The x-ray structure of dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase

The repeating unit of the glycan chain in the S-layer of the bacterium Aneurinibacillus thermoaerophilus L420-91(T) is composed of four alpha-d-rhamnose molecules and two 3-acetamido-3,6-dideoxy-alpha-d-galactose moieties (abbreviated as Fucp3NAc). Formation of the glycan layer requires nucleotide-activated sugars as the donor molecules. Whereas the enzymes involved in the synthesis of GDP-rhamnose have been well characterized, less is known regarding the structures and enzymatic mechanisms of the enzymes required for the production of dTDP-Fucp3NAc. One of the enzymes involved in the biosynthesis of dTDP-Fucp3NAc is a 3,4-ketoisomerase, hereafter referred to as FdtA. Here we describe the first three-dimensional structure of this sugar isomerase complexed with dTDP and solved to 1.5 A resolution. The FdtA dimer assumes an almost jellyfish-like appearance with the sole alpha-helices representing the tentacles. Formation of the FdtA dimer represents a classical example of domain swapping whereby beta-strands 2 and 3 from one subunit form part of a beta-sheet in the second subunit. The active site architecture of FdtA is characterized by a cluster of three histidine residues, two of which, His(49) and His(51), appear to be strictly conserved in the amino acid sequences deposited to date. Site-directed mutagenesis experiments, enzymatic assays, and x-ray crystallographic analyses suggest that His(49) functions as an active site base.     

Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required for the production of dTDP-desosamine. The focus of this X-ray crystallographic analysis is the third enzyme in the pathway, a PLP-dependent aminotransferase referred to as DesI. The structure of DesI was solved in complex with its product, dTDP-4-amino-4,6-dideoxyglucose, to a nominal resolution of 2.1 A. Each subunit of the dimeric enzyme contains 12 alpha-helices and 14 beta-strands. Three cis-peptides are observed in each subunit, Phe 330, Pro 332, and Pro 339. The two active sites of the enzyme are located in clefts at the subunit/subunit interface. Electron density corresponding to the bound product clearly demonstrates a covalent bond between the amino group of the product and C-4' of the PLP cofactor. Interestingly, there are no hydrogen-bonding interactions between the protein and the dideoxyglucosyl group of the product (within 3.2 A). The only other sugar-modifying aminotransferase whose structure is known in the presence of product is PseC from Helicobacter pylori. This enzyme, as opposed to DesI, catalyzes amino transfer to the axial position of the sugar. A superposition of the two active sites for these proteins reveals that the major differences in ligand binding occur in the orientations of the deoxyglucosyl and phosphoryl groups. Indeed, the nearly 180 degrees difference in hexose orientation explains the equatorial versus axial amino transfer exhibited by DesI and PseC, respectively.     

Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.     

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.     

The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis

An essential component of normal development is controlling the transition from cell proliferation to differentiation. One such transition occurs during Drosophila oogenesis. In early oogenesis, germ cells undergo mitotic proliferation and contain a specialized organelle called a fusome, whereas later post-mitotic cells differentiate and lose the fusome as F-actin-rich ring canals form. The hts gene encodes the only Drosophila Adducin, and is a female-sterile mutant that affects both the fusome and ring canals. We show that one Hts protein, Ovhts, is a polyprotein that is cleaved to produce two products, Ovhts-Fus and Ovhts-RC. Whereas Ovhts-Fus localizes to the fusome in mitotic cells, Ovhts-RC localizes to ring canals throughout later oogenesis. We demonstrate that an uncleavable version of Ovhts delays the transition from fusome-containing cells to those that have ring canals. Ovhts is the first polyprotein shown to produce proteins that function in separate structures.     

New tools suggest local variation in tool use by a montane community of the rare Nigeria–Cameroon chimpanzee, <Emphasis Type="Italic">Pan troglodytes ellioti</Emphasis>, in Nigeria

Regional variations in tool use among chimpanzee subspecies and between populations within the same subspecies can often be explained by ecological constraints, although cultural variation also occurs. In this study we provide data on tool use by a small, recently isolated population of the endangered Nigeria–Cameroon chimpanzee Pan troglodytes ellioti, thus demonstrating regional variation in tool use in this rarely studied subspecies. We found that the Ngel Nyaki chimpanzee community has its own unique tool kit consisting of five different tool types. We describe a tool type that has rarely been observed (ant-digging stick) and a tool type that has never been recorded for this chimpanzee subspecies or in West Central Africa (food pound/grate stone). Our results suggest that there is fine- scale variation in tool use among geographically close communities of P. t. ellioti, and that these variations likely reflect both ecological constraints and cultural variation.     

Effectiveness of interventions designed to reduce the use of imaging for low-back pain: a systematic review

Background:Rates of imaging for low-back pain are high and are associated with increased health care costs and radiation exposure as well as potentially poorer patient outcomes. We conducted a systematic review to investigate the effectiveness of interventions aimed at reducing the use of imaging for low-back pain.Methods:We searched MEDLINE, Embase, CINAHL and the Cochrane Central Register of Controlled Trials from the earliest records to June 23, 2014. We included randomized controlled trials, controlled clinical trials and interrupted time series studies that assessed interventions designed to reduce the use of imaging in any clinical setting, including primary, emergency and specialist care. Two independent reviewers extracted data and assessed risk of bias. We used raw data on imaging rates to calculate summary statistics. Study heterogeneity prevented meta-analysis.Results:A total of 8500 records were identified through the literature search. Of the 54 potentially eligible studies reviewed in full, 7 were included in our review. Clinical decision support involving a modified referral form in a hospital setting reduced imaging by 36.8% (95% confidence interval [CI] 33.2% to 40.5%). Targeted reminders to primary care physicians of appropriate indications for imaging reduced referrals for imaging by 22.5% (95% CI 8.4% to 36.8%). Interventions that used practitioner audits and feedback, practitioner education or guideline dissemination did not significantly reduce imaging rates. Lack of power within some of the included studies resulted in lack of statistical significance despite potentially clinically important effects.Interpretation:Clinical decision support in a hospital setting and targeted reminders to primary care doctors were effective interventions in reducing the use of imaging for low-back pain. These are potentially low-cost interventions that would substantially decrease medical expenditures associated with the management of low-back pain.Current evidence-based clinical practice guidelines recommend against the routine use of imaging in patients presenting with low-back pain.^1–3 Despite this, imaging rates remain high,^4,5 which indicates poor concordance with these guidelines.^6,7Unnecessary imaging for low-back pain has been associated with poorer patient outcomes, increased radiation exposure and higher health care costs.⁸ No short- or long-term clinical benefits have been shown with routine imaging of the low back, and the diagnostic value of incidental imaging findings remains uncertain.^9–12 A 2008 systematic review found that imaging accounted for 7% of direct costs associated with low-back pain, which in 1998 translated to more than US$6 billion in the United States and £114 million in the United Kingdom.¹³ Current costs are likely to be substantially higher, with an estimated 65% increase in spine-related expenditures between 1997 and 2005.¹⁴Various interventions have been tried for reducing imaging rates among people with low-back pain. These include strategies targeted at the practitioner such as guideline dissemination,^15–17 education workshops,^18,19 audit and feedback of imaging use,^7,20,21 ongoing reminders⁷ and clinical decision support.^22–24 It is unclear which, if any, of these strategies are effective.²⁵ We conducted a systematic review to investigate the effectiveness of interventions designed to reduce imaging rates for the management of low-back pain.     

The impact of maternal infection with Mycobacterium tuberculosis on the infant response to bacille Calmette–Guérin immunization

Bacille Calmette–Guérin (BCG) immunization provides variable protection against tuberculosis. Prenatal antigen exposure may have lifelong effects on responses to related antigens and pathogens. We therefore hypothesized that maternal latent Mycobacterium tuberculosis infection (LTBI) influences infant responses to BCG immunization at birth. We measured antibody (n = 53) and cellular (n = 31) responses to M. tuberculosis purified protein derivative (PPD) in infants of mothers with and without LTBI, in cord blood and at one and six weeks after BCG. The concentrations of PPD-specific antibodies declined between birth (median [interquartile range (IQR)]) 5600 ng ml⁻¹ [3300–11 050] in cord blood) and six weeks (0.00 ng ml⁻¹ [0–288]). Frequencies of PPD-specific IFN-γ-expressing CD4⁺T cells increased at one week and declined between one and six weeks (p = 0.031). Frequencies of IL-2- and TNF-α-expressing PPD-specific CD4⁺T cells increased between one and six weeks (p = 0.019, p = 0.009, respectively). At one week, the frequency of PPD-specific CD4⁺T cells expressing any of the three cytokines, combined, was lower among infants of mothers with LTBI, in crude analyses (p = 0.002) and after adjusting for confounders (mean difference, 95% CI −0.041% (−0.082, −0.001)). In conclusion, maternal LTBI was associated with lower infant anti-mycobacterial T-cell responses immediately following BCG immunization. These findings are being explored further in a larger study.