Search PubMed⌕ Search

Biomedical subjects

W L Miller

Publications and source records attributed to W L Miller.

At least 73 records · Page 4Linked to original sources
PubMed1998

Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer.

In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17alpha-hydroxylase and trace 17,20-lyase activities toward both Delta4 and Delta5 steroids. Coexpression of human OR with P450c17 increases the Vmax of both the 17alpha-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the Vmax of the 17,20-lyase reactions but not of the 17alpha-hydroxylase reactions. Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the Vmax of the 17,20-lyase reaction without altering 17alpha-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17. These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17.OR complex to stimulate 17, 20-lyase activity.

Catalysis↗
PubMed1998

Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins.

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish.

Aeromonas↗
PubMed1998

Early steps in androgen biosynthesis: from cholesterol to DHEA.

Sex steroids, both androgens and oestrogens, are made from dehydroepiandrosterone (DHEA). The biosynthesis of DHEA from cholesterol entails four steps. First, cholesterol enters the mitochondria with the assistance of a recently described factor called the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia. Next, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc. Mutations in the gene for P450scc and for its electron transfer partners, ferredoxin reductase and ferredoxin, have not been described and are probably incompatible with term gestation. Third, pregnenolone undergoes 17 alpha-hydroxylation by microsomal P450c17. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. Isolated 17,20 lyase deficiency is rare, but the identification of its genetic basis and the study of P450c17 enzymology have recently clarified the mechanisms by which DHEA synthesis may be regulated in adrenarche, and have suggested that the lesion underlying polycystic ovary syndrome might involve a serine kinase.

Adrenal Hyperplasia, Congenital↗
PubMed1998

Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families.

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1998

Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1-binding sites and protein kinase C.

FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit. Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSHbeta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSHbeta expression. As GnRH is a major regulator of FSHbeta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHbeta transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHbeta. This system was used to show that expression of luciferase constructs containing either the -4741/+759 region of the oFSHbeta gene (-4741oFSHbeta-Luc) or the -846/+44 region of the human alpha gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHbeta. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHbeta and alpha-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSHbeta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSHbeta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.

Animals↗
PubMed1998

The value of a family physician.

BACKGROUND: Most efforts to improve health care have been made without a full understanding of the value of a primary care approach. METHODS: This article synthesizes the observations from the Direct Observation of Primary Care (DOPC) study. This multimethod study of 138 family physicians in 84 practices included direct observation of 4454 patients visits were used to describe aspects of family practice that may provide value for patients. RESULTS: Family physicians provide and coordinate care for a wide variety of patients problems, prioritizing these competing demands on the basis of relationships developed during multiple patient visits over time. They use acute and chronic illness visits as opportunities to integrate care for specific diseases, mental health, and preventive care in ways that are tailored to the specific needs of patients and families. Higher rates of delivery of core attributes of family practice are associated with patient satisfaction and preventive services delivery, and are diminished by forced discontinuity of care. CONCLUSIONS: Family physicians prioritize and deliver care according to a broad agenda based on patient needs. These needs are understood within ongoing relationships with the patient, family, larger health care system, and community. This integrative approach includes numerous avenues for affecting important patient outcomes that are unlikely to be optimally met by less integrated models of medical care. Expanding the value of family practice will require the development and application of new knowledge of the core structures, processes, and contexts of family practice, and their effects on patient outcomes.

Cross-Sectional Studies↗
PubMed1998

Understanding change in primary care practice using complexity theory.

BACKGROUND: Understanding the organization of primary care practices is essential for implementing changes related to delivery of preventive or other health care services. A theoretical model derived from complexity theory provides a framework for understanding practice change. METHODS: Data were reviewed from brief participant observation fieldnotes collected in the 84 practices of the Direct Observation of Primary Care (DOPC) study and in 27 practices from three similar studies investigating preventive services delivery. These data were synthesized with information from an extensive search of the social science, nursing, and health services literature concerning practice organization, and of the literature on complexity theory from the fields of mathematics, physics, biology, management, medicine, and family systems, to create a complexity model of primary care practice. RESULTS: Primary care practices are understood as complex adaptive systems consisting of agents, such as patients, office staff, and physicians, who enact internal models of income generation, patient care, and organizational operations. These internal models interact dynamically to create each unique practice. The particular shape of each practice is determined by its primary goals. The model suggests three strategies for promoting change in practice and practitioner behavior: joining, transforming, and learning. CONCLUSIONS: This model has important implications for understanding change in primary care practice. Practices are much more complex than present strategies for change assume. The complexity model identified why some strategies work in particular practices and others do not.

Family Practice↗
PubMed1998

Illuminating the 'black box'. A description of 4454 patient visits to 138 family physicians.

BACKGROUND: The content and context of family practice outpatient visits have never been fully described, leaving many aspects of family practice in a "black box," unseen by policymakers and understood only in isolation. This article describes community family practices, physicians, patients, and outpatient visits. METHODS: Practicing family physicians in northeast Ohio were invited to participate in a multimethod study of the content of primary care practice. Research nurses directly observed consecutive patient visits, and collected additional data using medical record reviews, patient and physician questionnaires, billing data, practice environment checklists, and ethnographic fieldnotes. RESULTS: Visits by 4454 patients seeing 138 physicians in 84 practices were observed. Outpatient visits to family physicians encompassed a wide variety of patients, problems, and levels of complexity. The average patient paid 4.3 visits to the practice within the past year. The mean visit duration was 10 minutes. Fifty-eight percent of visits were for acute illness, 24% for chronic illness, and 12% for well care. The most common uses of time were history-taking, planning treatment, physical examination, health education, feedback, family information, chatting, structuring the interaction, and patient questions. CONCLUSIONS: Family practice and patient visits are complex, with competing demands and opportunities to address a wide range of problems of individuals and families over time and at various stages of health and illness. Multimethod research in practice settings can identify ways to enhance the competing opportunities of family practice to improve the health of their patients.

Adult↗
PubMed1998

Primary care practice organization and preventive services delivery: a qualitative analysis.

BACKGROUND: Rapid developments within the health care environment have led to increased pressures for change among primary care physicians and their practices. Nevertheless, a lack of understanding of practice organization and function has limited the effectiveness of attempts to change practice behaviors. Recent attempts to increase the delivery of preventive health care services illustrate the limitations of current approaches. To assist physicians in their attempts at change, our study looked at the office as a whole system and at the competing demands within the primary care setting. METHODS: Qualitative fieldnotes were recorded by research nurses who observed 138 family physicians in 84 practices in northeast Ohio for 4 days each. These data were content-analyzed to identify features that are important for understanding how practices are organized. RESULTS: These data indicate that primary care practice is much more complex than research and transformation efforts generally acknowledge. The data identified a diverse set of features that describe how primary care practices are organized and function. These included cognitive and behavioral components of physician philosophy and style, and numerous features of the practice organization, such as office efficiency, clarity of staff roles, communication patterns among physicians and staff, and approaches to using office protocols. The data also suggest that some practices are more innovative than others and that some physicians or staff have special motivations that can support or inhibit a particular change. CONCLUSIONS: Physicians who want to change their practice, as well as those persons who want to stimulate change from the outside, need to have a more comprehensive approach than is now commonly used to assess practices that encompass a broad spectrum of variables.

Continuity of Patient Care↗
PubMed1998

Dobutamine effect on ankle-brachial pressure index in patients with peripheral arterial occlusive disease. New noninvasive test for evaluation of peripheral circulation?

BACKGROUND: This study was designed to evaluate the effect of short-term administration of graded dose of dobutamine on the circulation of the lower limbs in the patients with symptomatic peripheral arterial occlusive disease. METHODS: An ankle-brachial pressure index (ABI) was determined at the time of dobutamine stress echocardiography by measuring systolic pressure over the brachial artery and right dorsalis pedis artery using continuous-wave Doppler instrument. Setting. The study was conducted on all patients who had dobutamine stress echocardiography ordered by their referring physicians and performed in the Echocardiography Laboratory of the Mayo Clinic. Patients. All patients scheduled for dobutamine stress echocardiography were the subject of this study unless they had rigid vessels defined as ABI > 1.5, were on beta-blockers or did not agree to participate in the study. 20 patients, mean age 67 +/- 9 years (9 men and 11 women) without peripheral occlusive arterial disease and resting ABI > or = 1.0 (group A), and 18 patients, mean age 71 +/- 10 years (11 men and 7 women) with the evidence of peripheral occlusive arterial disease and ABI < 1.0 were examined. Measures. ABI was measured just prior to starting the dobutamine infusion, and then at the third minute of each increment in dobutamine dosage. RESULTS: In control group patients ABI rises at 5-10 micrograms/kg/min of dobutamine infusion and at higher doses (20-50 micrograms/kg/min) drops back to the baseline values. In the patients with peripheral arterial occlusive disease low doses of dobutamine do not increase ABI, but higher doses cause decrease of pressure index from 0.6 to 0.3. There was no incidence of ischemic pain or any other kind of discomfort in the lower extremities. CONCLUSIONS: Low doses of dobutamine have no decremental effect on peripheral circulation. High doses of dobutamine cause a profound decrease of blood pressure in the lower limbs of patients with peripheral occlusive arterial disease, and the extent of decrease was proportional to the degree of ischemia. Peripheral blood pressure changes registered in the study over dorsalis pedis artery at the time of dobutamine infusion resemble those that occurred at the time of exercise-walking test. ABI measurement at the time of dobutamine echocardiography might be a useful test for the evaluation of peripheral circulation. Further studies are necessary for the assessment of a clinical usefulness of dobutamine-ankle-brachial test.

Adult↗
PubMed1998

Steroid hormone biosynthesis and actions in the materno-feto-placental unit.

Human pregnancy is associated with profound changes in the mother's steroid hormone milieu because of steroids produced by the corpus luteum of pregnancy and by the fetoplacental unit. The precise roles of these hormones in the maintenance of pregnancy, in fetal development, and in parturition have been controversial, but recent developments have clarified many of these areas.

Female↗
PubMed1997

Pathophysiology, genetics, and treatment of hyperandrogenism.

Presenting symptoms of hirsutism and virilism often signal a disorder of androgen biosynthesis, especially one of the forms of adrenal hyperplasia. The genetics and physiology of the various disorders are reviewed, emphasizing those that results in increased adrenal androgen production. All of these disorders can be diagnosed genetically, permitting family counseling, and all can be treated successfully with appropriate hormonal replacement therapy. Premature adrenarche is not caused by an enzymatic disorder; its origins remain obscure but may be an early harbinger of the polycystic ovary syndrome.

Female↗
PubMed1997

The regulation of 17,20 lyase activity.

P450c17 is a single microsomal enzyme that catalyzes two distinct steroid biosynthetic activities: 17 alpha-hydroxylase and 17,20 lyase. Human beings have only one gene that encodes only one form of P450c17. Three clinical observations indicated that these were independently regulated activities. First, several cases of isolated 17,20 lyase deficiency were reported, in which 17 alpha-hydroxylase activity was spared. Second, most adrenal steroidogenesis in children stops after 17 alpha-hydroxylation, thus permitting the synthesis of cortisol, whereas most gonadal steroidogenesis proceeds to C19 sex steroids as a result of both activities. Third, the 17,20 lyase activity of the human adrenal is developmentally activated during adrenarche. To catalyze these two activities, P450c17 must receive reducing equivalents from electron donors (redox partners). Previous observations showed that the molar ratio of P450 oxidoreductase to P450c17 was 3-fold higher in the testis than in the adrenal, and that increasing the molar ratio of the redox partner to P450c17 would increase the ratio of 17,20 lyase activity to 17 alpha-hydroxylase. We have recently shown that P450c17 must be phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase to acquire 17,20 lyase activity. We have also recently found two cases of isolated 17,20 lyase deficiency that have mutations of residues in the proposed redox partner binding site. Together, these studies suggest a unified view of the regulation of 17,20 lyase activity. The ratio of 17,20 lyase to 17 alpha-hydroxylase activity of P450c17 is regulated by the availability of reducing equivalents flowing to the enzyme. This can be increased by increasing the molar concentration of electron-donating redox partners, such as P450 oxidoreductase or possibly cytochrome b5, as appears to be the case in the gonads. Alternatively, the affinity of P450c17 for redox partners may be selectively increased by Ser/Thr phosphorylation, or selectively decreased by certain mutations in the redox partner binding site, in either case altering an electrostatic interaction between P450c17 and the redox partner. This model is consistent with all present observations about the biochemistry, genetics, enzymology, and clinical phenomenology of P450c17.

Adrenal Hyperplasia, Congenital↗
PubMed1997

Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.

The tenascins are a family of large extracellular matrix proteins with at least three members: tenascin-X (TNX), tenascin-C (TNC, or cytotactin) and tenascin-R (TN-R, or restrictin). Although the tenascins have been implicated in a number of important cellular processes, no function has been clearly established for any tenascin. We describe a new contiguous-gene syndrome, involving the CYP21B and TNX genes, that results in 21-hydroxylase deficiency and a connective-tissue disorder consisting of skin and joint hyperextensibility, vascular fragility and poor wound healing. The connective tissue findings are typical of the Ehlers-Danlos syndrome (EDS). The abundant expression of TNX in connective tissues is consistent with a role in EDS, and our patient's skin fibroblasts do not synthesize TNX protein in vitro or in vivo. His paternal allele carries a novel deletion arising from recombination between TNX and its partial duplicate gene, XA, which precludes TNX synthesis. Absence of TNX mRNA and protein in the proband, mapping of the TNX gene and HLA typing of this family suggest recessive inheritance of TNX deficiency and connective-tissue disease. Although the precise role of TNX in the pathogenesis of EDS is uncertain, this patient's findings suggest a unique and essential role for TNX in connective-tissue structure and function.

Adult↗
PubMed1997

The genetic and functional basis of isolated 17,20-lyase deficiency.

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.

Adolescent↗