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.

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

Nucleotide analogs facilitate base conversion with 3' mismatch primers.

We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced. Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.

Location of aluminium and gallium in human neuroblastoma cells treated with metal-chelating agent complexes.

The subcellular location of aluminium is unknown, probably because of difficulties in investigating aluminium biochemistry and the use of varied experimental approaches of uncertain sensitivity. We have studied levels of uptake and the localization of gallium and of aluminium in cultured human neuroblastoma cells treated with soluble metal complexes (mainly Al- or Ga-EDTA), radiolabeled with 26Al or 67Ga, respectively. Crude nuclei and cytoplasm were obtained by two separate methods, and DNA, RNA, and proteins were prepared from the nuclei by centrifugation in high salt; also, cytosol and noncytosol were separated using a nondissociating method. Levels of uptake were of similar order for the two metals-on average about 50 pmol/10(6) cells for aluminium and 120 pmol/10(6) cells for gallium, after 4 to 8 days treatment at 250 microM, and approximately 50 to 70% of the metal was found in the cytosol. About 20% of the aluminium and 10 to 25% of the gallium was associated with nuclear protein. A lower proportion was bound to DNA and to nuclear RNA. In cells treated with gallium-citrate/transferrin mixtures, 30 to 35% of the gallium in the cytosol was bound to protein, at least 35 being loosely bound; the main gallium-associated protein was probably intracellular transferrin. The remaining 65 to 70% of the metal in the cytosol was in low-molecular-weight form, and we suggest that the latter metal could affect structures such as the cytoskeleton and also metabolic processes in the cytoplasm. The similarity in distribution of the two metals supports the use of gallium as a "surrogate" for aluminium, at least in cell culture studies.

Uptake by man of aluminium in a public water supply.

1. After overnight fasting, two young male adults each received a single oral dose of 100 Bq 26Al in tap water. Coincidence gamma-ray spectrometry and accelerator mass spectrometry were used to determine the 26Al content of excretion collections and of blood samples. 2. Close to 100% of the intake was recovered in faeces during the first 7 days. Gastro-intestinal uptake, determined by comparing urinary excretion with patterns previously established following intravenous administration of 26Al, averaged 0.22% in the two subjects. 3. Uptake fractions based on comparisons of blood concentration following ingestion and injection were much lower, but were judged to be unreliable. It is concluded that aluminium present in most water supplies is unlikely to contribute as much as 1% of a typical daily uptake of 10 microg from food.

Kinetics of uptake and elimination of silicic acid by a human subject: a novel application of 32Si and accelerator mass spectrometry.

Silicon is possibly important in human physiology in protecting against the toxic effects of aluminium, but the kinetics of uptake and excretion of silicic acid, the bioavailable form, are not well characterised. We have used 32Si as a tracer in a human uptake experiment to determine a gastrointestinal uptake factor for silicic acid, and to elucidate the kinetics of renal elimination. Urine collections were made for extending intervals from 2 to 12 h over 2 days following ingestion by a single human subject of a neutral silicic acid solution containing tracer levels of 32Si (t1/2 approximately 150 y). Silicon was isolated as SiO2 and the 32Si content determined by accelerator mass spectrometry (AMS), using a gas-filled magnet technique to eliminate a prolific isobaric interference from 32S. Silicon uptake appears to have been essentially complete within 2 h of ingestion. Elimination occurred by two simultaneous first-order processes with half-lives of 2.7 and 11.3 h, representing around 90% and 10%, respectively, of the total output. The rapidly eliminated 32Si was probably retained in the extracellular fluid volume, whilst the slower component may represent intracellular uptake and release. Elimination of absorbed 32Si was essentially complete after 48 h and was equivalent to 36% of the ingested dose. This establishes only a lower limit for gastrointestinal absorption as, although there was no evidence for longer term retention of additional 32Si, the possibility could not be excluded by these results.

Lead concentrations and isotope ratios in street dust determined by electrothermal atomic absorption spectrometry and inductively coupled plasma mass spectrometry.

A major source of environmental lead, particularly in urban areas, has been from the combustion of leaded petrol. Street dust has previously been used to assess urban lead contamination, and the dust itself can also be a potential source of lead ingestion, particularly to children. The progressive reduction of lead in petrol, in recent years, would be expected to have been reflected in a reduction of lead in urban dust. We have tested this hypothesis by repeating an earlier survey of Manchester street dust and carrying out a comparable survey in Paris. Samples were collected from streets and parks, lead was extracted by digestion with concentrated nitric acid and determined by electrothermal atomic absorption spectrometry. Lead isotope ratios were measured by inductively coupled plasma mass spectrometry. Results for Manchester show that lead concentrations have fallen by about 40% (street dust averages, 941 micrograms g-1 (ppm) in 1975 down to 569 ppm in 1997). In Paris, the lead levels in street dust are much higher and significant differences were observed between types of street (not seen in Manchester). Additionally, lead levels in parks were much lower than in Manchester. Samples collected under the Eiffel Tower had very high concentrations and lead isotope ratios showed that this was unlikely to be fallout from motor vehicles but could be due to the paint used on the tower. Isotope ratios measurements also revealed that lead additives used in France and the UK come from different sources.

Induction of chromosome aberrations and delayed genomic instability by photochemical processes.

Exponentially growing cells cultured in medium containing bromodeoxyuridine, then exposed to UVA light in the presence of the dye Hoechst 33258, show significant levels of DNA strand breaks and base damage. This dye-bromodeoxyuridine-UVA photolysis treatment is markedly cytotoxic. We now demonstrate that exposure of cells to the agents used in photolysis leads directly to the formation of chromosome aberrations. Furthermore, we demonstrate that this photochemical treatment induces delayed chromosomal instability in clonal populations derived from single progenitor cells surviving photolysis. These results suggest that photolysis-induced DNA damage leads to chromosome rearrangements that could account for the observed cytotoxicity. Furthermore, in those cells surviving photolysis, the delayed effects of this treatment can be observed several generations after exposure and are manifested as compromised genomic integrity.

A new protocol for the measurement of picomole quantities of magnesium in rat renal tubular fluid.

The analysis of picomolar quantities of magnesium by electrothermal atomic absorption spectrophotometry (EAAS) was studied using a Perkin-Elmer-Zeeman 3030 spectrophotometer. The absorbance signal was not heavily dependent on the atomization temperature, but was greatly reduced when ashing temperatures in excess of 1200 degrees C were applied. The magnesium signal was significantly depressed in the presence of excess chloride in the sample matrix. However, use of NH4NO3 as a matrix modifier was sufficient to overcome this artefact. The analytical sensitivity was 0.15 absorbance units pmol-1 and the detection limit was 0.04 pmol. Using nanolitre constriction pipettes to dispense standards, the mean coefficient of variation was 5%. Measurement of magnesium handling in the rat proximal convoluted tubule revealed a significant correlation between the tubular fluid-to-plasma ultrafiltrate (TF/UF) concentration ratio for magnesium and the tubular fluid-to-plasma (TF/P) concentration ratio for [3H]inulin (r2 = 0.56, n = 17). This indicated that magnesium is concentrated during its passage along the proximal tubule. In contrast, this was not the case for sodium (r2 = 0.11, n = 16). Mean (TF/UF)Mg (1.16 +/- 0.07, n = 17) for random punctures was significantly greater than that for sodium ((TF/UF)Na = 1.02 +/- 0.02, n = 16). Despite concentration of magnesium in the lumen, significant net reabsorption of magnesium was observed along the length of the tubule (fractional reabsorption, FRMg = 19.4 +/- 3.0%, n = 17). In conclusion, EAAS provides a highly sensitive, reproducible and technically simple method for measuring picomolar quantities of magnesium in renal tubular fluid.

Recombination involving interstitial telomere repeat-like sequences promotes chromosomal instability in Chinese hamster cells.

The physical termini of mammalian chromosomes are capped with tandem repeats of the telomere sequence (TTAGGG)n. After fluorescence in situ hybridization with a labeled (TTAGGG)n probe, telomere-repeat-like sequences are seen as discrete bands at distinct intrachromosomal sites in a variety of vertebrate species. There is increasing evidence that these sites may be hot-spots for chromosomal rearrangements, fragility and neoplasia. We have investigated whether the interstitial telomere bands found in hamster chromosomes from a human hamster hybrid cell line are hot-spots for chromosome rearrangements induced by DNA-damaging agents. Our data indicate that the interstitial telomere bands are involved in chromosomal rearrangements observed at the first mitosis after G1 exposure of cells to X-rays or restriction endonucleases at a four- to fivefold higher frequency than expected based on their size. In addition, we have extended these observations to demonstrate for the first time that these interstitial telomere-repeat-like sequences participate in the delayed chromosomal instability observed in the progeny of cells surviving X-ray-exposure at multiple generations after irradiation. In two highly unstable clones showing multiple populations of rearranged chromosomes, interstitial telomere bands were observed at the site of recombination between the human and hamster chromosomes at a five- to sixfold higher frequency than expected. There were also rearrangement and amplification of the interstitial telomere bands within the hamster chromosomes. These rearrangements occur during clonal expansion of cells surviving treatment with DNA-damaging agents and suggest a role for the interstitial telomere band in driving chromosomal instability. We conclude from the observed data that interstitial telomere bands function as recombinational hot-spots that participate in generating the diverse chromosome rearrangements observed both immediately and as a delayed effect of cellular exposure to DNA damaging agents.

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice.

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of 26Al and 67Ga, has been compared with that of gallium, a proposed tracer for aluminium. 26Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7 ml 100 g-1; renal cortex 102.9 ml 100 g-1; liver 13.0 ml 100 g-1; spleen 8.4 ml 100 g-1 and brain 0.8 ml 100 g-1. 67Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for 26Al, respectively. In the hypotransferrinaemic mice, uptake of 67Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. 26Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced 67Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on 26AI uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.