Structural characterization of aspartate-semialdehyde dehydrogenase from Pseudomonas aeruginosa and Neisseria gonorrhoeae.

Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent.

A bimodal burst energy distribution of a repeating fast radio burst source.

The event rate, energy distribution and time-domain behaviour of repeating fast radio bursts (FRBs) contain essential information regarding their physical nature and central engine, which are as yet unknown1,2. As the first precisely localized source, FRB 121102 (refs. 3-5) has been extensively observed and shows non-Poisson clustering of bursts over time and a power-law energy distribution6-8. However, the extent of the energy distribution towards the fainter end was not known. Here we report the detection of 1,652 independent bursts with a peak burst rate of 122 h-1, in 59.5 hours spanning 47 days. A peak in the isotropic equivalent energy distribution is found to be approximately 4.8 × 1037 erg at 1.25 GHz, below which the detection of bursts is suppressed. The burst energy distribution is bimodal, and well characterized by a combination of a log-normal function and a generalized Cauchy function. The large number of bursts in hour-long spans allows sensitive periodicity searches between 1 ms and 1,000 s. The non-detection of any periodicity or quasi-periodicity poses challenges for models involving a single rotating compact object. The high burst rate also implies that FRBs must be generated with a high radiative efficiency, disfavouring emission mechanisms with large energy requirements or contrived triggering conditions.

Structural characterization of a GNAT family acetyltransferase from Elizabethkingia anophelis bound to acetyl-CoA reveals a new dimeric interface.

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine ß-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure-activity relationship studies.

Structure of tyrosine aminotransferase from Leishmania infantum.

The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding L-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 Å resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis.

A millisecond pulsar in a stellar triple system.

Gravitationally bound three-body systems have been studied for hundreds of years and are common in our Galaxy. They show complex orbital interactions, which can constrain the compositions, masses and interior structures of the bodies and test theories of gravity, if sufficiently precise measurements are available. A triple system containing a radio pulsar could provide such measurements, but the only previously known such system, PSR B1620-26 (refs 7, 8; with a millisecond pulsar, a white dwarf, and a planetary-mass object in an orbit of several decades), shows only weak interactions. Here we report precision timing and multiwavelength observations of PSR J0337+1715, a millisecond pulsar in a hierarchical triple system with two other stars. Strong gravitational interactions are apparent and provide the masses of the pulsar M[Symbol: see text](1.4378(13), where M[Symbol: see text]is the solar mass and the parentheses contain the uncertainty in the final decimal places) and the two white dwarf companions (0.19751(15)M[Symbol: see text] and 0.4101(3))M[Symbol: see text], as well as the inclinations of the orbits (both about 39.2°). The unexpectedly coplanar and nearly circular orbits indicate a complex and exotic evolutionary past that differs from those of known stellar systems. The gravitational field of the outer white dwarf strongly accelerates the inner binary containing the neutron star, and the system will thus provide an ideal laboratory in which to test the strong equivalence principle of general relativity.

Pulsar discovery by global volunteer computing.

Einstein@Home aggregates the computer power of hundreds of thousands of volunteers from 192 countries to mine large data sets. It has now found a 40.8-hertz isolated pulsar in radio survey data from the Arecibo Observatory taken in February 2007. Additional timing observations indicate that this pulsar is likely a disrupted recycled pulsar. PSR J2007+2722's pulse profile is remarkably wide with emission over almost the entire spin period; the pulsar likely has closely aligned magnetic and spin axes. The massive computing power provided by volunteers should enable many more such discoveries.

A bright millisecond radio burst of extragalactic origin.

Pulsar surveys offer a rare opportunity to monitor the radio sky for impulsive burst-like events with millisecond durations. We analyzed archival survey data and found a 30-jansky dispersed burst, less than 5 milliseconds in duration, located 3 degrees from the Small Magellanic Cloud. The burst properties argue against a physical association with our Galaxy or the Small Magellanic Cloud. Current models for the free electron content in the universe imply that the burst is less than 1 gigaparsec distant. No further bursts were seen in 90 hours of additional observations, which implies that it was a singular event such as a supernova or coalescence of relativistic objects. Hundreds of similar events could occur every day and, if detected, could serve as cosmological probes.

Tests of general relativity from timing the double pulsar.

The double pulsar system PSR J0737-3039A/B is unique in that both neutron stars are detectable as radio pulsars. They are also known to have much higher mean orbital velocities and accelerations than those of other binary pulsars. The system is therefore a good candidate for testing Einstein's theory of general relativity and alternative theories of gravity in the strong-field regime. We report on precision timing observations taken over the 2.5 years since its discovery and present four independent strong-field tests of general relativity. These tests use the theory-independent mass ratio of the two stars. By measuring relativistic corrections to the Keplerian description of the orbital motion, we find that the "post-Keplerian" parameter s agrees with the value predicted by general relativity within an uncertainty of 0.05%, the most precise test yet obtained. We also show that the transverse velocity of the system's center of mass is extremely small. Combined with the system's location near the Sun, this result suggests that future tests of gravitational theories with the double pulsar will supersede the best current solar system tests. It also implies that the second-born pulsar may not have formed through the core collapse of a helium star, as is usually assumed.

A periodically active pulsar giving insight into magnetospheric physics.

PSR B1931+24 (J1933+2421) behaves as an ordinary isolated radio pulsar during active phases that are 5 to 10 days long. However, when the radio emission ceases, it switches off in less than 10 seconds and remains undetectable for the next 25 to 35 days, then switches on again. This pattern repeats quasi-periodically. The origin of this behavior is unclear. Even more remarkably, the pulsar rotation slows down 50% faster when it is on than when it is off. This indicates a massive increase in magnetospheric currents when the pulsar switches on, proving that pulsar wind plays a substantial role in pulsar spin-down. This allows us, for the first time, to estimate the magnetospheric currents in a pulsar magnetosphere during the occurrence of radio emission.

Transient radio bursts from rotating neutron stars.

The radio sky is relatively unexplored for transient signals, although the potential of radio-transient searches is high. This was demonstrated recently by the discovery of a previously unknown type of source, varying on timescales of minutes to hours. Here we report a search for radio sources that vary on much shorter timescales. We found eleven objects characterized by single, dispersed bursts having durations between 2 and 30 ms. The average time intervals between bursts range from 4 min to 3 h with radio emission typically detectable for <1 s per day. From an analysis of the burst arrival times, we have identified periodicities in the range 0.4-7 s for ten of the eleven sources, suggesting origins in rotating neutron stars. Despite the small number of sources detected at present, their ephemeral nature implies a total Galactic population significantly exceeding that of the regularly pulsing radio pulsars. Five of the ten sources have periods >4 s, and the rate of change of the pulse period has been measured for three of them; for one source, we have inferred a high magnetic field strength of 5 x 10(13) G. This suggests that the new population is related to other classes of isolated neutron stars observed at X-ray and gamma-ray wavelengths.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

A double-pulsar system: a rare laboratory for relativistic gravity and plasma physics.

The clocklike properties of pulsars moving in the gravitational fields of their unseen neutron-star companions have allowed unique tests of general relativity and provided evidence for gravitational radiation. We report here the detection of the 2.8-second pulsar J0737-3039B as the companion to the 23-millisecond pulsar J0737-3039A in a highly relativistic double neutron star system, allowing unprecedented tests of fundamental gravitational physics. We observed a short eclipse of J0737-3039A by J0737-3039B and orbital modulation of the flux density and the pulse shape of J0737-3039B, probably because of the influence of J0737-3039A's energy flux on its magnetosphere. These effects will allow us to probe magneto-ionic properties of a pulsar magnetosphere.

An increased estimate of the merger rate of double neutron stars from observations of a highly relativistic system.

The merger of close binary systems containing two neutron stars should produce a burst of gravitational waves, as predicted by the theory of general relativity. A reliable estimate of the double-neutron-star merger rate in the Galaxy is crucial in order to predict whether current gravity wave detectors will be successful in detecting such bursts. Present estimates of this rate are rather low, because we know of only a few double-neutron-star binaries with merger times less than the age of the Universe. Here we report the discovery of a 22-ms pulsar, PSR J0737-3039, which is a member of a highly relativistic double-neutron-star binary with an orbital period of 2.4 hours. This system will merge in about 85 Myr, a time much shorter than for any other known neutron-star binary. Together with the relatively low radio luminosity of PSR J0737-3039, this timescale implies an order-of-magnitude increase in the predicted merger rate for double-neutron-star systems in our Galaxy (and in the rest of the Universe).

In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR).

Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progression to androgen independence usually results in relapse within 2 years. The molecular mechanisms underlying the clinically important transition from androgen dependence to androgen independence are poorly described. Several lines of investigation have suggested that insulin-like growth factors (IGFs) are involved in the biology of prostate cancer, but little is known about their relevance to progression to androgen independence. We used three in vivo models of androgen-dependent (AD) human prostate cancer to study this issue. Progression to androgen-independent (AI) growth was associated with a 60-fold increase in expression of IGF-I mRNA in LAPC-9 xenografts and a 28-fold increase in IGF-I expression in LNCAP xenografts, relative to the initial AD neoplasms. IGF type I receptor (IGF-IR) mRNA levels were approximately 2.5-fold and approximately 5-fold higher, respectively, in AI LAPC-9 and LNCaP tumors compared with the original AD neoplasms. AI growth of these xenografts was also associated with significant reductions in IGF binding protein-3 expression. LAPC-4 xenografts, which previously have been shown to exhibit molecular pathology related to HER-2/neu expression with progression to AI, showed relatively minor changes in expression of the genes investigated, but we nevertheless found evidence of increased IGF-IR phosphorylation with progression to androgen independence in this model. Taken together with prior observations, our results suggest that deregulation of expression of genes related to any one of several critical receptor tyrosine kinase regulatory systems, including IGF signaling, may confer androgen independence.

Cloning and characterization of a novel human histone deacetylase, HDAC8.

Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation [Brehm, Miska, McCance, Reid, Bannister and Kouzarides (1998) Nature (London) 391, 597-600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia [Grignani, De Matteis, Nervi, Tomassoni, Gelmetti, Cioce, Fanelli, Ruthardt, Ferrara, Zamir et al. (1998) Nature (London) 391, 815-818; Lin, Nagy, Inoue, Shao, Miller and Evans (1998), Nature (London) 391, 811-814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8 product contains a shorter C-terminal extension relative to other members of its class. After expression in two cell systems, immunopurified HDAC8 is shown to possess trichostatin A- and sodium butyrate-inhibitable HDAC activity on histone H4 peptide substrates as well as on core histones. Expression profiling reveals the expression of HDAC8 to various degrees in every tissue tested and also in several tumour lines. Mutation of two adjacent histidine residues within the predicted active site severely decreases activity, confirming these residues as important for HDAC8 enzyme activity. Finally, linkage analysis after radiation hybrid mapping has localized HDAC8 to chromosomal position Xq21.2-Xq21.3. These results confirm HDAC8 as a new member of the HDAC family.

Binary and Millisecond Pulsars.

Our knowledge of binary and millisecond pulsars has greatly increased in recent years. This is largely due to the success of large-area surveys which have brought the known population of such systems in the Galactic disk to around 50. As well as being interesting as a population of astronomical sources, many pulsars turn out to be superb celestial clocks. In this review we summarise the main properties of binary and millisecond pulsars and highlight some of their applications to relativistic astrophysics. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.12942/lrr-1998-10.

Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability.

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cells expressing mutant MLC20. These data demonstrate that MLC20 phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.

Location and characterization of the human GRP receptor expressed by gastrointestinal epithelial cells.

The exact location of normal gastrin-releasing peptide (GRP) receptor expression by epithelial cells lining the human gastrointestinal (GI) tract is not known; yet this receptor is found on upwards of 50% of GI cancers. Furthermore, the pharmacology reported for GRP receptors expressed by GI cancers varies considerably. Therefore, the purpose of this study was to determine the normal distribution of GRP receptor expression by cells lining the human GI tract, and then determine the normal pharmacology of the human receptor when ectopically expressed by the nonmalignant human colon epithelial cell line NCM460. We obtained endoscopic pinch biopsies of, and extracted the RNA from, epithelial cells lining the esophagus, stomach, jejunum, ileum, and proximal and descending colon, RT-PCR demonstrated that GRP-R expression is limited to cells lining the gastric antrum, indicating that this receptor is aberrantly expressed by GI cancers. To determine the normal pharmacology of this receptor when expressed by nonmalignant human tissues for the first time, we transfected NCM460 cells with the cDNA for the human GRP receptor. By studying three stable NCM460 cell lines expressing varying numbers of receptors, we demonstrate that agonist and antagonist binding affinity, binding kinetics, and G-protein coupling are all independent of receptor number. Finally, by comparing GRP receptors expressed by GI cancers with those on NCM460-transfected cells, we show that the pharmacology of the aberrantly expressed receptors is significantly altered. Thus, these data demonstrate that GI cancers aberrantly express GRP receptors that then behave abnormally.

Cloning, chromosomal location, and transcriptional regulation of the human galanin-1 receptor gene (GALN1R).

Galanin is a neuroendocrine peptide that modulates many different normal physiological effects including memory, weight, and pain perception. To better understand galanin receptor function, we cloned and characterized the galanin-1 receptor (GALN1R) gene isolated from a human P1 library. We determined that this gene contains 2 introns of approximately 2.1 and 2.9 Kb, both of which are located in the 3rd intracellular loop. We identified transcriptional initiation sites 61 and 63 bp upstream of the translation initiation codon ATG. Sequencing of the GALN1R gene 5' flanking region revealed it to be GC rich and devoid of TATA or CCAAT boxes, features of housekeeping genes. To identify potential sites regulating promoter activity, variable lengths of the 5' flanking region were fused to a CAT gene and studied in Bowes human melanoma cells. Significant losses in CAT activity were observed only with the elimination of 2 NF-kappa B sites, located -269 and -809 bp upstream from the translational start site, respectively. These findings suggest the novel possibility that GALN1R gene expression may be regulated as a consequence of inflammatory conditions. Finally fluorescent in situ hybridization (FISH) assigned this gene to chromosome 15q24.