P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Tissue neogenesis and STRO-1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin-O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Flotillins interact with PSGL-1 in neutrophils and, upon stimulation, rapidly organize into membrane domains subsequently accumulating in the uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.

Nasolabial esthetics in children with complete unilateral cleft lip and palate after 1- versus 3-stage treatment protocols

PURPOSE: Facial esthetics play an important role in social interactions. However, children with a repaired complete unilateral cleft lip and palate usually show some disfigurement of the nasolabial area. To date, few studies have assessed the nasolabial appearance after different treatment protocols. The aim of the present study was to compare the nasolabial esthetics after 1- and 3-stage treatment protocols. MATERIALS AND METHODS: Four components of the nasolabial appearance (nasal form, nasal deviation, mucocutaneous junction, and profile view) were assessed by 4 raters in 108 consecutively treated children who had undergone either 1-stage closure (Warsaw group, 41 boys and 19 girls, mean age 10.8 years, SD 2.0) or 3-stage (Nijmegen group, 30 boys and 18 girls, mean age 8.9 years, SD 0.7). A 5-grade esthetic index of Asher-McDade was used, in which grade 1 indicates the most esthetic and grade 5 the least esthetic outcome. RESULTS: The nasal form was judged the least esthetic in both groups and graded 3.1 (SD 1.1) and 3.2 (SD 1.1). The nasal deviation, mucocutaneous junction, and profile view were scored from 2.1 (SD 0.8) to 2.3 (SD 1.0) in both groups. The treatment outcome after the Warsaw and Nijmegen protocols was comparable. Neither overall nor any of the 4 components of the nasolabial appearance showed intercenter differences (P > .1). CONCLUSIONS: The nasolabial appearance after the Warsaw (1-stage) and Nijmegen (3-stage) protocols was comparable. The technique of lip repair (triangular flap in Warsaw and Millard rotation advancement in Nijmegen) gave comparable results for the esthetics of the nasolabial area.

Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade initiated by IL-5 in eosinophils

BACKGROUND: Eosinophil differentiation, activation, and survival are largely regulated by IL-5. IL-5-mediated transmembrane signal transduction involves both Lyn-mitogen-activated protein kinases and Janus kinase 2-signal transducer and activator of transcription pathways. OBJECTIVE: We sought to determine whether additional signaling molecules/pathways are critically involved in IL-5-mediated eosinophil survival. METHODS: Eosinophil survival and apoptosis were measured in the presence and absence of IL-5 and defined pharmacologic inhibitors in vitro. The specific role of the serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim) 1 was tested by using HIV-transactivator of transcription fusion proteins containing wild-type Pim-1 or a dominant-negative form of Pim-1. The expression of Pim-1 in eosinophils was analyzed by means of immunoblotting and immunofluorescence. RESULTS: Although pharmacologic inhibition of phosphatidylinositol-3 kinase (PI3K) by LY294002, wortmannin, or the selective PI3K p110delta isoform inhibitor IC87114 was successful in each case, only LY294002 blocked increased IL-5-mediated eosinophil survival. This suggested that LY294002 inhibited another kinase that is critically involved in this process in addition to PI3K. Indeed, Pim-1 was rapidly and strongly expressed in eosinophils after IL-5 stimulation in vitro and readily detected in eosinophils under inflammatory conditions in vivo. Moreover, by using specific protein transfer, we identified Pim-1 as a critical element in IL-5-mediated antiapoptotic signaling in eosinophils. CONCLUSIONS: Pim-1, but not PI3K, plays a major role in IL-5-mediated antiapoptotic signaling in eosinophils.

Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor activities

Vitamin A is a nutrient with remarkable effects on adipose tissue and skeletal muscles, and plays a role in controlling energy balance. Retinoic acid (RA), the carboxylic form of vitamin A, has been associated with improved glucose tolerance and insulin sensitivity. In contrast, elevated glucocorticoids have been implicated in the development of insulin resistance and impaired glucose tolerance. Here, we investigated whether RA might counteract glucocorticoid effects in skeletal muscle cells by lowering 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local glucocorticoid activation and/or activation of glucocorticoid receptor (GR). We found a dose-dependent down-regulation of 11beta-HSD1 mRNA expression and activity upon incubation of fully differentiated mouse C2C12 myotubes with RA. In addition, RA inhibited GR transactivation by an 11beta-HSD1-independent mechanism. The presence of RA during myogenesis did not prevent myotube formation but resulted in relatively glucocorticoid-resistant myotubes, exhibiting very low 11beta-HSD1 expression and GR activity. The use of selective retinoic acid receptor (RAR) and retinoid X receptor ligands provided evidence that these effects were mediated through RARgamma. Importantly, short hairpin RNA against RARgamma abolished the effect of RA on 11beta-HSD1 and GR. In conclusion, we provide evidence for an important role of RA in the control of glucocorticoid activity during myogenesis and in myotubes. Disturbances of the nutrient and hormonal regulation of glucocorticoid action in skeletal muscles might be relevant for metabolic diseases.

Buttress form of the central African rain forest tree Microberlinia bisulcata, and its possible role in nutrient acquisition

Buttressing is a trait special to tropical trees but explanations for its occurrence remain inconclusive. The two main hypotheses are that they provide structural support and/or promote nutrient acquisition. Studies of the first are common but the second has received much less attention. Architectural measurements were made on adult and juvenile trees of the ectomycorrhizal species Microberlinia bisulcata, in Korup (Cameroon). Buttressing on this species is highly distinctive with strong lateral extension of surface roots of the juveniles leading to a mature buttress system of a shallow spreading form on adults. This contrasts with more vertical buttresses, closer to the stem, found on many other tropical tree species. No clear relationship between main buttress and large branch distribution was found. Whilst this does not argue against the essential structural role of buttresses for these very large tropical trees, the form on M. bisulcata does suggest a likely second role, that of aiding nutrient acquisition. At the Korup site, with its deep sandy soils of very low phosphorus status, and where most nutrient cycling takes place in a thin surface layer of fine roots and mycorrhizas, it appears that buttress form could develop from soil-surface root exploration for nutrients by juvenile trees. It may accordingly allow M. bisulcata to attain the higher greater competitive ability, faster growth rate, and maximum tree size that it does compared with other co-occurring tree species. For sites across the tropics in general, the degree of shallowness and spatial extension of buttresses of the dominant species is hypothesized to increase with decreasing nutrient availability.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895–1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

Base pairing and miscoding properties of 1,N(6)-ethenoadenine- and 3,N(4)-ethenocytosine-containing RNA oligonucleotides

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.