Lithium pollution of a white dwarf records the accretion of an extrasolar planetesimal.

Tidal disruption and subsequent accretion of planetesimals by white dwarfs can reveal the elemental abundances of rocky bodies in exoplanetary systems. Those abundances provide information on the composition of the nebula from which the systems formed, which is analogous to how meteorite abundances inform our understanding of the early Solar System. We report the detection of lithium, sodium, potassium, and calcium in the atmosphere of the white dwarf Gaia DR2 4353607450860305024, which we ascribe to the accretion of a planetesimal. Using model atmospheres, we determine abundance ratios of these elements, and, with the exception of lithium, they are consistent with meteoritic values in the Solar System. We compare the measured lithium abundance with measurements in old stars and with expectations from Big Bang nucleosynthesis.

K-edge imaging with the XPAD3 hybrid pixel detector, direct comparison of CdTe and Si sensors.

We investigate the improvement from the use of high-Z CdTe sensors for pre-clinical K-edge imaging with the hybrid pixel detectors XPAD3. We compare XPAD3 chips bump bonded to Si or CdTe sensors in identical experimental conditions. Image performance for narrow energy bin acquisitions and contrast-to-noise ratios of K-edge images are presented and compared. CdTe sensors achieve signal-to-noise ratios at least three times higher than Si sensors within narrow energy bins, thanks to their much higher detection efficiency. Nevertheless Si sensors provide better contrast-to-noise ratios in K-edge imaging when working at equivalent counting statistics, due to their better estimation of the attenuation coefficient of the contrast agent. Results are compared to simulated data in the case of the XPAD3/Si detector. Good agreement is observed when including charge sharing between pixels, which have a strong impact on contrast-to-noise ratios in K-edge images.

A wireless beta-microprobe based on pixelated silicon for in vivo brain studies in freely moving rats.

The investigation of neurophysiological mechanisms underlying the functional specificity of brain regions requires the development of technologies that are well adjusted to in vivo studies in small animals. An exciting challenge remains the combination of brain imaging and behavioural studies, which associates molecular processes of neuronal communications to their related actions. A pixelated intracerebral probe (PIXSIC) presents a novel strategy using a submillimetric probe for beta(+) radiotracer detection based on a pixelated silicon diode that can be stereotaxically implanted in the brain region of interest. This fully autonomous detection system permits time-resolved high sensitivity measurements of radiotracers with additional imaging features in freely moving rats. An application-specific integrated circuit (ASIC) allows for parallel signal processing of each pixel and enables the wireless operation. All components of the detector were tested and characterized. The beta(+) sensitivity of the system was determined with the probe dipped into radiotracer solutions. Monte Carlo simulations served to validate the experimental values and assess the contribution of gamma noise. Preliminary implantation tests on anaesthetized rats proved PIXSIC's functionality in brain tissue. High spatial resolution allows for the visualization of radiotracer concentration in different brain regions with high temporal resolution.

Imaging performance of the hybrid pixel detectors XPAD3-S.

Hybrid pixel detectors, originally developed for tracking particles in high-energy physics experiments, have recently been used in material sciences and macromolecular crystallography. Their capability to count single photons and to apply a threshold on the photon energy suggests that they could be optimal digital x-ray detectors in low energy beams such as for small animal computed tomography (CT). To investigate this issue, we have studied the imaging performance of photon counting hybrid pixel detectors based on the XPAD3-S chip. Two detectors are considered, connected either to a Si or to a CdTe sensor, the latter being of interest for its higher efficiency. Both a standard 'International Electrotechnical Commission' (IEC) mammography beam and a beam used for mouse CT results published in the literature are employed. The detector stability, linearity and noise are investigated as a function of the dose for several imaging exposures ( approximately 0.1-400 microGy). The perfect linearity of both detectors is confirmed, but an increase in internal noise for counting statistics higher than approximately 5000 photons has been found, corresponding to exposures above approximately 110 microGy and approximately 50 microGy for the Si and CdTe sensors, respectively. The noise power spectrum (NPS), the modulation transfer function (MTF) and the detective quantum efficiency (DQE) are then measured for two energy threshold configurations (5 keV and 18 keV) and three doses ( approximately 3, 30 and 300 microGy), in order to obtain a complete estimation of the detector performances. In general, the CdTe sensor shows a clear superiority with a maximal DQE(0) of approximately 1, thanks to its high efficiency ( approximately 100%). The DQE of the Si sensor is more dependent on the radiation quality, due to the energy dependence of its efficiency its maximum is approximately 0.4 with respect to the softer radiation. Finally, we compare the XPAD3-S DQE with published curves of other digital devices in a similar radiation condition. The XPAD3-S/CdTe detector appears to be the best with the highest DQE at low frequency, although some improvements are expected to reduce the increase of noise with the counts statistics and to guarantee a better stability of the detector response.

Application of a hybrid pixel detector to powder diffraction.

Results obtained using a hybrid pixel photon-counting detector in powder diffraction experiments are presented. The detector works at room temperature and its dynamic response ranges from 0.01 photons pixel(-1) s(-1) up to 10(6) photons pixel(-1) s(-1). The pixel sizes are 0.33 mm x 0.33 mm for a total area of 68 mm x 68 mm. On recording high-resolution diffraction patterns of powders, a reduction of the experimental time by more than a factor of 20 is obtained without loss of data quality. The example of an X-zeolite shows that such detectors can be used for very demanding anomalous experiments. In situ experiments of quenching liquid oxides show that frames of 0.01 s can be achieved for studying such processes.

A photometric redshift of z = 6.39 +/- 0.12 for GRB 050904.

Gamma-ray bursts (GRBs) and their afterglows are the most brilliant transient events in the Universe. Both the bursts themselves and their afterglows have been predicted to be visible out to redshifts of z approximately 20, and therefore to be powerful probes of the early Universe. The burst GRB 000131, at z = 4.50, was hitherto the most distant such event identified. Here we report the discovery of the bright near-infrared afterglow of GRB 050904 (ref. 4). From our measurements of the near-infrared afterglow, and our failure to detect the optical afterglow, we determine the photometric redshift of the burst to be z = 6.39 - 0.12 + 0.11 (refs 5-7). Subsequently, it was measured spectroscopically to be z = 6.29 +/- 0.01, in agreement with our photometric estimate. These results demonstrate that GRBs can be used to trace the star formation, metallicity, and reionization histories of the early Universe.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.

Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

Differential accumulation of DPTP61F alternative transcripts: regulation of a protein tyrosine phosphatase by segmentation genes.

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. We have examined the spatial and temporal expression of the two alternative transcripts of dptp61F during Drosophila embryogenesis. Our observations indicate that the two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression in the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation in the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes.

A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase.

The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.

Overproduction and one-step purification of Escherichia coli 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and oxygen transfer studies during catalysis using isotopic-shifted heteronuclear NMR.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase catalyzes the condensation of D-arabinose 5-phosphate with phosphoenolpyruvate to give the unique 8-carbon acidic sugar 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) found only in Gram-negative bacteria and required for lipid A maturation and cellular growth. The Escherichia coli gene kdsA that encodes KDO 8-P synthase has been amplified by polymerase chain reaction methodologies and subcloned into the expression vector, pT7-7. A simple one-step purification yields 200 mg of homogeneous KDO 8-P synthase per liter of cell culture. [2-13C,18O]Phosphoenolpyruvate (PEP) was prepared by first, exchange of [2-13C]-3-bromopyruvate with 2H2 18O followed by reaction of the labeled bromopyruvate with trimethylphosphite. The fate of the enolic oxygen in this multilabeled PEP, during the course of the KDO 8-P synthase-catalyzed reaction with D-arabinose 5-phosphate, was monitored by 13C and 31P NMR spectroscopy. The inorganic phosphate formed during the reaction was further analyzed via mass spectral analysis of its trimethyl ester derivative. The 13C NMR spectrum of an incubation mixture of [2-13C]PEP and D-arabinose 5-phosphate in 2H2 18O in the presence of KDO 8-P synthase was also recorded. [2-13C]KDO 8-P was utilized to determine the extent of nonenzymatic incorporation of 18O into the C-2 position of KDO 8-P. The results indicate that the enolic oxygen of the PEP is recovered with the inorganic phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent, H2O.

Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis.

The Ser/Thr phosphoprotein phosphatases (PPases) display similarities in amino acid sequence and biochemical properties. Most members of this family require transition metal ions for activity. The smallest family member, the bacteriophage lambda PPase (lambda-PPase), has been successfully overexpressed in Escherichia coli, purified, and characterized (Zhuo, S., Clemens, J.C., Hakes, D.J., Barford, D., and Dixon, J. E. (1993) J. Biol. Chem. 268, 17754-17761). Site-directed mutagenesis has now been employed to define amino acid residues in lambda-PPase required for metal ion binding and catalysis. Conservative amino acid substitutions at residues Asp20, His22, Asp49, His76, and Glu77 affected lambda-PPase catalysis and metal ion binding, whereas substitutions at residues Arg53 and Arg73 affected catalysis and substrate binding. Each of these residues is invariant in all phosphoprotein phosphatases, suggesting that these residues may play important roles in binding and catalysis in all of the PPases. Computer-assisted sequence alignment further revealed that lambda-PPase residues Asp20, His22, Asp49, His76, Arg53, and Arg73 lie within three larger regions of PPase sequence identity with the consensus sequence (DXH-(approximately 25)-GDXXD-(approximately 25)-GNHD/E). This motif can be found in a wide variety of phosphoesterases unrelated to the PPases and defines structural and catalytic features utilized by a diverse group of enzymes for the hydrolysis of phosphate esters.

Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase.

A protein phosphatase (PPase) from the bacteriophage lambda was overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity yielding approximately 17 mg of enzyme from a single liter of bacterial culture. Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an activator. The recombinant enzyme was active toward serine, threonine, and tyrosine phosphoproteins and phosphopeptides. Surprisingly, the bacterial histidyl phosphoprotein, NRII, was also dephosphorylated by the lambda-PPase. The lambda-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies. Crystallization of the recombinant purified lambda-PPase yielded monoclinic crystals. The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation.

The Yersinia tyrosine phosphatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage.

YopH is a plasmid-encoded protein tyrosine phosphatase (PTPase) secreted by pathogenic Yersinia. Although the enzyme likely acts to dephosphorylate eukaryotic proteins during Yersinia infection of the mammalian host, the targets of YopH have not been identified. We infected the murine macrophage-like cell line J774A.1 with Yersinia pseudotuberculosis and investigated the specificity of YopH and YopHC403A, a catalytically inactive mutant derivative, for eukaryotic phosphoproteins. Upon infection, YopH specifically and rapidly dephosphorylated a macrophage protein of 120 kD. The 120-kD protein and a previously detected 55-kD substrate of YopH coprecipitated with YopHC403A. Coprecipitation of these proteins required tyrosine phosphorylation and could be competitively inhibited with excess phosphotyrosine. The 120- and 55-kD proteins that coprecipitate with YopHC403A exhibited the in vitro activity of protein tyrosine kinases (PTKases), suggesting that YopH dephosphorylates activated tyrosine kinases in vivo.

Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase.

The Yersinia protein tyrosine phosphatase (PTPase) Yop51, a C235R point mutation (Yop51*), and a protein lacking the first 162 amino acids at the NH2 terminus (Yop51*delta 162) have been overexpressed in Escherichia coli and purified to homogeneity through the use of CM Sephadex C25 cation exchange chromatography followed by Sephadex G-100 gel filtration. Greater than 50 mg of homogeneous Yop51* and Yop51*delta 162 can be obtained from a single liter of bacterial culture, whereas the same procedure yields only 5 mg of pure Yop51. Large, diffraction-quality crystals have been obtained for Yop51*delta 162. Size exclusion chromatography, sedimentation equilibrium, and enzyme concentration dependence experiments have established that the Yersinia PTPases exist and function as monomers in solution. Yop51 and Yop51* display identical UV, CD, and fluorescence spectra and have identical kinetic and structural stability properties. These full-length Yersinia PTPases have 31% alpha-helix, an emission maximum of 342 nm, a turn-over number of 1200 s-1 at pH 5.0, 30 degrees C, and an unfolding delta G value of 6 kcal/mol at 25 degrees C. Yop51*delta 162 has very similar kinetic and fluorescence characteristics to the full-length molecules, whereas its CD and UV spectra show noticeable differences due to the elimination of 162 NH2-terminal residues. The Yersinia PTPases are by far the most active PTPases known, and their kinetic parameters are extremely sensitive to the ionic strength of reaction medium.

Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.

Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on Mössbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial pathogenesis and tyrosine dephosphorylation: surprising 'bedfellows'.

The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with the normal function of protein tyrosine phosphatase in eukaryotic cells.