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931.
Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected α-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.Pathogens are detected by the host when antigenic molecules directly contact immune receptors during the early stages of infection. The strategy of intracellular infection allows viruses, certain bacteria and protozoa to partially cloak themselves from the immune response by physically encapsulating their antigens within host cells. Intracellular residence also takes advantage of immune tolerance mechanisms that prevent autoimmune destruction of self. T cells play a central role in immunity to intracellular pathogens because they can respond to antigens that are generated inside cells and then transported to the surface of infected cells after binding to antigen-presenting molecules. The antigen-presenting molecules encoded in the major histocompatibility complex are widely known for presenting peptide fragments of proteins (1). More recently, human and mouse members of the CD1 (cluster of differentiation 1) system have been shown to present small amphipathic molecules, including a variety of membrane lipids, glycolipids, and lipopeptides, greatly expanding the molecular structures recognized by the cellular immune system (2, 3).Among human CD1 proteins (CD1a, CD1b, CD1c, CD1d, and CD1e), each CD1 isoform is expressed on a different spectrum of antigen-presenting cells. Human CD1a proteins are distinguished from other CD1 proteins by high expression levels on the surface of intradermal Langerhans cells, which play a role in barrier immune function (4). Human T cell clones have been shown to directly recognize CD1a proteins in the presence of exogenous foreign antigens (5) or in the presence of sulfatide and other self lipids (6, 7), suggesting a role for CD1a in T cell activation. In addition, mycobacteria and other intracellular pathogens have been shown to increase CD1a expression in lesions found in leprosy and tuberculosis patients, implying a possible role for CD1a in the response to infection, especially at mucosal or skin sites (810). Analysis of the molecular target recognized by CD1a-restricted T cell clone (CD8-2) allowed the identification of a foreign antigen presented by CD1a as dideoxymycobactin (DDM) (11).2Mycobactin binds iron to promote Mycobacterium tuberculosis survival. DDM was initially isolated (11) from antigenic lipid extracts of M. tuberculosis, a pathogen that kills ∼1.7 million humans annually on a worldwide basis (12). The determination of DDM structure was based on mass spectrometric and NMR studies of limiting amounts of natural material derived from the pathogenic organisms, so that not all elements of its chemical structure could be formally determined. Instead, its assigned structure was facilitated by obvious parallels of dideoxymycobactin with mycobactin, a lipopeptide siderophore (13, 14). Iron is required for reduction-oxidation reactions involving respiration and other basic metabolic pathways in bacterial pathogens (13). Environmental mycobacteria have at least two iron uptake pathways, but mycobactin and the related molecule carboxymycobactin represent the only known dedicated iron uptake pathway for pathogenic species like M. tuberculosis (15, 16). Highlighting the physiological importance of the mycobactin pathway, deletion of mycobactin synthase B limits M. tuberculosis survival in cells (13, 14). Also, mammalian innate immune systems produce siderocalin, a 20-kDa lipocalin that binds both ferric and apo siderophores, preventing their uptake and subsequent iron delivery to microbes (1720). The small available yields of natural material highlighted the need for a straightforward method to synthesize DDM for studies of its role in mycobacterial iron acquisition and testing T cell responses in human populations, as well as to provide authentic standards to investigate unknown aspects of natural DDM stereochemistry. Here we report two syntheses for production of DDM in solution phase and solid phase. Comparison of synthetic and natural DDMs gives unexpected insight into the stereochemical structures of the methylserine, oxazoline, and butyrate moieties of DDM and provides direct evidence that the T cell response is highly specific for a unique aspect of DDM structure that protrudes from the surface of the CD1a-DDM complexes.  相似文献   
932.
933.
934.
Parkin is an ubiquitin‐protein ligase (E3), mutations of which cause juvenile onset – autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2‐DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (±2‐fold change; p<0.05) using 2‐DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2‐DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.  相似文献   
935.
Ralstonia eutropha H16 is an H2‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H2 and CO2, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H2‐oxidizing and CO2‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H2‐CO2‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.  相似文献   
936.
Multicellular magnetotactic prokaryotes (MMPs) represent highly organized, spherical and motile aggregates of 10–40 bacterial cells containing magnetosomes. Although consisting of different cells, each with its own magnetosomes and flagellation, MMPs orient themselves within a magnetic field and exhibit magnetotaxis. So far, MMPs have only been found in several North and South American coastal lagoons and salt marshes. In the present study, a novel type of MMP was discovered in coastal tidal sand flats of the North Sea. High‐resolution scanning electron microscopy revealed the presence of bullet‐shaped magnetosomes which were aligned in several parallel chains. Within each aggregate, the magnetosome chains of individual cells were oriented in the same direction. Energy dispersive X‐ray (EDX) analysis showed that the magnetosomes are composed of iron sulfide. This particular morphology and arrangement of magnetosomes has previously not been reported for other MMPs. 16S rRNA gene sequence analysis revealed a single phylotype which represented a novel phylogenetic lineage with ≥ 4% sequence divergence to all previously described MMP sequences and was related to the dissimilatory sulfate‐reducing Desulfosarcina variabilis within the family Desulfobacteraceae of the subphylum Deltaproteobacteria. Fluorescence in situ hybridization with a specific oligonucleotide probe revealed that all MMPs in the tidal flat sediments studied belonged to the novel phylotype. Within each MMP, all bacterial cells showed a hybridization signal, indicating that the aggregates are composed of cells of the same phylotype. Genes for dissimilatory sulfite reductase (dsrAB) and dissimilatory adenosine‐5′‐phosphate reductase (aprA) could be detected in purified MMP samples, suggesting that MMPs are capable of sulfate reduction. Chemotaxis assays with 41 different test compounds yielded strong responses towards acetate and propionate, whereas other organic acids, alcohols, sugars, sugar alcohols or sulfide did not elicit any response. By means of its coordinated magnetotaxis and chemotaxis, the novel type of MMP is well adapted to the steep chemical gradients which are characteristic for intertidal marine sediments.  相似文献   
937.

Background

Phthalates are synthetic compounds with a widespread field of applications. For example, they are used as plasticizers in PVC plastics and food packaging, or are added to personal care products. Diethyl phthalate (DEP) may be used to denature alcohol, e.g., for cosmetic purposes. Public health concerns of phthalates include carcinogenic, teratogenic, hepatotoxic and endocrine effects. The aim of this study was to develop and validate a method for determining phthalates in alcohol samples and to provide a risk assessment for consumers of such products.

Methodology/Principal Findings

A liquid-liquid extraction procedure was optimized by varying the following parameters: type of extraction solvent (cyclohexane, n-hexane, 1,1,2-trichlorotrifluoroethane), the ratio extraction solvent/sample volume (1∶1 to 50∶1) and the number of extraction repetitions (1–10). The best extraction yield (99.9%) was achieved with the solvent 1,1,2-trichlorotrifluoroethane, an extraction solvent volume/sample volume ratio of 10∶1 and a double extraction. For quantification, gas chromatography/mass spectrometry with deuterated internal standards was used. The investigated samples were alcoholic beverages and unrecorded alcohol products from different countries (n = 257). Two unrecorded alcohol samples from Lithuania contained diethyl phthalate in concentrations of 608 mg/L and 210 mg/L.

Conclusions/Significance

The consumption of the phthalate-positive unrecorded alcohols would exceed tolerable daily intakes as derived from animal experiments. Both positive samples were labelled as cosmetic alcohol, but had clearly been offered for human consumption. DEP seems to be unsuitable as a denaturing agent as it has no effect on the organoleptic properties of ethanol. In light of our results that DEP might be consumed by humans in unrecorded alcohols, the prohibition of its use as a denaturing agent should be considered.  相似文献   
938.
In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.  相似文献   
939.
940.
The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.  相似文献   
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