Cytochrome b5 (b5) in the developing rhesus adrenal.
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Adrenal Glands↗
Biomedical subjects
A Conley
Publications and source records attributed to A Conley.
The Sp1-like protein BTEB3 inhibits transcription via the basic transcription element box by interacting with mSin3A and HDAC-1 co-repressors and competing with Sp1.
Sp1-like proteins are characterized by three conserved C-terminal zinc finger motifs that bind GC-rich sequences found in promoters of numerous genes essential for mammalian cell homeostasis. These proteins behave as transcriptional activators or repressors. Although significant information has been reported on the molecular mechanisms by which Sp1-like activators function, relatively little is known about mechanisms for repressor proteins. Here we report the functional characterization of BTEB3, a ubiquitously expressed Sp1-like transcriptional repressor. GAL4 assays show that the N terminus of BTEB3 contains regions that can act as direct repressor domains. Immunoprecipitation assays reveal that BTEB3 interacts with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. Gel shift assays demonstrate that BTEB3 specifically binds the BTE site, a well characterized GC-rich DNA element, with an affinity similar to that of Sp1. Reporter and gel shift assays in Chinese hamster ovary cells show that BTEB3 can also mediate repression by competing with Sp1 for BTE binding. Thus, the characterization of this protein expands the repertoire of BTEB-like members of the Sp1 family involved in transcriptional repression. Furthermore, our results suggest a mechanism of repression for BTEB3 involving direct repression by the N terminus via interaction with mSin3A and HDAC-1 and competition with Sp1 via the DNA-binding domain.
Amino Acid Motifs↗
Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation.
Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.
Animals↗
A comparative approach to structure-function studies of mammalian aromatases.
To date, structure--function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B' and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.
Animals↗
Mammalian aromatases.
Aromatase is the enzyme complex that catalyses the synthesis of oestrogens from androgens, and therefore it has unique potential to influence the physiological balance between the sex steroid hormones. Both aromatase cytochrome P450 (P450arom) and NADPH-cytochrome P450 reductase (reductase), the two essential components of the enzyme complex, are highly conserved among mammals and vertebrates. Aromatase expression occurs in the gonads and brain, and is essential for reproductive development and fertility. Of interest are the complex mechanisms involving alternative promoter utilization that have evolved to control tissue-specific expression in these tissues. In addition, in a number of species, including humans, expression of aromatase has a broader tissue distribution, including placenta, adipose and bone. The relevance of oestrogen synthesis and possibly androgen metabolism in these peripheral sites of expression is now becoming clear from studies in P450arom knockout (ArKO) mice and from genetic defects recognized recently in both men and women. Important species differences in the physiological roles of aromatase expression are also likely to emerge, despite the highly conserved nature of the enzyme system. The identification of functionally distinct, tissue-specific isozymes of P450arom in at least one mammal, pigs, and several species of fish indicates that there are additional subtle, but physiologically significant, species-specific roles for aromatase. Comparative studies of mammalian and other vertebrate aromatases will expand understanding of the role played by this ancient enzyme system in the evolution of reproduction and the adaptive influence of oestrogen synthesis on general health and well being.
Amino Acid Sequence↗
The primate adrenal zona reticularis is defined by expression of cytochrome b5, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) and NADPH-cytochrome P450 reductase (reductase) but not 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3beta-HSD).
Biochemical studies suggest that 17,20-lyase activity, and thus efficient synthesis of androgens by human P450c17, requires both reductase and the accessory protein cytochrome b5. Since the human and primate zona reticularis (ZR) secrete androgens, the expression of these proteins, and of 3beta-HSD, was investigated by immunocytochemistry in the adrenal cortex of the mature rhesus macaque. Cytochrome b5 expression was restricted to the cells of the ZR which appeared deficient in 3beta-HSD. However, both P450c17 and reductase were evident throughout the zona fasciculata. These data provide essential evidence in support of a functional role for cytochrome b5 in the regional control of 17alpha-hydroxylase and 17,20-lyase activities of P450c17 and thereby adrenal C19 steroid secretion by the primate adrenal gland.
Animals↗
Patellofemoral design influences function following total knee arthroplasty.
The functional influence of patellofemoral design was evaluated by testing two cohorts of patients with total knee arthroplasty while walking, climbing stairs, and rising from a chair. The two cohorts received one of two different designs of total knee arthroplasty. The designs differed primarily in the curvature of the femoral trochlea. These differences in curvature have been shown in in vitro studies to influence the tracking of the patella. One design had a smaller radius on the patellar flange, which caused the patella to articulate more anteriorly and distally than the second design, which had a larger radius in this region. The second design more closely replicated the curvature of the femoral trochlear anatomy. There was a significant functional difference during stairclimbing between the patient groups, whereas there was no difference during walking or chair rising. The group with the design that had nonanatomic tracking of the patella had a higher than normal moment tending to flex the knee (net quadriceps moment) during late stance phase. The increased net quadriceps moment during late stance was concurrent with an increased knee flexion. The abnormal function occurred when the patellar was tracking over the nonanatomic portion of the trochlea. Patients in the group with the anatomic trochlea did not have abnormal function. These results suggest a relationship between a nonanatomic trochlea and abnormal function during stair-climbing.
Aged↗
Porcine aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene?
Aromatase cytochrome P450 (P450arom) is expressed in a variety of tissues. Pigs express P450arom as bilaminar blastocysts in utero, and thereafter in the gonads, adrenal glands and placenta. Our studies also demonstrate the existence of porcine isozymes of P450arom which differ substantially in their amino acid composition and function. The placental isoform, most similar to P450arom in other mammals, consists of 503 amino acids. The ovarian isoform, expressed in both theca and granulosa cells, is a 501 amino acid protein exhibiting less than 20% of the activity of the placental isozyme. Furthermore, it is inhibited not only by CGS16949A but also by etomidate which does not inhibit the placental P450arom. Partial sequences generated by the rapid amplification of the cDNA ends (RACE) procedure indicate that the expression of a third isoform in the blastocyst is switched to the placental isozyme during differentiation of the fetal membranes. In addition, these transcripts, and others from the theca, granulosa, testes, adrenal glands and placenta demonstrate differences in the 5'-untranslated region (putative exon I) suggestive of tissue-specific alternative splicing. An identical 5'-untranslated sequence was obtained from transcripts expressed in the theca and granulosa. Testes and adrenal transcripts also have identical 5' ends, which differ substantially from the ovarian sequence. Blastocyst and placenta 5'-untranslated sequences differ from each other and from those expressed in the gonads and adrenals. Several tissue-specific transcripts thus encode porcine P450arom. Interestingly, distinct 5' sequences exist for ovarian and testes P450arom mRNAs, suggesting different promoters and therefore regulation in the male and female gonads. The molecular origins of the functional isoforms and the tissue-specific transcripts are uncertain, however partial genomic sequence and other genetic analyses suggest the existence of multiple genes. However, sequence alignment of the placental and ovarian isoforms indicates complete conservation of putative exon III, so that complex splicing remains a possibility. Clearly, the regulation of P450arom expression is more complex in the pig than in other vertebrates investigated to date.
Amino Acid Sequence↗
Complications of a cable grip system.
From a group of 643 total hip arthroplasties, 98 patients required trochanteric osteotomies, and in 68 the trochanteric osteotomies were repaired with the Dall-Miles cable grip system (Howmedica, Rutherford, NJ). Trochanteric nonunion occurred in 17 of these patients (25%), with fraying and fragmentation of the cable present in 15 (88%). Of the 51 patients with radiographic union, 18 (35%) also had signs of fraying and fragmentation. Bone destruction around the cable in the area of the lesser trochanter was seen in seven patients (10%). Large deposits of metal debris at the inferior border of the acetabulum were seen in eight hips (12%). Multifilament cable did not appear to offer significant advantages over standard monofilament wire, and the potential problems of fraying, fragmentation, and free-floating metallic debris must be considered.
Adult↗
Participatory decision making: issues and guidelines.
The concept of shared responsibility in decision making through the use of participatory decision making has received considerable support in nursing education. There are important omissions in the discussions and applications of this concept. Problems in the use of participatory decision making as related to information transmission; conflict, consensus, and coalitions; and faculty/administrator role differences are analyzed. Criteria and guidelines for the appropriate use of participatory decision making are provided.
Conflict, Psychological↗
Comments on reports on hormone synthesis and regulation.
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Animals↗
Relationship of demographic characteristics of interviewers to blood pressure measurements.
This report describes findings from the Hypertension Detection and Follow-Up Program (HDFP) on the relationship between traits of interviewers and outcome of blood pressure measurements taken during home interviews. Mean diastolic blood pressure readings and prevalence data for 137,417 respondents taken by 617 interviewers are analyzed. Findings from regression analysis show that the magnitude of the absolute or relative difference in outcome of blood pressure measurements is associated much more positively with the characteristics of race and sex of the respondent than these characteristics in the interviewer.
Adult↗
Axoplasmic flow of proteolipids and lipids in the hypoglossal nerve of the rabbit.
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Animals↗