FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system.

Disrupted in schizophrenia 1 (DISC1) is a multi-functional scaffolding protein that has been associated with neuropsychiatric disease. The role of DISC1 is to assemble protein complexes that promote neural development and signaling, hence tight control of the concentration of cellular DISC1 in neurons is vital to brain function. Using structural and biochemical techniques, we show for we believe the first time that not only is DISC1 turnover elicited by the ubiquitin proteasome system (UPS) but that it is orchestrated by the F-Box protein, FBXW7. We present the structure of FBXW7 bound to the DISC1 phosphodegron motif and exploit this information to prove that disruption of the FBXW7-DISC1 complex results in a stabilization of DISC1. This action can counteract DISC1 deficiencies observed in neural progenitor cells derived from induced pluripotent stem cells from schizophrenia patients with a DISC1 frameshift mutation. Thus manipulation of DISC1 levels via the UPS may provide a novel method to explore DISC1 function.

Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)-HSP20 complex attenuates the ß-agonist induced hypertrophic response in cardiac myocytes.

The small heat shock protein HSP20 is known to be cardioprotective during times of stress and the mechanism underlying its protective abilities depends on its phosphorylation on Ser16 by PKA (protein kinase A). Although the external stimuli that trigger Ser16 phosphorylation have been well studied, the events that modulate spatial and temporal control of this modification remain to be clarified. Here, we report that inhibition of cAMP phosphodiesterase-4 (PDE4) induces the phosphorylation of HSP20 in resting cardiac myocytes and augments its phosphorylation by PKA following ß-adrenergic stimulation. Moreover, using peptide array technology, in vitro binding studies, co-immunoprecipitation techniques and immunocytochemistry, we show that HSP20 binds directly to PDE4 within a region of the conserved catalytic domain. We also show that FRET-based, genetically-encoded cAMP reporters anchored to HSP20 exhibit a larger response to PDE4 inhibition compared to free cytosolic cAMP reporters, suggesting that the interaction with PDE4 is crucial in modulating the highly localised pool of cAMP to which HSP20 is exposed. Using information gleaned from peptide array analyses, we developed a cell-permeable peptide that serves to inhibit the interaction of PDE4 with HSP20. Disruption of the HSP20-PDE4 complex, using this peptide, suffices to induce phosphorylation of HSP20 by PKA and to protect against the hypertrophic response measured in neonatal cardiac myocytes following chronic ß-adrenergic stimulation.

ß-Arrestin 1 inhibits the GTPase-activating protein function of ARHGAP21, promoting activation of RhoA following angiotensin II type 1A receptor stimulation.

Activation of the small GTPase RhoA following angiotensin II stimulation is known to result in actin reorganization and stress fiber formation. Full activation of RhoA, by angiotensin II, depends on the scaffolding protein ß-arrestin 1, although the mechanism behind its involvement remains elusive. Here we uncover a novel partner and function for ß-arrestin 1, namely, in binding to ARHGAP21 (also known as ARHGAP10), a known effector of RhoA activity, whose GTPase-activating protein (GAP) function it inhibits. Using yeast two-hybrid screening, a peptide array, in vitro binding studies, truncation analyses, and coimmunoprecipitation techniques, we show that ß-arrestin 1 binds directly to ARHGAP21 in a region that transects the RhoA effector GAP domain. Moreover, we show that the level of a complex containing ß-arrestin 1 and ARHGAP21 is dynamically increased following angiotensin stimulation and that the kinetics of this interaction modulates the temporal activation of RhoA. Using information gleaned from a peptide array, we developed a cell-permeant peptide that serves to inhibit the interaction of these proteins. Using this peptide, we demonstrate that disruption of the ß-arrestin 1/ARHGAP21 complex results in a more active ARHGAP21, leading to less-efficient signaling via the angiotensin II type 1A receptor and, thereby, attenuation of stimulated stress fiber formation.

cGMP signalling in a transporting epithelium.

The biochemical aspects of cGMP signalling are well known, although in vivo roles of cGMP have only been recently discovered through work in genetic model organisms. The Drosophila melanogaster Malpighian (renal) tubule has been used to address the roles of cGMP in epithelial function. Here, we describe some of this work and outline recent progress in understanding the organotypic function of novel phosphodiesterases encoded by the D. melanogaster genome.

Determination of U-236 in sediment samples by accelerator mass spectrometry.

236U is produced only by neutron irradiation of uranium and therefore is potentially useful as a marker for anthropogenic uranium in the environment. Accelerator mass spectrometry (AMS) provides a technique for the determination of very low concentrations of actinide nuclides, and has now been applied to the determination of 236U:235U ratios in an intertidal sediment core collected from the North Irish Sea. Combining measurements of the 238U mass concentrations calculated from alpha spectrometry with 238U:235U ratios from ICP-MS and 236U:235U ratios from AMS has allowed the estimation of the mass concentrations of 236U in the sediments. 236U mass concentrations are in the range 10(-8) to 10(-9) g kg-1, and 236U:238U atom ratios in the range from 10(-5) to 10(-6), well above natural baseline levels. Uncertainties based on propagation of measurement errors were less than +/- 10% although +/- 15% is perhaps a more realistic estimate of overall uncertainty.

Measurement of 237Np in environmental water samples by accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) was used to measure 237Np in environmental water samples extracted from Irish Sea sediments. The samples were of limited volume (approximately 700 ml) and of low activity (0.06-0.79 mBq l-1; 2.30-30.3 pg l-1). AMS proved to have the required sensitivity for measuring these samples, and was in principle capable of measuring much smaller amounts, as low as 0.4 microBq (3.9 x 10(7) atoms). However, the background level in the procedural blanks showed that there was a systematic low level 237Np contamination of each sample, arising from the 239Np yield monitor used in the separations procedure, which effectively increased the detection limit of these analyses.

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2.

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.

Absorption of aluminium-26 in Alzheimer's disease, measured using accelerator mass spectrometry.

Although chromosomal abnormalities underpin some early onset cases of familial Alzheimer's disease (AD), most cases are sporadic and not associated with such abnormalities. Aluminium (Al) is a significant but controversial risk factor for sporadic AD, and studies have reported associations between Al and the principal pathological features of AD, senile plaques and neurofibrillary tangles. The present study measured gastrointestinal (GI) absorption of Al under normal dietary conditions using (26)Al tracer and accelerator mass spectrometry (AMS). Following overnight fast, 13 AD patients (aged 63-76 years) and 13 age-matched controls (aged 62-76 years) ingested a fruit drink containing 27 ng (26)Al. Plasma samples were obtained before and 1 h after the drink and from these the fraction of (26)Al absorbed across the GI tract was estimated. The GI tract rigorously excludes Al with only 0.06-0.1% of the ingested Al being absorbed. The mean fraction absorbed by AD subjects exceeded controls by a factor of 1.64 (p

Oligomeric but not monomeric silica prevents aluminum absorption in humans.

BACKGROUND: Soluble silica, a ubiquitous component of the diet, may be the natural ligand for dietary aluminum and may prevent its accumulation and toxicity in animals. However, previous studies on the inhibition of aluminum absorption and toxicity by soluble silica have produced conflicting results. We recently identified a soluble silica polymer, oligomeric silica, that has a much higher affinity for aluminum than does monomeric silica and that may be involved in the sequestration of aluminum. OBJECTIVE: By using (26)Al as a tracer, we investigated the effects of oligomeric and monomeric silica on the bioavailability of aluminum (study 1) and compared the availability of silicon from oligomeric and monomeric silica in the human gastrointestinal tract (study 2). DESIGN: In study 1, three healthy volunteers each ingested aluminum alone (control), aluminum with oligomeric silica (17 mg), and aluminum with monomeric silica (17 mg). In study 2, five healthy volunteers ingested both the oligomeric and monomeric forms of silica (34 mg). Serum and urine samples were analyzed for aluminum and silicon. RESULTS: Oligomeric silica reduced the availability of aluminum by 67% (P = 0.01) compared with the control, whereas monomeric silica had no effect (P = 0.40). Monomeric silica was readily taken up from the gastrointestinal tract and then excreted in urine (53%), whereas oligomeric silica was not detectably absorbed or excreted. CONCLUSIONS: The oligomeric, high-aluminum-affinity form of soluble silica reduces aluminum availability from the human gastrointestinal tract. Its potential role in the amelioration of aluminum toxicity in other biological systems requires attention.