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc [cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving); EC 1.14.15.6]. Several subsequent steroidal conversions occur in the endoplasmic reticulum (ER), but the last step in the production of glucocorticoids and mineralocorticoids again occurs in the mitochondria. Although cellular compartmentalization of steroidogenic enzymes appears to be a feature of all steroidogenic pathways, some reports indicate that cholesterol can be converted to pregnenolone outside the mitochondria. To investigate whether P450scc can function outside the mitochondria, we constructed vectors producing P450scc and various fusion enzymes of P450scc with electron-transport proteins and directed their expression to either the ER or the mitochondria. Whether targeted to mitochondria or to the ER, plasmid vectors encoding P450scc and fusion proteins of P450scc with either mitochondrial or microsomal electron-transport proteins produced immunodetectable protein. When expressed in mitochondria, all of these constructions converted 22-hydroxycholesterol to pregnenolone, but when expressed in the ER none of them produced pregnenolone. These results show that P450scc can function only in the mitochondria. Furthermore, it appears to be the mitochondrial environment that is required, rather than the specific mitochondrial electron-transport intermediates.

Animals↗
PubMed1994

Gonadal hormones and gonadotropin-releasing hormone (GnRH) alter messenger ribonucleic acid levels for GnRH receptors in sheep.

GnRH regulates the synthesis and secretion of pituitary gonadotropins. The number of receptors for GnRH (GnRH-rec) can vary from 500 to 15,000-20,000/gonadotrope in ovine pituitary cultures after treatment with physiologically relevant combinations of gonadal hormones. This large range suggests that regulation of GnRH-rec expression may be an important control point in GnRH action at the pituitary level. Reported here are the changes in GnRH-rec mRNA associated with pituitary treatments (48 h) of 17 beta-estradiol (E), progesterone (P), and an enriched preparation of porcine follicular inhibin (IN). Northern blot analysis was used to detect 3 species of GnRH-rec mRNA in primary ovine pituitary culture [5.5 kilobases (kb; 32%), 3.6 kb (51%), and 1.4 kb (17%)]; all were changed in parallel by E, P, and IN. GnRH-rec mRNAs were increased 190% over control levels after treatment with either E or IN, and 400% with E and IN combined; when E and IN were added along with P, the increase was only 50% (P caused an 87% inhibition of E plus IN induction). The addition of P in the absence of any other treatment reduced levels of GnRH-rec mRNA by 50%. Studies were also conducted with GnRH agonists (GnRH-A) due to their widespread clinical use for down-regulating reproductive function in men and women. The addition of GnRH-A to cultures was as effective as P in blocking E plus IN induction of GnRH-rec mRNA. In vivo studies in wethers showed that 7 days of chronic treatment with GnRH-A decreased all sizes of ovine GnRH-rec mRNA by 84-89%. These data indicate that E, P, and IN change GnRH-rec levels at least in part by changing levels of GnRH-rec mRNAs. They also show that GnRH-A can almost entirely block E plus IN induction of GnRH-rec mRNA in vitro and decrease levels of GnRH-rec mRNA in vivo in wethers.

Animals↗
PubMed1994

Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17 alpha-hydroxylase deficiency.

Genetic disorders in the gene encoding P450c17 cause 17 alpha-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralocorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1 cells showed that the mutant P450c17 protein was produced, but it lacked both 17 alpha-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17 alpha-hydroxylase deficiency, indicating that mutations in this gene are due to random events.

Adolescent↗
PubMed1994

Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice.

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Modeling and mutagenesis of the active site of human P450c17.

Cytochrome P450c17 is the single enzyme having steroid 17 alpha-hydroxylase and 17,20 lyase activities. We sought to model the active site of this enzyme to identify residues contributing to its catalytic activities, and to test the roles of the identified amino acids by altering them via site-directed mutagenesis. Using the MIDAS-plus program, we modeled P450c17 and the structurally related steroid 21-hydroxylase, P450c21, on the crystallographically determined structure of bacterial P450cam. By positioning the progesterone substrate into each model, we identified five residues that appeared crucial for determining whether progesterone would undergo 17 alpha-hydroxylation (by P450c17) or 21-hydroxylation (by P450c21). Each identified residue in the P450c17 sequence was changed to the corresponding residue in the P450c21 sequence, yielding the four P450c17 mutants L102Y, G111D, G301l, and M369L + l371L. The mutants were transfected into COS-1 cells and their 17 alpha-hydroxylase, 17,20 lyase, and 21-hydroxylase activities were assayed by incubation with [14C]pregnenolone, [3H]17OH-pregnenolone, and [3H]17OH-progesterone and TLC. The L102Y and M369L + l371L mutants retained 50-80% of 17 alpha-hydroxylase and 70-100% of 17,20 lyase activity, while the G111D and G301l mutants lost both activities, but no mutants acquired detectable 21-hydroxylase activity (0.1% of wild type P450c21). Combination of the two mutants that retained partial activity (L102Y and M369L + l371L) yielded a single protein that retained 40% of 17 alpha-hydroxylase and 50% of 17,20 lyase activity, but none of the seven possible vectors expressing two, three, or all four of the mutations in a single enzyme yielded detectable 21-hydroxylase activity. The mutations D298V and D298S were predicted to ablate 17,20 lyase activity while retaining 17 alpha-hydroxylase activity, but were both inactive. These studies indicate that models based on the crystal structure of P450cam correctly predict many gross architectural features of steroidogenic enzymes and that many of the predicted residues are in or near the active site of P450c17. However, because enzymatic activity requires interactions between the enzyme and substrate at distances of less than 1 A, and modeling cannot predict atomic loci to greater than 1.5-2.0 A, it was not possible to design mutants that would confer 21-hydroxylase activity to P450c17. Currently available data cannot predict the structural and amino acid sequence requirements for a specific P450 activity.

Aldehyde-Lyases↗
PubMed1994

Qualitative analysis: how to begin making sense.

The clinical process used to make sense of patient concerns closely parallels the analysis process of qualitative research. This partly explains why qualitative research methods are appropriate for many family practice research questions. Unfortunately, the language used by qualitative researchers, especially with regards to analysis, is often obscure. This impedes family physicians from implementing qualitative research. This paper overviews qualitative analysis and introduces a language and means by which family physicians can begin to make sense of qualitative data. The concepts, "reflexivity," "iteration," "data saturation," and "text," are defined. Three core steps of qualitative analysis are identified and compared to the diagnostic process. They consist of choosing an organizing system, reducing the data, and making connections. Four idealized ways for conducting these steps, editing, template, quasistatistical, and immersion/crystallization, are presented and compared to four ways of approaching patient concerns. Finally, the process of creating an appropriate qualitative analysis strategy is described for a hypothetical research study and some pitfalls and principles of qualitative analysis are reviewed.

Family Practice↗
PubMed1994

Patients, family physicians, and pain: visions from interview narratives.

BACKGROUND: Family physicians reportedly underestimate their patients' pain severity. Explanations for this remain unexamined. This study explores the understanding family physicians and their patients have of the common experience of pain. METHODS: We studied six culturally homogeneous private practice family physician-patient pairs from Connecticut using a qualitative, cross-sectional long interview design. Interviews were taped in the office or home and analyzed by the research team using an editing style derived from the constant comparative method. RESULTS: The patients described pain as spiritually awakening, experienced in the body, and part of everyday life with conceptual, behavioral, functional, and spiritual dimensions. The patients all claimed that physicians don't listen. The family physicians expressed a personal understanding of pain similar to the patient and a professional one which was more biomedical, concerned about addiction, and related to control of and connection with patients. Patients and physicians described the role of the doctor as a four-stage process of listening, knowing, responding, and taking time, but they meant different things. CONCLUSIONS: This study reveals family physicians and their patients struggling to communicate about pain. The role of family physician socialization, strategies for listening and sharing power in the clinical encounter, and a new four-dimensional classification of pain are briefly discussed.

Adult↗
PubMed1993

Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.

We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17 alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11 beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373-->Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17 alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373-->Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.

Adolescent↗
PubMed1993

Abundant adrenal-specific transcription of the human P450c21A "pseudogene".

Human adrenal steroid 21-hydroxylase (P450c21) is encoded by the CYP21A1 (21B) gene located in the class III region of the HLA locus. A tandemly duplicated gene designated CYP21A1P (21A), which lies 30 kilobases upstream, contains several point mutations and an 8-base pair deletion so that it cannot encode P450c21 protein; as a result, it is generally considered to be a pseudogene. We previously showed that two additional genes, XA and XB, lie on the opposite strand of DNA overlapping the 3'-ends of the 21A and 21B genes. We have now identified a third pair of duplicated overlapping genes in this locus, termed YA and YB, whose transcriptional orientation is the same as 21A and 21B and opposite to XA and XB. YA transcripts use the 21A promoter, have 5'-ends that are similar to 21B mRNA, and have approximately 10-20% of the abundance of 21B transcripts, but have unique 3'-ends. The YA gene encodes a 7.5-kilobase RNA that overlaps XA completely and a 3.0-kilobase RNA that excludes most of XA. The YB gene appears to be similar in size and organization to YA. The YA and YB genes extend beyond the limit of the duplication in this locus; hence, their cDNAs are distinguishable by differences in their 3'-sequences. YA and YB transcripts are expressed only in the fetal and adult adrenal glands, but their cDNAs do not contain a long open reading frame. Although the function of these genes is not yet clear, the complex genetic organization of three overlapping genes (21/X/Y) appears to be unique among higher eukaryotes. As YA transcription is initiated by the 21A 5'-flanking DNA and includes 21A sequences, the designation of 21A as a "pseudogene" merits reconsideration.

Adrenal Glands↗
PubMed1993

Rainbow trout ovarian cholesterol side-chain cleavage cytochrome P450 (P450scc). cDNA cloning and mRNA expression during oogenesis.

A cDNA clone encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contains an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acids. The predicted amino acid sequence of trout P450scc shows 48% homology with that of human, and 46% homology with that of rat, bovine and pig. P450scc activity was confirmed by transfected COS-1 monkey kidney tumour cells with an expression vector for trout P450scc cDNA and subsequent detection of conversion from 25-hydroxycholesterol to pregnenolone by radioimmunoassay. The cDNA only hybridized to a single 1.8 kb RNA transcript. The transcript was not found in early vitellogenic follicles, barely detected in postvitellogenic follicles, and abundant in postovulatory follicles.

Amino Acid Sequence↗
PubMed1993

Congenital lipoid adrenal hyperplasia--genes for P450scc, side chain cleavage enzyme, are normal.

In the most severe form of congenital adrenal hyperplasia (CAH), termed lipoid CAH, both the adrenals and gonads fail to convert cholesterol to pregnenolone, so that no steroid hormones are made. Newborns have female external genitalia irrespective of karyotype, and suffer a severe salt-losing form of CAH. Previous studies have shown that adrenal or gonadal mitochondria from these patients also fail to convert cholesterol to pregnenolone in vitro, implicating a lesion in the single gene for P450scc, which is the sole enzyme converting cholesterol to pregnenolone. Two patients with XY karyotypes had female genitalia and unmeasurable steroids after stimulation with ACTH and hCG. ACTH stimulation tests of parents, obligate heterozygotes, showed normal stimulation of all precursor steroids. Southern blotting patterns of the P450scc gene were normal. Oligonucleotide-initiated enzymatic amplification (PCR) of all P450scc exons showed normal sequences on multiple amplifications and sequencing reactions, indicating normal P450scc genes. Northern blots of testicular RNA from a 6-month-old patient and from a control fetus showed normal P450scc mRNA, indicating a normal P450scc promoter. Reprobing of the blot with our cloned human cDNAs for adrenodoxin reductase and adrenodoxin showed that these electron transport cofactors used by P450scc were also normal. Similarly, probing with cDNAs for all three known factors involved in cholesterol transport to the mitochondria-sterol carrier protein 2, endozepine, and steroidogenesis activator peptide were also normal. These results suggest that the lesion in lipoid CAH is not in the P450scc system or in any known step upstream from P450scc.

Adrenal Hyperplasia, Congenital↗
PubMed1993

The human peripheral benzodiazepine receptor gene: cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia.

The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondria to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, we have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5' untranslated segment. The 5' flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of "house-keeping" genes. The encoded PBR mRNA is alternately spliced into two forms: "authentic" PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [3H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease.

Adrenal Hyperplasia, Congenital↗