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W L Miller

Publications and source records attributed to W L Miller.

At least 37 records · Page 2Linked to original sources
PubMed2001

NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells.

Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.

Adrenal Gland Neoplasms↗
PubMed2001

Adjunctive therapies in the treatment of acute coronary syndromes.

Survival after myocardial infarction has been improving steadily in recent decades, in part because of more effective adjunctive medical therapies. However, the issue of underutilization of effective medical therapies remains. Adjunctive therapy for acute myocardial infarction should include aspirin, beta-adrenergic blocking agents, angiotensin-converting enzyme inhibitors, and lipid-lowering agents, all of which improve survival in the treatment and secondary prevention of myocardial infarction. This review presents the current knowledge supporting the use of specific adjunctive pharmacologic agents and also discusses the current status of other agents that are emerging or controversial.

Acute Disease↗
PubMed2001

Developing the knowledge base of family practice.

Borrowed and adapted knowledge is insufficient to optimize the potential of a comprehensive, integrative, relationship-centered generalist approach to improve the health of individuals, families, and communities. The knowledge base for family practice must be expanded by integrating multiple ways of knowing. This involves (1) self-reflective practice by clinicians, (2) involving the patient voice in generating research questions and interpreting data, (3) inquiry into the systems affecting health care, and (4) investigation of disease phenomena and treatment effects in patients over time. A multimethod, transdisciplinary, participatory approach is needed to create knowledge that retains connections with its meaning and context and therefore is readily translated into practice. This research integrates quantitative and qualitative traditions and involves the active participation of both clinicians and patients. The generation of relevant knowledge should be supported through (a) developing a culture of reflective practice among clinicians, (b) expanding the infrastructure for practice-based research, (c) developing a multimethod, transdisciplinary, participatory research paradigm, (d) longitudinal study of the process and outcomes of broad, integrative, relationship-centered care, and (e) incorporating pursuit of new knowledge as a central feature of training programs and policy. The time has come for the generalist disciplines to commit to the generation of new knowledge based on the needs of patients, families, and communities for relationship-centered, integrated, prioritized health care. Development of a culture of learning and inquiry, and the necessary research methods and skills will require a long-term commitment, creation of partnerships, and a focus on core principles by individuals and organizations.

Algorithms↗
PubMed2001

Understanding practice from the ground up.

BACKGROUND: Our objective was to understand family practices from the ground up through intensive direct observation of the practice environment and patient care. METHODS: Eighteen practices were purposefully drawn from a random sample of Nebraska family practices that had earlier participated in a study of preventive service delivery. Each practice was studied intensely over a 4- to 12-week period using a comparative case study design that included extended direct observation of the practice environment and clinical encounters, formal and informal interviews of clinicians and staff, and medical record review. DESIGN: This multimethod assessment process (MAP) provided insights into a wide range of practice activities ranging from descriptions of the organization and patient care activities to quantitative documentation of physician- and practice-level delivery of a wide range of evidence-based preventive services. Initial insights guided subsequent data collection and analysis and led to the integration of complexity science concepts into the design. In response to the needs and wishes of the participants, practice meetings were initiated to provide feedback, resulting in a more collaborative model of practice-based research. CONCLUSIONS: Our multimethod assessment process provided rich data for describing multiple aspects of primary care practice, testing a priori hypotheses, discovering new insights grounded in the actual experience of practice participants, and fostering collaborative practice change.

Data Collection↗
PubMed2000

N-218 MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved similarly to StAR.

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane in adrenal and gonadal cells, fostering steroid biosynthesis. MLN64 is a 445-amino acid protein of unknown function. When 218 amino-terminal residues of MLN-64 are deleted, the resulting N-218 MLN64 has 37% amino acid identity with StAR and 50% of StAR's steroidogenic activity in transfected cells. Antiserum to StAR cross-reacts with N-218 MLN64, indicating the presence of similar epitopes in both proteins. Western blotting shows that MLN64 is proteolytically cleaved in the placenta to a size indistinguishable from N-218 MLN64. Bacterially expressed N-218 MLN64 exerts StAR-like activity to promote the transfer of cholesterol from the outer to inner mitochondrial membrane in vitro. CD spectroscopy indicates that N-218 MLN64 is largely alpha-helical and minimally affected by changes in ionic strength or the hydrophobic character of the solvent, although glycerol increases the beta-sheet content. However, decreasing pH diminishes structure, causing aggregation. Limited proteolysis at pH 8.0 shows that the C-terminal domain of N-218 MLN64 is accessible to proteolysis whereas the 244-414 domain is resistant, suggesting it is more compactly folded. The presence of a protease-resistant domain and a protease-sensitive carboxy-terminal domain in N-218 MLN64 is similar to the organization of StAR. However, as MLN64 never enters the mitochondria, the protease-resistant domain of MLN64 cannot be a mitochondrial pause-transfer sequence, as has been proposed for StAR. Thus the protease-resistant domain of N-218 MLN64, and by inference the corresponding domain of StAR, may have direct roles in their action to foster the flux of cholesterol from the outer to the inner mitochondrial membrane.

Amino Acid Sequence↗
PubMed2000

Cloning of factors related to HIV-inducible LBP proteins that regulate steroidogenic factor-1-independent human placental transcription of the cholesterol side-chain cleavage enzyme, P450scc.

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, we sought factors that bind to the -155/-131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 x 10(6) cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the -155/-131 DNA; antisera to LBP proteins supershifted the LBP-9.DNA complex and inhibited formation of the LBP-1b.DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the -155/-131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription.

Amino Acid Sequence↗
PubMed2000

Improved survival after acute myocardial infarction in patients with advanced Killip class.

BACKGROUND: The continuing applicability of the Killip classification system to the effective stratification of long-term and short-term outcome in patients with acute myocardial infarction (MI) and its influence on treatment strategy calls for reanalysis in the setting of today's primary reperfusion treatments. HYPOTHESIS: Our study sought to test the hypothesis that Killip classification, established on admission in patients with acute MI, is an effective tool for early prediction of in-hospital mortality and long-term survival. METHODS: A series of 909 consecutive Olmsted County patients admitted with acute MI to St. Marys Hospital, Mayo Clinic, between January 1988 and March 1998 was analyzed. Killip classification was the primary variable. Endpoints were in-hospital death, major in-hospital complications, and post-hospital death. RESULTS: Patients analyzed included 714 classified as Killip I, 170 classified as Killip II/III, and 25 classified as Killip IV. Increases in in-hospital mortality and prevalence of in-hospital complications correspond significantly with advanced Killip class (p < 0.01), with in-hospital mortality 7% in class I, 17.6% in classes II/III, and 36% in class IV patients (p < 0.001). Killip classification was strongly associated with mode of therapy administered within 24 h of admission (p < 0.01). Killip IV patients underwent primary angioplasty most commonly and were less likely to receive medical therapy. CONCLUSIONS: Killip classification remains a strong independent predictor of in-hospital mortality and complications, and of long-term survival. Early primary angioplasty has contributed to a decrease in mortality in Killip IV patients, but effective adjunctive medical therapy is underutilized.

Aged↗
PubMed2000

Human 25-hydroxyvitamin D-1alpha-hydroxylase: cloning, mutations, and gene expression.

The rate-limiting, hormonally regulated step in the bioactivation of vitamin D is the 1alpha-hydroxylation of 25-hydroxyvitamin D, which occurs in the kidney and other tissues and is catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1alpha. After many years of effort, the cDNA and gene encoding this enzyme were cloned from mouse, rat, and human tissue in late 1997. The human gene encoding the 1alpha-hydroxylase is 5 kb in length, located on chromosome 12, and comprises nine exons and eight introns; its intron/exon organization is very similar to that of the other four mitochondrial P450 enzymes cloned to date. Mutations in P450c1alpha cause 1alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1, a rare autosomal recessive disease characterized by rickets and impaired growth due to failure of renal synthesis of 1,25(OH)2D. To date, 31 patients have been studied and 20 distinct mutations in the gene identified, including 13 mis-sense mutations, none of which encode a protein with significant enzyme activity. Recent studies in animals demonstrate that regulation of P450c1alpha gene expression by parathyroid hormone (PTH), low calcium diet, low phosphorus diet, and 1,25(OH)2D occurs at the level of its mRNA. Transcriptional activity of the mouse and human P450c1alpha gene promoters can be stimulated by PTH, cAMP, and forskolin and suppressed by 1,25(OH)2D.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2000

Vitamin D 1 alpha-hydroxylase.

The rate-limiting, hormonally regulated step in the bioactivation of vitamin D is renal 1 alpha-hydroxylation by P450c1 alpha. In late 1997, we reported the cloning of the human cDNA and gene from keratinocytes, and established that P450c1 alpha mutations cause vitamin D-dependent rickets, type I, while three other groups reported the cloning of the rodent enzyme. The genetics of P450c1 alpha are well established, with studies of over 30 patients, but the molecular mechanisms for the hormonal regulation of P450c1 alpha are still under investigation.

Animals↗
PubMed2000

Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Adrenodoxin↗
PubMed2000

Fragmentation of 23S rRNA in strains of Proteus and Providencia results from intervening sequences in the rrn (rRNA) genes.

Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.

Base Sequence↗
PubMed2000

Distribution of intervening sequences in the genes for 23S rRNA and rRNA fragmentation among strains of the Salmonella reference collection B (SARB) and SARC sets.

Intervening sequences (IVSs) occur sporadically in several bacterial genera in the genes for 23S rRNA at relatively conserved locations. They are cleaved after transcription and lead to the presence of fragmented rRNA, which is incorporated into the ribosomes without religation but is nevertheless functional. The fragmentation of rRNA and the number of IVSs in all 72 strains of the Salmonella Reference Collection B set and 16 strains of the Salmonella Reference Collection C set, which have been established on the basis of multilocus enzyme electrophoresis (MLEE), were analyzed in the present study. Fragmentation of 23S rRNA was restricted to conserved cleavage sites located at bp 550 (helix 25) and bp 1170 (helix 45), locations where IVSs have been reported. Random cleavage at sites where IVSs could not be detected was not seen. Uncleaved IVSs were not detected in any case; thus, the IVSs invariably led to rRNA fragmentation, indicating a strong selection for maintenance of RNase III cleavage sites. The distribution of the number of IVSs carried by the different strains in the seven rrl genes is diverse, and the pattern of IVS possession could not be related to the MLEE pattern among the various Salmonella strains tested; this indicates that the IVSs are frequently exchanged between strains by lateral transfer. All eight subspecies of the genus Salmonella, including subspecies V represented by Salmonella bongori, have IVSs in both helix 25 and helix 45; this indicates that IVSs entered the genus after its divergence from Escherichia coli (more than 100 million years ago) but before separation of the genus Salmonella into many forms or that they were in the ancestor but have been lost from Escherichia.

Base Pairing↗
PubMed2000

'If you pay, we'll operate immediately'.

OBJECTIVES: To study the attitudes of health care staff in four postcommunist countries towards taking gifts from their clients--and their confessed experience of actually taking such gifts. DESIGN: Survey questionnaire administered to officials including health care staff, supplemented by focus-group discussions with the general public. SETTING: Ukraine, Bulgaria, Slovakia and the Czech Republic. PARTICIPANTS: A quota sample of 1,307 officials including 292 health care staff, supplemented by stratified national random samples of 4,778 ordinary members of the public and in-depth interviews or focus-group discussions involving another 323. MAIN MEASUREMENTS: Explicit justifications and willingness to accept offers, reported frequency of offers, and personal confessions to accepting "money and expensive presents" as well as smaller gifts. RESULTS: Health care staff were far more inclined than the average official or public servant to accept "money or an expensive present" if offered, far more inclined to justify asking clients for "extra payments", and far more inclined to confess that they had actually taken gifts from clients recently. Judged by their own confessions, hospital doctors were only rivalled by traffic police and customs officials for taking money or expensive gifts from their clients. CONCLUSIONS: Poor pay does not explain why doctors so often took large gifts from their clients. Moral self justification, opportunity, and bargaining power are much more effective explanations.

Attitude of Health Personnel↗
PubMed2000

Extent and role of multisegmental coupling in the Lamprey spinal locomotor pattern generator.

Timing of oscillatory activity along the longitudinal body axis is critical for locomotion in the lamprey and other elongated animals. In the lamprey spinal locomotor central pattern generator (CPG), intersegmental coordination is thought to arise from the pattern of extensive connections made by propriospinal interneurons. However, the mechanisms responsible for intersegmental coordination remain unknown, in large part because of the difficulty in obtaining quantitative information on these multisegmental fibers. System-level experiments were performed on isolated 50-segment preparations of spinal cord of adult silver lampreys, Ichthyomyzon unicuspis, to determine the dependence of CPG performance on multisegmental coupling. Coupling was manipulated through use of an experiment chamber with movable partitions, which allowed separate application of solution to rostral, middle, and caudal regions of the spinal cord preparation. During control trials, fictive locomotion, induced by bath application of D-glutamate in all three regions, was recorded extracellularly from ventral roots. Local synaptic activity in a variable number of middle segments was subsequently blocked with a low-Ca(2+), high-Mn(2+) saline solution in the middle compartment, whereas conduction in axons spanning the middle segments was unaffected. Spectral analysis was used to assess the effects of blocking propriospinal coupling on intersegmental phase lag, rhythm frequency, correlation, and variability. Significant correlation and a stable phase lag between the rostral and caudal regions of the spinal cord preparation were maintained during block of as many as 16 and sometimes 20 intervening segments. However, the mean value of this rostrocaudal phase decreased with increasing number of blocked segments from the control value of approximately 1% per segment. By contrast, phase lags within the rostral and caudal end regions remained unaffected. The cycle frequency in the rostral and caudal regions decreased with the number of blocked middle segments and tended to diverge when a large number of middle segments was blocked. The variability in cycle frequency and intersegmental phase both increased with increasing number of blocked segments. In addition, a number of differences were noted in the properties of the motor output of the rostral and caudal regions of the spinal cord. The results indicate that the maximal functional length of propriospinal coupling fibers is at least 16-20 segments in I. unicuspis, whereas intersegmental phase lags are controlled at a local level and are not dependent on extended multisegmental coupling. Other possible roles for multisegmental coupling are discussed.

Action Potentials↗
PubMed2000

Estradiol induces and hyperglycosylates the receptor for ovine gonadotropin-releasing hormone.

The crucial first link between GnRH and its pleiotropic stimulation of the reproductive system is its receptor (GnRHRec). In mammals, 17beta-estradiol is a major regulator of GnRH action, and part of its regulation occurs at the level of the GnRHRec. In ovine pituitary cultures, estradiol simultaneously increases GnRHRec and GnRH-stimulated LH secretion (the LH response), but after 6-15 h the effect of estradiol becomes paradoxical, and the LH response rapidly decreases to control levels (by 24 h), whereas GnRHRec remains elevated. A preliminary study used photoaffinity labeling of the GnRHRec to show that estradiol can induce 38- and 43-kDa GnRHRec. The photoaffinity technique has been used here to 1) further investigate estradiol-mediated induction of GnRHRec, 2) define the nature of the different sized GnRHRecs, and 3) determine whether the larger size is related to degradation of the LH response. The effect of estradiol is compared with that of inhibin, which only induces the 38-kDa GnRHRec and always increases the LH response to GnRH treatment. Receptors for GnRH in ovine pituitary cultures were photoaffinity labeled with [125I](azidobenzoyl-D-Lys6-des-Gly10)-GnRH-N-ethylamide and analyzed by SDS-PAGE. Treatment with estradiol or inhibin for 6-24 h induced a 38-kDa GnRHRec only. Further treatment with estradiol (>24 h), but not inhibin, shifted the apparent Mr of the GnRHRec to 43 kDa. Phosphatase treatment did not reverse this apparent Mr change. Analysis of receptor glycosylation using N-glycosidase F or tunicamycin showed that the 43-kDa GnRHRec was a hyperglycosylated form of the 38-kDa GnRHRec. The 38-kDa GnRHRec, in turn, was a glycosylated form of the 29-kDa GnRHRec. The studies presented here define several glycosylated intermediates of the ovine GnRHRec that are induced by estradiol and/or inhibin. The function of estrogen-mediated hyperglycosylation is unclear, but kinetic studies dissociate it from degeneration of the LH response to GnRH.

Animals↗
PubMed2000

Mutations in the steroidogenic acute regulatory protein (StAR) in six patients with congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (lipoid CAH), the most severe form of CAH, is caused by mutations in the steroidogenic acute regulatory protein (StAR). Lipoid CAH is common among the Japanese, Korean, and Palestinian Arab populations, but is rare elsewhere. We describe six patients with lipoid CAH: four Japanese, one Palestinian, and one Guatemalan Native American. All had classical clinical presentations of normal female external genitalia in both genetic sexes, with severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Quite atypically, one patient had small adrenal glands shown by computed tomographic scanning. The StAR genes were characterized in all six patients. Three of the Japanese patients were compound heterozygotes for the common Japanese mutation Q258X in association with three different novel frameshift mutations; the fourth Japanese patient was homozygous for the mutation R182L, which is common among Palestinian patients but has not been described previously in a Japanese patient. Our Palestinian and Native American patients were each homozygous for novel frameshift mutations. Thus we have found five new frameshift mutations, but no new amino acid replacement (missense) mutations. This would be consistent with the view that only a small number of residues in the StAR protein are crucial for biological activity. The tomographic finding of small adrenals in a patient with genetically proven lipoid CAH due to a StAR mutation suggests a substantially broader spectrum of clinical findings in this disease than has been appreciated previously.

Adrenal Hyperplasia, Congenital↗
PubMed2000

Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.

Adolescent↗