Histone deacetylase (HDAC) 1 and 2 complexes regulate both histone acetylation and crotonylation in vivo.

Proteomic analysis of histones has shown that they are subject to a superabundance of acylations, which extend far beyond acetylation, to include: crotonylation, propionylation, butyrylation, malonylation, succinylation, ß-hydroxybutyrylation and 2-hydroxyisobutyrylation. To date, much of the functional data has focussed on histone crotonylation which, similar to acetylation, has been associated with positive gene regulation and is added by the acyltransferase, p300. Although Sirtuins 1-3, along with HDAC3, have been shown to possess decrotonylase activity in vitro, there is relatively little known about the regulation of histone crotonylation in vivo. Here we show that Histone Deacetylase 1 and 2 (HDAC1/2), the catalytic core of numerous co-repressor complexes, are important histone decrotonylase enzymes. A ternary complex of HDAC1/CoREST1/LSD1 is able to hydrolyse both histone H3 Lys18-acetyl (H3K18ac) and H3 Lys18-crotonyl (H3K18cr) peptide substrates. Genetic deletion of HDAC1/2 in ES cells increases global levels of histone crotonylation and causes an 85% reduction in total decrotonylase activity. Furthermore, we mapped H3K18cr in cells using ChIP-seq, with and without HDAC1/2, and observed increased levels of crotonylation, which largely overlaps with H3K18ac in the vicinity of transcriptional start sites. Collectively, our data indicate that HDAC1/2 containing complexes are critical regulators of histone crotonylation in vivo.

New Antarctic Gravity Anomaly Grid for Enhanced Geodetic and Geophysical Studies in Antarctica.

Gravity surveying is challenging in Antarctica because of its hostile environment and inaccessibility. Nevertheless, many ground-based, airborne and shipborne gravity campaigns have been completed by the geophysical and geodetic communities since the 1980s. We present the first modern Antarctic-wide gravity data compilation derived from 13 million data points covering an area of 10 million km2, which corresponds to 73% coverage of the continent. The remove-compute-restore technique was applied for gridding, which facilitated levelling of the different gravity datasets with respect to an Earth Gravity Model derived from satellite data alone. The resulting free-air and Bouguer gravity anomaly grids of 10 km resolution are publicly available. These grids will enable new high-resolution combined Earth Gravity Models to be derived and represent a major step forward towards solving the geodetic polar data gap problem. They provide a new tool to investigate continental-scale lithospheric structure and geological evolution of Antarctica.

Novel fatty acid gentamicin salts as slow-release drug carrier systems for anti-infective protection of vascular biomaterials.

Infections of vascular prostheses are still a major risk in surgery. The current work presents an in vitro evaluation of novel slow release antibiotic coatings based on new gentamicin fatty acid salts for polytetrafluoroethylene grafts. These grafts were coated with gentamicin sodium dodecyl sulfate, gentamicin laurate and gentamicin palmitate. Drug release kinetics, anti-infective characteristics, biocompatibility and haemocompatibility of developed coatings were compared to commercially available gelatin sealed PTFE grafts (SEALPTFE™) and knitted silver coated Dacron(®) grafts (InterGard(®)). Each gentamicin fatty acid coating showed a continuous drug release in the first eight hours followed by a low continuous release. Grafts coated with gentamicin fatty acids reduced bacterial growth even beyond pathologically relevant high concentrations. Cytotoxicity levels depending on drug formulation bringing up gentamicin palmitate as the most promising biocompatible coating. Thrombelastography studies, ELISA assays and an amidolytic substrate assay confirmed haemocompatibility of developed gentamicin fatty acid coatings comparable to commercially available grafts.

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1.

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.

Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains.

Most mutations in the androgen receptor (AR) ligand-binding domain (LBD) disrupt binding of the natural ligands: dihydrotestosterone and testosterone. Some AR LBD mutations do not affect ligand binding but they disrupt androgen-induced interaction of the N-terminal motif FXXLF and C-terminal activation function 2 (AF2). As N-/C-terminal interaction requires binding of agonists that have androgen activity in vivo, it correlates well with the phenotype. To study this further, we searched the Cambridge intersex database for patients with a detected missense mutation in the AR LBD presenting with normal ligand binding. Six mutations (D695N, Y763C, R774H, Q798E, R855H and L907F) were selected and introduced by site-directed mutagenesis into the pSVAR and pM-LBD plasmids. The transactivational potential of the wild-type and mutant androgen receptors (pSVAR) was examined by dual-luciferase assay using pGRE-LUC as a reporter vector. N-/C-terminal interaction was studied by mammalian two-hybrid assay using wild-type and mutated AR LBD (pM-LBD), pVP16-rAR-(5-538) (encoding rat amino-terminal AR) and pCMX-UAS-TK-LUC as a reporter. AR LBD mutations D695N, R774H and L907F presented with minimal transactivational capacity and N-/C-terminal interaction was totally disrupted. Mutations Y763C and R885H had some residual dose-dependent transactivational potential and minimal N-/C-terminal interaction. Q798E presented with good transactivational potential and it showed only mild reduction in N-/C-terminal interaction. With the selected mutations, N-/C-terminal interaction correlated well with AR transactivation and the phenotype. Disrupted N-/C-terminal interaction is capable of providing the mechanism for androgen-insensitivity syndrome in most cases where the mutation in the LBD does not disrupt ligand binding. Furthermore, mutations leading to the disrupted N-/C-terminal interaction can be localized to certain critical regions in the three-dimensional structure of the AR LBD. Our study shows that apart from the previously reported regions, regions just before helix 3, between helices 5 and 6, and at helix 10 are also important for AR N-/C-terminal interaction.

Aplasia cutis congenita of the scalp: how much therapy is necessary in large defects?

AIM: To show that local antibiotic management and a regular inspection of aplasia cutis congenita of the skull can give an excellent result. METHOD: This case reports a girl born with aplasia cutis congenita of the skull presenting with a large aplasia of the epidermis, dermis, subcutaneous tissue and galea, including a bone defect without any additional risk factor, e.g. early eschar formation, cerebrospinal fluid leakage or uncommon dural blood vessels. RESULTS: A primarily conservative treatment with local wet and antibiotic dressings together with a systemic antibiotic treatment for the first 2 wk led to an excellent result and thus prevented untimely operative and peri-operative procedures. CONCLUSIONS: Here we have shown that conservative treatment might be an option, even if the wound diameter is greater than 1 cm(2), to prevent infants from any untimely operative procedure with an elevated operative risk if any additional risk factors are excluded.

A dominant negative human peroxisome proliferator-activated receptor (PPAR){alpha} is a constitutive transcriptional corepressor and inhibits signaling through all PPAR isoforms.

Several missense mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor (PPAR)gamma have been described in subjects with dominantly inherited severe insulin resistance associated with partial lipodystrophy, hypertension, and dyslipidemia. These mutant receptors behave as dominant-negative inhibitors of PPARgamma signaling when studied in transfected cells. The extent to which such dominant-negative effects extend to signaling through other coexpressed PPAR isoforms has not been evaluated. To examine these issues further, we have created a PPARalpha mutant harboring twin substitutions, Leu459Ala and Glu462Ala, within the ligand binding domain (PPARalpha(mut)), examined its signaling properties, and compared the effects of dominant-negative PPARalpha and PPARgamma mutants on basal and ligand-induced gene transcription in adipocytes and hepatocytes. PPARalpha(mut) was transcriptionally inactive, repressed basal activity from a PPAR response element-containing promoter, inhibited the coactivator function of cotransfected PPAR-gamma coactivator 1alpha, and strongly inhibited the transcriptional response to cotransfected wild-type receptor. In contrast to PPARgamma, wild-type PPARalpha failed to recruit the transcriptional corepressors NCoR and SMRT. However, PPARalpha(mut) avidly recruited these corepressors in a ligand-dissociable manner. In hepatocytes and adipocytes, both PPARalpha(mut) and the corresponding PPARgamma mutant were capable of inhibiting the expression of genes primarily regulated by PPARalpha, -gamma, or -delta ligands, albeit with some differences in potency. Thus, dominant-negative forms of PPARalpha and PPARgamma are capable of interfering with PPAR signaling in a manner that is not wholly restricted to their cognate target genes. These findings may have implications for the pathogenesis of human syndromes resulting from mutations in this family of transcription factors.

Papillary fibroelastoma of the right atrium: an atypical presentation. A case report and review of the literature.

Papillary fibroelastomas are a rare form of benign cardiac neoplasm. While the majority of these lesions are asymptomatic and found incidentally via echocardiography or cardiac catheterization, those occurring on left-sided structures may become clinically important producing symptoms of syncope, angina, myocardial infarction, and sudden death. These masses also have the propensity to embolize resulting in transient ischemic attacks and strokes. Most papillary fibroelastomas are found on valvular structures and currently there are only 4 published case reports of these lesions occurring in the right atrium. Of these reports, only 3 have been presented as arising on the right atrial free wall. This case report presents the 54th known case of a papillary fibroelastoma occurring in the right atrium and the 4th to be reported as developing from the right atrial free wall. A review of the literature as well as the histogenesis, diagnosis, and therapy of this rare entity are discussed.

Sexual precocity and recurrent beta-human chorionic gonadotropin upsurges preceding the diagnosis of a malignant mediastinal germ-cell tumor in a 9-year-old boy.

Numerous disorders are known to cause sexual precocity. Beta-human chorionic gonadotropin (beta-HCG)-secreting germ-cell tumors are one of the sources that have to be considered in the differential diagnosis of processes inducing a peripheral precocious puberty. Germ-cell tumors might be located in the ovaries or testes, retroperitoneum, mediastinum or the cranium. We present the case of a 9-year-old boy with sexual precocity and a recurrent transient beta-HCG elevation. After an interval of 2 years with repeated radiological examinations including the mediastinum, a mediastinal tumor was identified by magnetic resonance imaging. To our knowledge, this is the first case of a diagnosis of a mediastinal choriocarcinoma with a recurrent serum beta-HCG elevation. So far, factors that might be responsible for the repeated spontaneous beta-HCG decline are unknown.

The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation.

Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X receptor (RXR). RXR plays a uniquely important role in differentiation, development, and homeostasis through its ability to serve as a heterodimeric partner to many other nuclear receptors. RXR is able to influence the activity of its partner receptors through the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known high-affinity ligand and is thought to be a silent component in the heterodimeric complex with partner receptors such as the ecdysone receptor. Here we report the 2.4-A crystal structure of the USP ligand-binding domain. The structure shows that a conserved sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs, serves to lock USP in an inactive conformation. It also shows that USP has a large hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This cavity is larger than that seen previously for most other nuclear receptors. Intriguingly, this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural ligand for USP.

St John's wort, a herbal antidepressant, activates the steroid X receptor.

St John's wort (SJW), an extract of the medicinal plant Hypericum perforatum, is widely used as a herbal antidepressant. Recently, this agent has been found to adversely affect the metabolism of various coadministered drugs. Steroid X receptor (SXR), an orphan nuclear receptor, induces hepatic cytochrome P450 gene expression in response to diverse endogenous steroids, xenobiotics and drugs. Here, we report that, when coexpressed with SXR, a reporter construct derived from the cytochrome P450 3A promoter is activated by St John's wort. A GAL4-SXR ligand binding domain (LBD) fusion mediates concentration-dependent transactivation by SJW, whereas a mutant GAL4-SXR fusion, containing substitutions in key residues in a transactivation domain, is inactive. SJW recruits steroid receptor coactivator-1 to SXR in a two-hybrid assay and competes with radiolabelled ligand in binding studies, suggesting it interacts directly with the receptor LBD. Of two constituents of SJW, we find that hyperforin, but not hypericin, mediates both transactivation and coactivator recruitment by SXR. Our observations suggest that SXR activation by St John's wort mediates its adverse interaction with drugs metabolised via the CYP 3A pathway. Future development of SJW derivatives lacking SXR activation, may enable its antidepressant and drug-metabolising properties to be dissociated.

Transcriptional repression by nuclear receptors: mechanisms and role in disease.

Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that co-repressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.

A dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutant is a constitutive repressor and inhibits PPARgamma-mediated adipogenesis.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) promotes adipocyte differentiation, exerts atherogenic and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic thiazolidinedione ligands. As no complete PPARgamma antagonists have been described hitherto, we have constructed a dominant-negative mutant receptor to inhibit wild-type PPARgamma action. Highly conserved hydrophobic and charged residues (Leu(468) and Glu(471)) in helix 12 of the ligand-binding domain were mutated to alanine. This compound PPARgamma mutant retains ligand and DNA binding, but exhibits markedly reduced transactivation due to impaired coactivator (cAMP-response element-binding protein-binding protein and steroid receptor coactivator-1) recruitment. Unexpectedly, the mutant receptor silences basal gene transcription, recruits corepressors (the silencing mediator of retinoid and thyroid receptors and the nuclear corepressor) more avidly than wild-type PPARgamma, and exhibits delayed ligand-dependent corepressor release. It is a powerful dominant-negative inhibitor of cotransfected wild-type receptor action. Furthermore, when expressed in primary human preadipocytes using a recombinant adenovirus, this PPARgamma mutant blocks thiazolidinedione-induced differentiation, providing direct evidence that PPARgamma mediates adipogenesis. Our observations suggest that, as in other mutant nuclear receptor contexts (acute promyelocytic leukemia, resistance to thyroid hormone), dominant-negative inhibition by PPARgamma is linked to aberrant corepressor interaction. Adenoviral expression of this mutant receptor is a valuable means to antagonize PPARgamma signaling.

Three novel mutations at serine 314 in the thyroid hormone beta receptor differentially impair ligand binding in the syndrome of resistance to thyroid hormone.

The syndrome of resistance to thyroid hormone is associated with diverse mutations in the ligand-binding domain of the thyroid hormone beta receptor, localizing to three clusters around the hormone binding cavity. Here, we report three novel resistance to thyroid hormone mutations (S314C, S314F, and S314Y), due to different nucleotide substitutions in the same codon, occurring in six separate families. Functional characterization of these mutant receptors showed marked differences in their properties. S314F and S314Y receptor mutants exhibited significant transcriptional impairment in keeping with negligible ligand binding and were potent dominant negative inhibitors of wild-type receptor action. In contrast, the S314C mutant bound ligand with reduced affinity, such that its functional impairment and dominant negative activity manifest at low concentrations of thyroid hormone, but are more reversible at higher T3 concentrations. The degree of functional impairment of mutant receptors in vitro may correlate with the magnitude of thyroid dysfunction in vivo. Modelling these mutations using the crystal structure of thyroid hormone receptor beta shows why ligand binding is perturbed and why the phenylalanine/tyrosine mutations are more deleterious than cysteine.

Mechanism of corepressor binding and release from nuclear hormone receptors.

The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs. We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.

Dominant negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension.

Thiazolidinediones are a new class of antidiabetic agent that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type 2 diabetes. Although these agents can bind and activate an orphan nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), there is no direct evidence to conclusively implicate this receptor in the regulation of mammalian glucose homeostasis. Here we report two different heterozygous mutations in the ligand-binding domain of PPARgamma in three subjects with severe insulin resistance. In the PPARgamma crystal structure, the mutations destabilize helix 12 which mediates transactivation. Consistent with this, both receptor mutants are markedly transcriptionally impaired and, moreover, are able to inhibit the action of coexpressed wild-type PPARgamma in a dominant negative manner. In addition to insulin resistance, all three subjects developed type 2 diabetes mellitus and hypertension at an unusually early age. Our findings represent the first germline loss-of-function mutations in PPARgamma and provide compelling genetic evidence that this receptor is important in the control of insulin sensitivity, glucose homeostasis and blood pressure in man.

Signal transduction: fast lane to transcriptional activation.

Two newly reported structures of homodimeric 'STAT' transcription factors bound to DNA reveal at atomic resolution the elegant mechanism through which kinase activity at the cell membrane can be transduced into transcriptional activation within the cell nucleus.

Lhx2, a vertebrate homologue of apterous, regulates vertebrate limb outgrowth.

apterous specifies dorsal cell fate and directs outgrowth of the wing during Drosophila wing development. Here we show that, in vertebrates, these functions appear to be performed by two separate proteins. Lmx-1 is necessary and sufficient to specify dorsal identity and Lhx2 regulates limb outgrowth. Our results suggest that Lhx2 is closer to apterous than Lmx-1, yet, in vertebrates, Lhx2 does not specify dorsal cell fate. This implies that in vertebrates, unlike Drosophila, limb outgrowth can be dissociated from the establishment of the dorsoventral axis.