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Sample records for falciparum m17 aminopeptidase

  1. Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

    PubMed Central

    McGowan, Sheena; Oellig, Christine A.; Birru, Woldeamanuel A.; Caradoc-Davies, Tom T.; Stack, Colin M.; Lowther, Jonathan; Skinner-Adams, Tina; Mucha, Artur; Kafarski, Pawel; Grembecka, Jolanta; Trenholme, Katharine R.; Buckle, Ashley M.; Gardiner, Donald L.; Dalton, John P.; Whisstock, James C.

    2010-01-01

    Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy. PMID:20133789

  2. Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions.

    PubMed

    Drinkwater, Nyssa; Vinh, Natalie B; Mistry, Shailesh N; Bamert, Rebecca S; Ruggeri, Chiara; Holleran, John P; Loganathan, Sasdekumar; Paiardini, Alessandro; Charman, Susan A; Powell, Andrew K; Avery, Vicky M; McGowan, Sheena; Scammells, Peter J

    2016-03-01

    Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. Plasmodium falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics. PMID:26807544

  3. Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

    PubMed

    Aboge, Gabriel Oluga; Cao, Shinuo; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Goo, Younkyoung; AbouLaila, Mahmoud; Nishikawa, Yoshifumi; Igarashi, Ikuo; Suzuki, Hiroshi; Xuan, Xuenan

    2015-10-01

    The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 μM, respectively, while the Ki was 2210 ± 358 μM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 μM] than B. gibsoni [IC50 = 460.8 ± 114.45 μM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this. PMID:26057618

  4. Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

    PubMed Central

    Lee, Yu-Ran; Na, Byoung-Kuk; Moon, Eun-Kyung; Song, Su-Min; Joo, So-Young; Kong, Hyun-Hee; Goo, Youn-Kyoung; Chung, Dong-Il; Hong, Yeonchul

    2015-01-01

    Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba. PMID:26075721

  5. Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

    PubMed Central

    McGowan, Sheena; Porter, Corrine J.; Lowther, Jonathan; Stack, Colin M.; Golding, Sarah J.; Skinner-Adams, Tina S.; Trenholme, Katharine R.; Teuscher, Franka; Donnelly, Sheila M.; Grembecka, Jolanta; Mucha, Artur; Kafarski, Pawel; DeGori, Ross; Buckle, Ashley M.; Gardiner, Donald L.; Whisstock, James C.; Dalton, John P.

    2009-01-01

    Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite. PMID:19196988

  6. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    PubMed

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  7. Modeling of human M1 aminopeptidases for in silico screening of potential Plasmodium falciparum alanine aminopeptidase (PfA-M1) specific inhibitors

    PubMed Central

    Sahi, Shakti; Rai, Sneha; Chaudhary, Meenakshi; Nain, Vikrant

    2014-01-01

    Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1. PMID:25258488

  8. Exploration of Sitagliptin as a potential inhibitor for the M1 Alanine aminopeptidase enzyme in Plasmodium falciparum using computational docking

    PubMed Central

    Krishnamoorthy, Mohana; Achary, Anant

    2013-01-01

    Plasmodium falciparum has limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin to maintain protein metabolism and synthesis of proteins. M1 alanine aminopeptidase enzyme of the parasite involved in the terminal degradation of host hemoglobin was subjected to in silico screening with low molecular weight protease inhibitors. The km (avg) of the enzyme M1 alanine aminopeptidase for the substrate DL – Alanine β Napthylamide Hydrochloride was estimated as 322.05µM. The molecular interactions between the enzyme and the substrate and the mechanism of enzyme action were analyzed which paved way for inhibition strategies. Among all the inhibitors screened, Sitagliptin was found to be most potent inhibitor with ki of 0.152 µM in its best orientation whereas the ki(avg) was 2.0055 µM. The ki of Sitagliptin is lower than the km of M1 alanine aminopeptidase for the substrate DL – Alanine β Napthylamide Hydrochloride (322.05 µM) and Ki of the known inhibitor Bestatin. Therefore Sitagliptin may serve as a potent competitive inhibitor of the enzyme M1 alanine aminopeptidase of Plasmodium falciparum. PMID:23559748

  9. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput Screening

    PubMed Central

    Spicer, Timothy; Fernandez-Vega, Virneliz; Chase, Peter; Scampavia, Louis; To, Joyce; Dalton, John P; Da Silva, Fabio L; Skinner-Adams, Tina S; Gardiner, Donald L; Trenholme, Katharine R; Brown, Christopher L; Ghosh, Partha; Porubsky, Patrick; Wang, Jenna L; Whipple, David A; Schoenen, Frank J; Hodder, Peter

    2015-01-01

    The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP suggesting that it is a valid target for new anti-malaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network (MLPCN) collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository (MLSMR)). A Cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from HTS. Two structurally related compounds, CID 6852389 and CID 23724194, yield micromolar potency and are inactive in CTSL1 titration experiments (IC50 >59.6 μM). As measured by Ki assay, both compounds demonstrate micromolar non-competitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrate potency in malaria growth assays (IC50 4 μM and 1.3 μM, respectively). PMID:24619116

  10. The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

    PubMed Central

    Jones, Peter M.; Robinson, Mark W.; Dalton, John P.; George, Anthony M.

    2011-01-01

    Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1–3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 Å-long channel and at the opening of the 30 Å-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed. PMID:22205955

  11. Leucine aminopeptidase blood test

    MedlinePlus

    Serum leucine aminopeptidase ... Leucine aminopeptidase is a type of protein called an enzyme . This enzyme is normally found in cells ... check if your liver is damaged. Too much leucine aminopeptidase is released into your blood when you ...

  12. Leucine aminopeptidase - urine

    MedlinePlus

    Leucine aminopeptidase is a type of protein called an enzyme. It is normally found in liver cells ... Increased levels of leucine aminopeptidase can be seen in ... Hepatitis Liver cancer Liver ischemia (reduced blood flow to the ...

  13. Leucine aminopeptidase - urine

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003617.htm Leucine aminopeptidase - urine To use the sharing features on this page, please enable JavaScript. Leucine aminopeptidase is a type of protein called an ...

  14. The Aminopeptidase Inhibitor CHR-2863 Is an Orally Bioavailable Inhibitor of Murine Malaria

    PubMed Central

    Skinner-Adams, Tina S.; Peatey, Christopher L.; Anderson, Karen; Trenholme, Katharine R.; Krige, David; Brown, Christopher L.; Stack, Colin; Nsangou, Desire M. M.; Mathews, Rency T.; Thivierge, Karine; Dalton, John P.

    2012-01-01

    Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria. PMID:22450967

  15. Aminopeptidases of Bacillus subtilis.

    PubMed Central

    Desmond, E P; Starnes, W L; Behal, F J

    1975-01-01

    Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism. PMID:240808

  16. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii. PMID

  17. Two Molecular Clouds near M17

    NASA Astrophysics Data System (ADS)

    Wilson, T. L.; Hanson, M. M.; Muders, D.

    2003-06-01

    We present fully sampled images in the C18O J=2-1 line extending over 13'×23', made with the Heinrich Hertz Telescope (HHT) on Mount Graham, AZ. The HHT has a resolution of 35" at the line frequency. This region includes two molecular clouds. Cloud A, to the north, is more compact, while cloud B is to the west of the H II region M17. Cloud B contains the well-known source M17SW. In C18O we find 13 maxima in cloud A and 39 in cloud B. Sixteen sources in cloud B are in M17SW, mapped previously with higher resolution. In cloud B, sources outside M17SW have line widths comparable to those in M17SW. In comparison, cloud A has lower C18O line intensities and smaller line widths but comparable densities and sizes. Maps of the cores of these clouds were also obtained in the J=5-4 line of CS, which traces higher H2 densities. Our images of the cores of clouds A and B show that for VLSR<=20 km s-1, the peaks of the CS emission are shifted closer to the H II region than the C18O maxima, so higher densities are found toward the H II region. Our CS data give additional support to the already strong evidence that M17SW and nearby regions are heated and compressed by the H II region. Our data show that cloud A has a smaller interaction with the H II region. We surmise that M17SW was an initially denser region, and the turn-on of the H II region will make this the next region of massive star formation. Outside of M17SW, the only other obvious star formation region may be in cloud A, since there is an intense millimeter dust continuum peak found by Henning et al. (1998) but no corresponding C18O maximum. If the CO/H2 ratio is constant, the dust must have a temperature of ~100 K or the H2 density is greater than 106 cm-3 or both to reconcile the C18O and dust data. Alternatively, if the CO/H2 ratio is low, perhaps much of the CO is depleted.

  18. An irradiated jet in M 17

    NASA Astrophysics Data System (ADS)

    Comerón, F.; Pasquali, A.; Alves de Oliveira, C.

    2013-04-01

    Context. M 17 is one of the best studied giant HII regions in our galactic neighborhood. It should also provide a suitable environment for the class of fully ionized jets externally irradiated by the presence of nearby hot stars. However, no such jets have been observed thus far in M 17. Aims: We report on a visible imaging survey of the M 17 nebula with the goal of identifying likely shock-excited nebulosities. Methods: We imaged M 17 through narrow-band filters centered on the most intense visible lines of [OIII], Hα+[NII], [SII], and Hβ. We obtained follow-up spectroscopy of the only jet-like structure identified in the images. We also used published X-ray observations of M 17 obtained with the Chandra X-ray Observatory, as well as infrared data obtained with the Very Large Telescope and with the Spitzer Space Telescope, to look for evidence of a jet-driving source. Results: We have detected what appears to be the first jet identified in M 17 visible HII region. The jet is composed of a set of knots, two of which have significant radial velocities with respect to the HII region, and a distant arc-like bright nebulosity that may represent an early episode of intense mass loss by the jet-driving source. The follow-up spectra of the structures composing the jet, including the arc, support this interpretation by revealing intense forbidden lines of [NII] and [SII] due to enhanced collisional excitation in shocks. The presence of a X-ray source, most likely a young stellar object, at a position where the jet launching source should be expected to lie reinforces the interpretation. We identify a tentative near-infrared counterpart of the X-ray source, although it is offset by 1".9 from the nominal position of the X-ray source, which is almost three times the radius of its positional uncertainty. The very weak [OI] emission in the spectra of the jet knots and the arc suggests that they are nearly fully ionized, in agreement with the environment in which the jet is

  19. THE SUBMILLIMETER POLARIZATION SPECTRUM OF M17

    SciTech Connect

    Zeng Lingzhen; Jimenez-Serra, Izaskun; Bennett, Charles L.; Chapman, Nicholas L.; Novak, Giles; Chuss, David T.; Vaillancourt, John E.

    2013-08-10

    We present 450 {mu}m polarimetric observations of the M17 molecular cloud obtained with the SHARP polarimeter at the Caltech Submillimeter Observatory. Across the observed region, the magnetic field orientation is consistent with previous submillimeter and far-infrared polarization measurements. Our observations are centered on a region of the molecular cloud that has been compressed by stellar winds from a cluster of OB stars. We have compared these new data with previous 350 {mu}m polarimetry and find an anti-correlation between the 450 and 350 {mu}m polarization magnitude ratio and the ratio of 21 cm to 450 {mu}m intensity. The polarization ratio is lower near the east end of the studied region where the cloud is exposed to stellar winds and radiation. At the west end of the region, the polarization ratio is higher. We interpret the varying polarization spectrum as evidence supporting the radiative alignment torque model for grain alignment, implying higher alignment efficiency in the region that is exposed to a higher anisotropic radiation field.

  20. Gingipain aminopeptidase activities in Porphyromonas gingivalis

    PubMed Central

    Veillard, Florian; Potempa, Barbara; Poreba, Marcin; Drag, Marcin; Potempa, Jan

    2014-01-01

    Bestatin, a specific inhibitor of metalloaminopeptidases, inhibits the growth of Porphyromonas gingivalis. To identify its target enzyme, a library of fluorescent substrates was used but no metalloaminopeptidase activity was found. All aminopeptidase activity of P. gingivalis was bestatin-insensitive and directed exclusively toward N-terminal arginine and lysine substrates. Class-specific inhibitors and gingipain-null mutants showed that gingipains were the only enzymes responsible for this activity. The kinetic constants obtained for Rgps were comparable to those of human aminopeptidases but Kgp aminopeptidase activity was weaker. This finding reveals a new role for gingipains as aminopeptidases in degradation of proteins and peptides P. gingivalis. PMID:23667904

  1. The peculiar radio source M17 JVLA 35

    SciTech Connect

    Rodríguez, L. F.; Carrasco-González, C.; Montes, G.; Tapia, M.

    2014-07-01

    M17 JVLA 35 is a radio source detected in projection against the M17 H II region. In recent observations, its spectrum between 4.96 and 8.46 GHz was found to be positive and very steep, with α ≥ 2.9 ± 0.6 (S {sub ν}∝ν{sup α}). Here we present Very Large Array observations made in the 18.5 to 36.5 GHz region that indicate a spectral turnover at ∼13 GHz and a negative spectral index (α ≅ –2.0) at higher frequencies. The spectrum is consistent with that of an extragalactic high frequency peaker (HFP). However, M17 JVLA 35 has an angular size of ∼0.''5 at 8.46 GHz, while HFPs have extremely compact, milliarcsecond dimensions. We discuss other possible models for the spectrum of the source and do not find them feasible. Finally, we propose that M17 JVLA 35 is indeed an HFP but that its angular size becomes broadened by plasma scattering as its radiation travels across M17. If our interpretation is correct, accurate measurements of the angular size of M17 JVLA 35 across the centimeter range should reveal the expected ν{sup –2} dependence.

  2. Endoplasmic reticulum aminopeptidases: biochemistry, physiology and pathology.

    PubMed

    Hattori, Akira; Tsujimoto, Masafumi

    2013-09-01

    The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized. PMID:23946506

  3. Star formation in the M17 SW giant molecular cloud

    NASA Technical Reports Server (NTRS)

    Jaffe, D. T.; Fazio, G. G.

    1982-01-01

    The first high-sensitivity, high-resolution far-IR survey of an entire molecular cloud complex is presented. The 20 km/s M17 SW complex, in addition to the three luminous M17 sources, contains 10 sources spread over 110 pc. The 10 lower luminosity sources divide into two groups: small blister sources powered by late O stars and compact sources powered by clusters of early B stars. No compact far-IR sources with luminosities between the detection limit and 10,000 solar luminosities were detected. Three possible formation mechanisms for the stars that power the far-IR sources in the M17 SW complex are examined. Sequential formation cannot explain the sources seen throughout the complex. Some type of stochastic formation mechanism or collapse induced by a spiral density wave could explain the observations.

  4. [Endocellular aminopeptidase from Astasia longa].

    PubMed

    Rudenskaia, Iu A; Aseev, V V; Rudensaia, G N

    2008-01-01

    A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase. PMID:18672681

  5. Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

    PubMed Central

    Martinez, Josefina; Azam, Farooq

    1993-01-01

    Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments. PMID:16349084

  6. The Young Stellar Population in M17 Revealed by Chandra

    NASA Astrophysics Data System (ADS)

    Broos, Patrick S.; Feigelson, Eric D.; Townsley, Leisa K.; Getman, Konstantin V.; Wang, Junfeng; Garmire, Gordon P.; Jiang, Zhibo; Tsuboi, Yohko

    2007-04-01

    We report here results from a Chandra ACIS observation of the stellar populations in and around the M17 H II region. The field reveals 886 sources with observed X-ray luminosities (uncorrected for absorption) between ˜29.3 ergs s-1< log LX<32.8 ergs s-1, 771 of which have stellar counterparts in infrared images. In addition to comprehensive tables of X-ray source properties, several results are presented: 1. The X-ray luminosity function is calibrated to that of the Orion Nebula Cluster population to infer a total population of roughly 8000-10,000 stars in M17, one-third lying in the central NGC 6618 cluster. 2. About 40% of the ACIS sources are heavily obscured with AV>10 mag. Some are concentrated around well-studied star-forming regions -- IRS 5/UC1, the Kleinmann-Wright Object, and M17-North -- but most are distributed across the field. As previously shown, star formation appears to be widely distributed in the molecular clouds. X-ray emission is detected from 64 of the hundreds of Class I protostar candidates that can be identified by near- and mid-infrared colors. These constitute the most likely protostar candidates known in M17. 3. The spatial distribution of X-ray stars is complex: in addition to the central NGC 6618 cluster and well-known embedded groups, we find a new embedded cluster (designated M17-X), a 2 pc long arc of young stars along the southwest edge of the M17 H II region, and 0.1 pc substructure within various populations. These structures may indicate that the populations are dynamically young. 4. All (14/14) of the known O stars but only about half (19/34) of the known B0-B3 stars in the M17 field are detected. These stars exhibit the long-reported correlation between X-ray and bolometric luminosities of LX˜10-7Lbol. While many O and early-B stars show the soft X-ray emission expected from microshocks in their winds or moderately hard emission that could be caused by magnetically channeled wind shocks, six of these stars exhibit very hard

  7. Novel selective inhibitors of aminopeptidases that generate antigenic peptides.

    PubMed

    Papakyriakou, Athanasios; Zervoudi, Efthalia; Theodorakis, Emmanuel A; Saveanu, Loredana; Stratikos, Efstratios; Vourloumis, Dionisios

    2013-09-01

    Endoplasmic reticulum aminopeptidases, ERAP1 and ERAP2, as well as Insulin regulated aminopeptidase (IRAP) play key roles in antigen processing, and have recently emerged as biologically important targets for manipulation of antigen presentation. Taking advantage of the available structural and substrate-selectivity data for these enzymes, we have rationally designed a new series of inhibitors that display low micromolar activity. The selectivity profile for these three highly homologous aminopeptidases provides a promising avenue for modulating intracellular antigen processing. PMID:23916253

  8. Aminopeptidase N1 (EtAPN1), an M1 Metalloprotease of the Apicomplexan Parasite Eimeria tenella, Participates in Parasite Development

    PubMed Central

    Gras, Simon; Byzia, Anna; Gilbert, Florence B.; McGowan, Sheena; Drag, Marcin; Niepceron, Alisson; Lecaille, Fabien; Lalmanach, Gilles; Brossier, Fabien

    2014-01-01

    Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs. PMID:24839124

  9. Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake

    PubMed Central

    Tsuboi, Shun; Yamamura, Shigeki; Imai, Akio; Iwasaki, Kazuhiro

    2016-01-01

    We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla—Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis. PMID:26936797

  10. Deep Near-Infrared Survey toward the M17 Region

    NASA Astrophysics Data System (ADS)

    Jiang, Zhibo; Yao, Yongqiang; Yang, Ji; Ando, Minoru; Kato, Daisuke; Kawai, Toshihide; Kurita, Mikio; Nagata, Tetsuya; Nagayama, Takahiro; Nakajima, Yasushi; Nagashima, Chie; Sato, Shuji; Tamura, Motohide; Nakaya, Hidehiko; Sugitani, Koji

    2002-09-01

    We conducted a deep JHKs-band imaging survey of the M17 region, using a near-infrared camera, the Simultaneous 3-color InfraRed Imager for Unbiased Survey (SIRIUS), mounted on the InfraRed Survey Facility (IRSF) 1.4 m telescope at the South African Astronomical Observatory. This survey covers an area of ~200 arcmin2 with 10 σ limiting magnitudes of J~18.7, H~18.2, and Ks~17.5. The near-infrared (NIR) images reveal an unprecedented view of the region. The NIR nebulae are highly structured, with two nebular bars corresponding to, but a little larger than, the H II region defined by Felli, Massi, & Churchwell, constructing a conical shape. Fine structures are found all over the nebular area. The central region contains a congregation of intermediate- to high-mass stars. From the slope of the Ks-band luminosity function and the frequency of young stellar objects (YSOs) we infer that the central cluster has an age less than 3 Myr. The central OB cluster provides tremendous energy that heats and ionizes its surrounding materials, triggering the star formation of second-generation in the nebular bars. The second generation stars are so numerous that could they affect the star formation efficiency in the whole region. To the southwest of the central cluster and the nebular bars, where a giant molecular cloud core is located, a large number of red stars are detected. We argue that these red stars are most probably associated YSOs with intrinsic color excesses, not normal field stars reddened by the molecular cloud in front of them. Being located beyond the photodissociation region, the star-forming process in the molecular region could be independent of the impact of the central cluster.

  11. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1460 Leucine aminopeptidase...

  12. Purification and characterization of an immunogenic aminopeptidase of Brucella melitensis.

    PubMed

    Contreras-Rodriguez, Araceli; Ramirez-Zavala, Bernardo; Contreras, Andrea; Schurig, Gerhardt G; Sriranganathan, Nammalwar; Lopez-Merino, Ahide

    2003-09-01

    An immunogenic aminopeptidase was purified from Brucella melitensis strain VTRM1. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. This procedure resulted in a yield of 29% and a 144-fold increase in specific activity. The aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kDa and an isoelectric point of 4.8. Its activity was optimal at pH 7.0 at 40 degrees C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn(2+) and Hg(2+)), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. The K(m) values for L-alanine-p-nitroanilide (Ala-pNA) and Lys-pNA were 0.35 and 0.18 mM, respectively. The N-terminal sequence of aminopeptidase was used for a homologous search in the genomes of B. melitensis 16M and Brucella suis 1330. The analysis revealed an exact match of the probe sequence (36 bp) with an open reading frame of 2,652 bp encoding a protein predicted to be alanyl aminopeptidase (aminopeptidase N). Collectively, these data suggest designation of the B. melitensis enzyme as an aminopeptidase N. The aminopeptidase was recognized by sera from patients with acute and chronic brucellosis, suggesting that the enzyme may have important diagnostic implications. PMID:12933870

  13. Stereospecificity of amino acid hydroxamate inhibition of aminopeptidases.

    PubMed

    Wilkes, S H; Prescott, J M

    1983-11-25

    Hydroxamates of amino acids and aliphatic acids are effective inhibitors of Aeromonas proteolytica amino-peptidase (EC 3.4.11.10) and of both the cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases of swine kidney. Cytosolic leucine aminopeptidase and the Aeromonas enzyme were inhibited to a greater extent by D isomers than by the L enantiomorphs, manganese-activated kidney cytosolic leucine aminopeptidase being inhibited 10 times more effectively by D-leucine and D-valine hydroxamic acids than by the L isomers. The D isomers of these two compounds inhibited Aeromonas aminopeptidase to an even greater extent with Ki values of 2 X 10(-9) and 5 X 10(-9), respectively, whereas the corresponding L isomers were bound 150 times less tightly. With the Aeromonas enzyme, a comparison of inhibition by racemic mixtures with that of the corresponding L isomers indicated that in all cases the contribution of the D isomer was predominant. Isocaproic hydroxamic acid inhibited this enzyme equally well as L-leucine hydroxamic acid, indicating that the amino group orientation in the D isomer contributes to the binding efficacy. Swine kidney microsomal aminopeptidase was also inhibited by D isomers of leucine and valine hydroxamic acids but in contrast to the other two enzymes, the inhibition was 10-fold less than that observed for the corresponding L isomers. Cytosolic leucine aminopeptidase with either 6 g atoms of zinc per mol or 12 g atoms of zinc per mol was inhibited only slightly by any of the hydroxamic acid compounds; evidently enzyme-bound manganese (or magnesium) is specific for hydroxamate binding to this aminopeptidase. PMID:6643439

  14. New aminopeptidase from "microbial dark matter" archaeon.

    PubMed

    Michalska, Karolina; Steen, Andrew D; Chhor, Gekleng; Endres, Michael; Webber, Austen T; Bird, Jordan; Lloyd, Karen G; Joachimiak, Andrzej

    2015-09-01

    Marine sediments host a large population of diverse, heterotrophic, uncultured microorganisms with unknown physiologies that control carbon flow through organic matter decomposition. Recently, single-cell genomics uncovered new key players in these processes, such as the miscellaneous crenarchaeotal group. These widespread archaea encode putative intra- and extracellular proteases for the degradation of detrital proteins present in sediments. Here, we show that one of these enzymes is a self-compartmentalizing tetrameric aminopeptidase with a preference for cysteine and hydrophobic residues at the N terminus of the hydrolyzed peptide. The ability to perform detailed characterizations of enzymes from native subsurface microorganisms, without requiring that those organisms first be grown in pure culture, holds great promise for understanding key carbon transformations in the environment as well as identifying new enzymes for biomedical and biotechnological applications. PMID:26062601

  15. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  16. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  17. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  18. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  19. A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases.

    PubMed

    Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Joachimiak, Andrzej; Mucha, Artur

    2016-07-19

    N'-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki = 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region. PMID:27100031

  20. Adipocyte aminopeptidases in obesity and fasting.

    PubMed

    Alponti, Rafaela Fadoni; Silveira, Paulo Flavio

    2015-11-01

    This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands. PMID:26257241

  1. Structure and function of the methionine aminopeptidases.

    PubMed

    Lowther, W T; Matthews, B W

    2000-03-01

    The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family. PMID:10708856

  2. A High-Resolution VLA Study of M17-UC1

    NASA Astrophysics Data System (ADS)

    Johnson, C. Olivia; De Pree, C. G.; Goss, W. M.

    1998-06-01

    Using the Very Large Array, we have observed the H66α radio recombination line (RRL) emission at 1.3 cm and the H52α RRL emission at 7 mm toward the ultracompact H II region M17-UC1 and the nearby arc-shaped structure to the east. The angular resolution of the data is 1"-2". The compact H II region (deconvolved size ~0.6" or 0.006 pc) is one of a number of H II regions with broad (>=35 km s-1) RRLs. The line parameters of the nearby arc of ionized emission (hereafter the Arc), about 1" or 0.01 pc to the east of UC1, are remarkably constant over an area of 16.5" × 3" (0.17 pc × 0.03 pc). The derived LTE electron temperature from the H66α line (T*H66α) is 6600 K in M17-UC1 and 8200 K in the Arc region. H2O maser emission at 1.3 cm from the region of M17-UC1 was observed in the D array with a beam of 4". Observations of the H2O maser emission toward M17-UC1 reveal four masers within 30" of M17-UC1 two of these sources were previously known. We discuss the region near M17-UC1 as an example of shock-induced star formation consisting of a hot, young, massive star surrounded by ionized material, perhaps resulting from a stellar wind outflow. We compare M17-UC1 with the six other known examples of broad-line RRL emission from ultracompact H II regions.

  3. EVIDENCE FOR DELAYED MASSIVE STAR FORMATION IN THE M17 PROTO-OB ASSOCIATION

    SciTech Connect

    Povich, Matthew S.; Whitney, Barbara A. E-mail: bwhitney@spacescience.or

    2010-05-10

    Through analysis of archival images and photometry from the Spitzer GLIMPSE and MIPSGAL surveys combined with Two Micron All Sky Survey and MSX data, we have identified 488 candidate young stellar objects (YSOs) in the giant molecular cloud M17 SWex, which extends {approx}50 pc southwest from the prominent Galactic H II region M17. Our sample includes >200 YSOs with masses >3 M {sub sun} that will become B-type stars on the main sequence. Extrapolating over the stellar initial mass function (IMF), we find that M17 SWex contains >1.3 x 10{sup 4} young stars, representing a proto-OB association. The YSO mass function is significantly steeper than the Salpeter IMF, and early O stars are conspicuously absent from M17 SWex. Assuming M17 SWex will form an OB association with a Salpeter IMF, these results reveal the combined effects of (1) more rapid circumstellar disk evolution in more massive YSOs and (2) delayed onset of massive star formation.

  4. Nobeyama 45m CO Galactic Plane Survey: Filament properties and star formation in M17

    NASA Astrophysics Data System (ADS)

    Nishimura, Atsushi; Umemoto, Tomofumi; Minamidani, Tetsuhiro; Kuno, Nario; Tosaki, Tomoka; Fujita, Shinji; Matsuo, Mitsuhiro; Tsuda, Yuya; Ohashi, Satoshi

    2015-08-01

    We present the 12CO(J=1-0), 13CO(J=1-0), and C18O(J=1-0) maps of M17 molecular clouds obtained as part of the Nobeyama 45m CO Galactic Plane Survey. The observations cover the entire area of M17 Cloud A and M17 SW with an angular resolution of ~15" which corresponds to ~0.15 pc, and they can be used to trace the formation and evolution of filamentary structure of molecular clouds in GMC scale. The Cloud A consists of a couple of twisted filaments, they are extended in parallel toward the HII region. The typicall width of the filaments is ~0.4 pc in 13CO intensity map. They are twisted with an interval of ~5 pc, and an amplitude of ~2 pc. Some filaments have a bright rim structure in 8μm at the filament edge facing the HII region. Therefore, the filaments might be formed by the feedback of the HII region. The mass distribution have a gradient depending on the distance of M17 HII region. Most of the filaments have points where the line mass exceed the critical value of 16 M⊙ pc-1. This indicates that the high-density cores can be formed on the most of the filaments in the Cloud A. In addition, YSOs distribution from MYStIX infrared excess source catalog shows that the most of YSOs are on the filaments in the Cloud A. Hence the filamentally structure plays an important role to form stars in Cloud A. However, the fact that most of the OB stars are located away from filaments suggests that the Cloud A filaments could not trigger the formation of the M17 cluster including OB stars. We found high-velocity clumps (Vlsr~23 km sec-1) which are associated with OB stars. The distribution of high-velocity clumps is anticorrelated with Cloud A and M17 SW. The Cloud A filaments (Vlsr~20 km sec-1) are corresponding to IRDCs identified by Spitzer, while the high-velocity clumps have no IRDC counterpart. Therefore, Cloud A filaments are located near side of the HII region and the high-velocity clumps are located far side of the HII region. One possibility which satisfy the

  5. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  6. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  7. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  8. 21 CFR 862.1460 - Leucine aminopeptidase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leucine aminopeptidase test system. 862.1460 Section 862.1460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  9. Prospective evaluation of aminopeptidase activities in plasma and peripheral organs of streptozotocin-induced diabetic rats.

    PubMed

    Zambotti-Villela, L; Yamasaki, S C; Villarroel, J S; Alponti, R F; Silveira, P F

    2008-06-01

    The cleavage of peptides by aminopeptidase enzyme types could be among the mechanisms related to certain disruptions on mediator and modulatory functions in diabetes mellitus. In order to examine this hypothesis, we measured representative aminopeptidase activities in tissues of peripheral organs of control and streptozotocin-diabetic rats. None of the examined aminopeptidase activities differed between diabetics and controls in plasma, ileum, stomach or lung. Soluble and membrane-associated alanyl, and membrane-associated cystyl aminopeptidase activities were higher in the kidney of diabetics. Decreased activity was observed in soluble and membrane-associated aspartyl and soluble dipeptidyl-peptidase IV, while increased activity was observed in soluble alanyl, arginyl, and cystyl aminopeptidases in the pancreas of diabetics. In the jejunum, soluble cystyl aminopeptidase increased in diabetics. Soluble arginyl and type-1-pyroglutamyl aminopeptidase and membrane-associated dipeptidyl-peptidase IV activities increased in the liver of diabetics. Membrane-associated dipeptidyl-peptidase IV and alanyl aminopeptidase activities in the spleen were higher in diabetics than in controls. Membrane-associated alanyl aminopeptidase activity also increased in the heart of diabetics. All these changes in streptozotocin-treated rats were avoided by the administration of insulin. Our comparative analysis of a diverse array of aminopeptidase activities supported the proposal that the regulation of peptide cleavage by these enzyme types is associated with the effects of streptozotocin-diabetes mellitus on peripheral organs. PMID:18591879

  10. Isoprenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Guggisberg, Ann M.; Amthor, Rachel E.

    2014-01-01

    Malaria kills nearly 1 million people each year, and the protozoan parasite Plasmodium falciparum has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in P. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In P. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in P. falciparum has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in P. falciparum, in order to identify valuable directions for future research. PMID:25217461

  11. Genetic associations and functional characterization of M1 aminopeptidases and immune-mediated diseases.

    PubMed

    Agrawal, N; Brown, M A

    2014-12-01

    Endosplasmic reticulum aminopeptidase 1 (ERAP1), endoplasmic reticulum aminopeptidase 2 (ERAP2) and puromycin-sensitive aminopeptidase (NPEPPS) are key zinc metallopeptidases that belong to the oxytocinase subfamily of M1 aminopeptidase family. NPEPPS catalyzes the processing of proteosome-derived peptide repertoire followed by trimming of antigenic peptides by ERAP1 and ERAP2 for presentation on major histocompatibility complex (MHC) Class I molecules. A series of genome-wide association studies have demonstrated associations of these aminopeptidases with a range of immune-mediated diseases such as ankylosing spondylitis, psoriasis, Behçet's disease, inflammatory bowel disease and type I diabetes, and significantly, genetic interaction between some aminopeptidases and HLA Class I loci with which these diseases are strongly associated. In this review, we highlight the current state of understanding of the genetic associations of this class of genes, their functional role in disease, and potential as therapeutic targets. PMID:25142031

  12. The abundance of atomic carbon near the ionization fronts in M17 and S140

    NASA Technical Reports Server (NTRS)

    Keene, J.; Blake, G. A.; Phillips, T. G.; Huggins, P. J.; Beichman, C. A.

    1985-01-01

    The 492 GHz ground-state line of atomic carbon in the edge-on ionization fronts in M17 and S140 were observed. It was found that, contrary to expectation, the C I emission peaks farther into the molecular cloud from the ionization front than does the CO. In fact the peak C I abundance in M17 occurs more than 60 mag of visual extinction into the cloud from the ionization front. Calculations of the ratio of C I to CO column densities yield values of 0.1-0.2. These observations do not support chemical models which predict that neutral atomic carbon should be found only near the edges of molelcular clouds. Other models are discussed which may explain the observations.

  13. The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

    PubMed

    Cadel, Sandrine; Darmon, Cécile; Pernier, Julien; Hervé, Guy; Foulon, Thierry

    2015-02-01

    Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases. PMID:25530263

  14. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available from the National Academy Press, 2101... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Aminopeptidase enzyme preparation derived from... Listing of Specific Substances Affirmed as GRAS § 184.1985 Aminopeptidase enzyme preparation derived...

  15. Structural analysis of Anopheles midgut aminopeptidase N reveals a novel malaria transmission-blocking vaccine B-cell epitope

    PubMed Central

    Atkinson, Sarah C.; Armistead, Jennifer S.; Mathias, Derrick K.; Sandeu, Maurice M.; Tao, Dingyin; Borhani-Dizaji, Nahid; Tarimo, Brian B.; Morlais, Isabelle; Dinglasan, Rhoel R.; Borg, Natalie A.

    2015-01-01

    Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission remains elusive. Here we present the 2.65 Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profile of three AnAPN1 monoclonal antibodies (mAb), including mAb 4H5B7, which effectively block transmission of natural strains of Plasmodium falciparum. Utilizing the AnAPN1 structure we map the conformation-dependent 4H5B7 neo-epitope to a previously uncharacterized region on domain 1, and further demonstrate that non-human primate neo-epitope-specific IgG also block parasite transmission. We discuss the prospect of a novel biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV. PMID:26075520

  16. In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis.

    PubMed

    Fitzpatrick, Leo R; Small, Jeffrey; Hoerr, Robert A; Bostwick, Eileen F; Maines, Lynn; Koltun, Walter A

    2008-09-01

    We examined the in vitro and in vivo effects of a probiotic, Escherichia coli strain M-17 (EC-M17), on NF-kappaB signalling, cytokine secretion and efficacy in dextran sulfate sodium (DSS)-induced murine colitis. NF-kappaB signalling was assessed using an NF-kappaB luciferase reporter cell line that was stimulated with TNF-alpha (100 ng/ml). p65 Nuclear binding and cytokine secretion (TNF-alpha, IL-1beta and IL-6) were evaluated using a RAW 264.7 macrophage cell line that was exposed to lipopolysaccharide (LPS; 5 microg/ml). Mice were administered vehicle, EC-M17, metronidazole, or EC-M17 plus metronidazole for 13 d. During the final 6 d, mice also received 2 % DSS. Parameters evaluated included disease activity index (DAI), histology, myeloperoxidase and NF-kappaB p65. EC-M17 dose dependently inhibited TNF-alpha-induced NF-kappaB signalling. At 5 x 109 colony-forming units/ml, EC-M17 inhibited NF-kappaB by >95 %. LPS-induced nuclear p65 binding was significantly inhibited (78 %; P 90 %) the LPS-induced secretion of TNF-alpha, IL-1beta and IL-6. In mice with DSS-induced colitis, EC-M17, metronidazole, and EC-M17 plus metronidazole significantly reduced DAI and colonic histology scores. Both EC-M17 and metronidazole reduced colonic IL-12, IL-6, IL-1beta and interferon-gamma. The combination of EC-M17 plus metronidazole resulted in more substantial cytokine reductions than were found with either treatment alone, and combination therapy significantly (P < 0.05 in both cases) reduced IL-1beta compared with EC-M17 and colonic histology scores compared with metronidazole. Alone, and in combination with metronidazole, EC-M17 improved murine colitis, probably due to an inhibitory effect on NF-kappaB signalling. PMID:18279557

  17. Disentangling the excitation conditions of the dense gas in M17 SW

    NASA Astrophysics Data System (ADS)

    Pérez-Beaupuits, J. P.; Güsten, R.; Spaans, M.; Ossenkopf, V.; Menten, K. M.; Requena-Torres, M. A.; Wiesemeyer, H.; Stutzki, J.; Guevara, C.; Simon, R.

    2015-11-01

    Context. Stars are formed in dense molecular clouds. These dense clouds experience radiative feedback from UV photons, and X-ray from stars, embedded prestellar cores, young stellar objects (YSOs), and ultra compact H II regions. This radiative feedback affects the chemistry and thermodynamics of the gas. Aims: We aim to probe the chemical and energetic conditions created by radiative feedback through observations of multiple CO, HCN, and HCO+ transitions. We measure the spatial distribution and excitation of the dense gas (n(H2) > 104 cm-3) in the core region of M17 SW and aim to investigate the influence of UV radiation fields. Methods: We used the dual band receiver GREAT on board the SOFIA airborne telescope to obtain a 5.7 arcmin × 3.7 arcmin map of the J = 16 → 15, J = 12 → 11, and J = 11 → 10 transitions of 12CO in M17 SW. We compare these maps with corresponding APEX and IRAM 30 m telescope data for low- and mid-J CO, HCN, and HCO+ emission lines, including maps of the HCN J = 8 → 7 and HCO+J = 9 → 8 transitions. The excitation conditions of 12CO, HCO+, and HCN are estimated with a two-phase non-LTE radiative transfer model of the line spectral energy distributions (LSEDs) at four selected positions. The energy balance at these positions is also studied. Results: We obtained extensive LSEDs for the CO, HCN, and HCO+ molecules toward M17 SW. These LSEDs can be fit simultaneously using the same density and temperature in the two-phase models and to the spectra of all three molecules over a ~12 square arc minute size region of M17 SW. Temperatures of up to 240 K are found toward the position of the peak emission of the 12CO J = 16 → 15 line. High densities of 106 cm-3 were found at the position of the peak HCN J = 8 → 7 emission. Conclusions: We found HCO+/HCN line ratios larger than unity, which can be explained by a lower excitation temperature of the higher-J HCN lines. The LSED shape, particularly the high-J tail of the CO lines observed

  18. Astrometry and expanding bubble of a deeply embedded young stellar object in M17

    NASA Astrophysics Data System (ADS)

    Chibueze, James O.; Kamezaki, Tatsuya; Omodaka, Toshihiro; Handa, Toshihiro; Nagayama, Takumi; Baba, Tatsuya; Sunada, Kazuyoshi; Shizugami, Makoto; Burns, Ross A.; Honma, Mareki; Ubachukwu, Augustine A.; Chukwude, Augustine E.; Alhassan, Jibrin A.

    2016-08-01

    We measured the trigonometric parallax of the H2O maser source in the M17 cluster-forming region to be 0.491 ± 0.041 mas, corresponding to a distance of 2.04^{+0.16}_{-0.17} kpc with 8 per cent accuracy. This result is consistent with the Very Long Baseline Array (VLBA) previous parallax measurement of 12.2 GHz CH3OH masers in M17-UC1. We performed the first measurement of the relative proper motions of the H2O maser feature 2, and its morphology traces an expanding arcuate shell with a mean motion of ˜ 19.4 km s-1. The redshifted maser component also shows expanding motion. Fitting a uniformly expanding spherical flow model, we estimate the most probable position and motion of the driving source and we propose that the motion of the masers traces an expanding bubble from a central source, with a dynamical time-scale of ˜ 12.5 yr. This is the first evidence that the maser 2 cluster is driven internally. There is no known counterpart Two-Micron All-Sky Survey (2MASS), Wide-Field Infrared Survey Explorer (WISE) or radio source around this H2O maser. However, the Spitzer point source catalogue has a source that is spatially coincident with the position of the H2O masers. This was detected only in the Spitzer 4.5- and 5.8-μm bands (with magnitudes 9.97 ± 0.12 and 7.82 ± 0.10 mag, respectively). The non-detection at other wavelengths could indicate that the source is still at a very early formative stage, and/or very deeply embedded and obscured in its formative environment.

  19. Astrometry and Expanding Bubble of a Deeply Embedded YSO in M17

    NASA Astrophysics Data System (ADS)

    Chibueze, James O.; Kamezaki, Tatsuya; Omodaka, Toshihiro; Handa, Toshihiro; Nagayama, Takumi; Baba, Tatsuya; Sunada, Kazuyoshi; Shizugami, Makoto; Burns, Ross A.; Honma, Mareki; Ubachukwu, Augustine A.; Chukwude, Augustine E.; Alhassan, Jibrin

    2016-04-01

    We measured the trigonometric parallax of H2O maser source in M17 cluster forming region to be 0.491±0.041 mas, corresponding to a distance of 2.04^{+0.16}_{-0.17} kpc with 8% accuracy. This result is consistent with VLBA previous parallax measurement of 12.2 GHz CH3OH masers in M17-UC1. We performed the first measurement of the relative proper motions of the H2O maser feature 2 (of Johnson et al. 1998), and its morphology trace an expanding arcuate shell with mean motion of ˜ 19.4 km s-1. The redshifted maser component also show expanding motion. Fitting a uniformly expanding spherical flow model, we estimated the most probable position and motion of the driving source and propose that the motion of the masers trace and expanding bubble from a central source, with a dynamical time-scale of ˜ 12.5 years. This is the first evidence that maser 2 cluster is driven internally. There is no known counterpart 2MASS, WISE or radio source around this H2O masers. But Spitzer point source catalog has a source that is spatially coincident with the position of the H2O masers. The was detected only in the Spitzer's 4.5 and 5.8 μm (magnitudes as 9.97±0.12 and 7.82±0.10 mag, respectively) bands. The non-detection at other wavelength could indicate that the source is yet at a very early formative stage, and/or very deeply embedded and obscured in its formative environment.

  20. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  1. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  2. Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca.

    PubMed

    Alponti, Rafaela Fadoni; Zambotti-Villela, Leonardo; Murena-Nunes, Cristiane; Marinho, Camila Eduardo; do Amaral Olivo, Renata; Silveira, Paulo Flavio

    2005-07-01

    The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species. PMID:16006161

  3. Purification and identification of a novel leucine aminopeptidase from Bacillus thuringiensis israelensis.

    PubMed

    Cahan, Rivka; Hetzroni, Efrat; Nisnevitch, Marina; Nitzan, Yeshayahu

    2007-11-01

    A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65 degrees C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65 degrees C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase's effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin. PMID:17682820

  4. New role for leucyl aminopeptidase in glutathione turnover.

    PubMed Central

    Cappiello, Mario; Lazzarotti, Alessandra; Buono, Francesca; Scaloni, Andrea; D'Ambrosio, Chiara; Amodeo, Pietro; Méndez, Blanca L; Pelosi, Paolo; Del Corso, Antonella; Mura, Umberto

    2004-01-01

    A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme-substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K(m)=0.57 mM, kcat=6.0x10(3) min(-1) at pH 7.4 and 25 degrees C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed. PMID:14583094

  5. Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii.

    PubMed

    Yang, Mingfa; Zheng, Jun; Jia, Honglin; Song, Mingxin

    2016-09-01

    In the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a K m value of 0·255 µ m and a k cat value of 35·6 s-1. The overall catalytic efficiency (k cat/K m) of the rTgAPP was 139·6 × 105 M-1 s-1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP. PMID:27220680

  6. Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

    PubMed Central

    Sanz, Yolanda; Toldrá, Fidel

    2002-01-01

    An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment. PMID:11916721

  7. CEN 34 - high-mass YSO in M 17 or background post-AGB star?

    NASA Astrophysics Data System (ADS)

    Chen, Zhiwei; Nürnberger, Dieter E. A.; Chini, Rolf; Liu, Yao; Fang, Min; Jiang, Zhibo

    2013-09-01

    We investigate the proposed high-mass young stellar object (YSO) candidate CEN 34, thought to be associated with the star-forming region M 17. Its optical to near-infrared (550-2500 nm) spectrum reveals several photospheric absorption features, such as Hα, the Ca ii triplet, and the CO bandhead, but lacks emission lines. The spectral features in the range 8375-8770 Å are used to constrain an effective temperature Teff = 5250 ± 250 K (early-/mid-G) and a log g = 2.0 ± 0.3 (supergiant). The spectral energy distribution (SED) displays a faint infrared excess that resembles that of a high-mass YSO or an evolved star of intermediate mass. Moreover, the observed temperature and surface gravity are identical for high-mass YSOs and evolved stars. The radial velocity of CEN 34 relative to the local standard of rest (VLSR) as obtained from various photospheric lines is of the order of -60 km s-1 and thus distinct from the +25 km s-1 found for several OB stars in the cluster and for the associated molecular cloud. The SED modeling yields 10-4 M⊙ of circumstellar material, which contributes only a tiny fraction to the total visual extinction (11 mag). The distance of CEN 34 is between 2.0 kpc and 4.5 kpc. In the case of a YSO, a dynamical ejection process is proposed to explain the VLSR difference between CEN 34 and M 17. Additionally, to match the temperature and luminosity, we speculate that CEN 34 had accumulated the bulk of its mass with an accretion rate >4 × 10-3M⊙/yr over a very short time span (~103 yrs), and it is currently undergoing a phase of gravitational contraction without any further mass gain. However, all the aforementioned characteristics of CEN 34 are compatible with an evolved star of 5-7 M⊙ and an age of 50-100 Myr, so it is most likely a background post-AGB star with a distance between 2.0 kpc and 4.5 kpc. We consider the latter classification as the more likely interpretation. Further discrimination of the two possible scenarios should come

  8. Hydroxamate-induced spectral perturbations of cobalt Aeromonas aminopeptidase.

    PubMed

    Wilkes, S H; Prescott, J M

    1987-06-25

    The absorption spectrum of cobalt(II)-substituted Aeromonas aminopeptidase is markedly perturbed by the presence of equimolar concentrations of D-amino acid hydroxamates and acyl hydroxamates that have previously been shown to be powerful inhibitors of this enzyme (Wilkes, S. H., and Prescott, J. M. (1983) J. Biol. Chem. 258, 13517-13521). D-Valine hydroxamate produces the most distinctive perturbation, splitting the characteristic 527 nm absorption peak of the cobalt enzyme to form peaks at 564, 520, and 487 nm with molar extinction values of 126, 98, and 67 M-1 cm-1, respectively. A qualitatively similar perturbation, albeit with lower extinction values, results from the addition of D-leucine hydroxamate, whereas D-alanine hydroxamate perturbs the spectrum, but does not evoke the peak at 564 nm. In contrast, hydroxamates of L-valine and L-leucine in concentrations equi-molar to that of the enzyme produce only faint indications of change in the spectrum, but the hydroxamates of several other L-amino acids perturb the spectrum essentially independently of the identity of the side chain and in a qualitatively different manner from that of D-valine hydroxamate and D-leucine hydroxamate. At the high enzyme:substrate ratios used in the spectral experiments, L-leucine hydroxamate and L-valine hydroxamate proved to be rapidly hydrolyzed, hence their inability to perturb the spectrum of the cobalt-substituted enzyme during the time course of a spectral experiment. Values of kcat for L-amino acid hydroxamates, all of which are good reversible inhibitors of the hydrolysis of L-leucine-p-nitroanilide by Aeromonas aminopeptidase, were found to range from 0.01 min-1 to 5.6 min-1 for the native enzyme and from 0.27 min-1 to 108 min-1 for the cobalt-substituted enzyme; their km values toward the cobalt aminopeptidase range from 1.2 X 10(-7) M to 1.9 X 10(-5) M. The mutual exclusivity of binding for hydroxamate inhibitors and 1-butaneboronic acid, previously shown by kinetics

  9. Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure.

    PubMed

    Lai, Amy; Hosseini-Tabatabaei, Azadeh; Hartwell, Ryan; Rahmani-Neishaboor, Elham; Kilani, Ruhangiz Taghi; Ghahary, Aziz

    2013-01-01

    Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal-dermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction. PMID:23054189

  10. Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library.

    PubMed

    Byzia, Anna; Szeffler, Agata; Kalinowski, Leszek; Drag, Marcin

    2016-03-01

    Aminopeptidases are exopeptidases that process peptide bonds at the N-terminus of protein substrates, and they are involved in controlling several metabolic pathways. Due to their involvement in diseases such as cancer or rheumatoid arthritis, their presence can also be used as a predictive biomarker. Here, we used a library of fluorogenic substrates containing natural and unnatural amino acids to reliably measure the aminopeptidase N (APN) activity in cell lysates obtained from human, pig and rat kidneys. We compared our results to the substrate specificity profile of isolated APN. Our data strongly support the observation that fluorogenic substrates can be successfully used to identify aminopeptidases and to measure their activity in cell lysates. Moreover, in contrast to assays using single substrates, which can result in overlapping specificity due to cleavage by several aminopeptidases, our library fingerprint can provide information about single enzymes. PMID:26449746

  11. Development of bestatin-based activity-based probes for metallo-aminopeptidases

    PubMed Central

    Harbut, Michael B.; Velmourougane, Geetha; Reiss, Gilana; Chandramohanadas, Rajesh; Greenbaum, Doron C.

    2009-01-01

    A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be amenable to ‘click’ chemistry labeling for possible use in live cells. Finally, we demonstrate that the ABPs are able to label an aminopeptidase in a complex proteome. Thus, these bestatin-based probes should have wide utility to functionally profile aminopeptidases in many biological systems. PMID:18823778

  12. Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema1

    PubMed Central

    Paige, Mikell; Wang, Kan; Burdick, Marie; Park, Sunhye; Cha, Josiah; Jeffrey, Erin; Sherman, Nicholas; Shim, Y. Michael

    2014-01-01

    The leukotriene A4 hydrolase (LTA4H) is a bi-functional enzyme with an epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing Proline-Glycine-Proline (PGP), a tri-amino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild type mice to CS over five months while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bio-production of leukotriene B4 (LTB4). Emphysematous phenotypes were assessed by pre mortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the BALF and infiltration of neutrophils into the lungs were significant reduced without affecting the levels of LTB4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the LTA4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema. PMID:24771855

  13. Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase.

    PubMed

    Rose, Jonathan A; Lahiri, Sushmita D; McKinney, David C; Albert, Rob; Morningstar, Marshall L; Shapiro, Adam B; Fisher, Stewart L; Fleming, Paul R

    2015-08-15

    With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections. PMID:26099541

  14. Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis.

    PubMed

    Kenna, Tony J; Robinson, Philip C; Haroon, Nigil

    2015-09-01

    There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS. PMID:26070942

  15. Substrate-dependent nitric oxide synthesis by secreted endoplasmic reticulum aminopeptidase 1 in macrophages.

    PubMed

    Goto, Yoshikuni; Ogawa, Kenji; Nakamura, Takahiro J; Hattori, Akira; Tsujimoto, Masafumi

    2015-06-01

    In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)-γ and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis. PMID:25577645

  16. Endoplasmic reticulum aminopeptidase 1 and rheumatic disease: functional variation

    PubMed Central

    Tran, Tri M.; Colbert, Robert A.

    2015-01-01

    Purpose of review To review recent developments in our understanding of endoplasmic reticulum (ER) aminopeptidase-1 (ERAP1) function in relation to its role in MHC class I peptide presentation and HLA class I-associated diseases. Recent findings ERAP1 polymorphisms exhibiting loss-of-function have been associated with protection from ankylosing spondylitis (AS). The aminopeptidase function of ERAP1 optimizes peptides for binding and presentation by MHC class I. Most studies have revealed reduced MHC class I expression in situations of reduced ERAP1 function. Under these circumstances the presented peptides are often N-terminally extended, and cell surface complexes are unstable and fall apart more readily. In contrast, peptides presented by HLA-B*27:05 when ERAP1 is silenced are frequently extended on the C-terminus. Recent work has emphasized the importance of assessing the function of allotypes encoded by ERAP1 haplotypes, rather than effects of single amino acid substitutions. The allotypes found in a series of AS patients were poorer at restoring HLA-B27 expression than allotypes found in unaffected controls, which may seem contrary to the genetic data linking loss-of-function to protection. Summary More work is needed to understand how ERAP1 variants associated with risk and protection influence the quality and quantity of peptides available for binding to HLA class I molecules in the ER. Moreover, we need to determine allele-specific effects of ERAP1 variants in the context of HLA-B*51 and HLA-Cw*6, which are associated with Behçet’s disease and psoriasis, respectively. PMID:26002027

  17. VLT/X-shooter spectroscopy of massive pre-main-sequence stars in M17

    NASA Astrophysics Data System (ADS)

    Ramirez-Tannus, Maria Claudia; Kaper, Lex

    2015-08-01

    The formation process of massive stars is still poorly understood. Formation timescales are short, the corresponding accretion rates very high, and the forming stars are hidden from view due to vast amounts of interstellar extinction. On top of that, massive stars are rare, are located at relatively large distances, and play a major role in shaping the interstellar medium due to their strong UV radiation fields and stellar winds. Although massive stars show most spectral features in the UV and optical range, so far only for a handful of massive Young Stellar Objects (mYSOs) optical and near-infrared spectra have been obtained. For some of these their pre-main-sequence (PMS) nature has now been firmly established (e.g. Ochsendorf et al. 2011, Ellerbroek et al. 2013). The objective of our project is to determine the physical properties of mYSOs, to search for signatures remnant of their formation process and to better understand the feedback on their environment.To this aim the optical to near-infrared (300-2500 nm) spectra of six candidate mYSOs (Hanson et al. 1997), deeply embedded in the massive star forming region M17, have been obtained with X-Shooter on the ESO Very Large Telescope. These mYSO candidates have been identified based on their infrared excess and spectral features (double-peaked emission lines, CO band-head emission) indicating the presence of a disk. In most cases, we detect a photospheric spectrum allowing us to measure the physical properties of the mYSO and to confirm its PMS nature. We also uncover many emission features, including forbidden lines, providing information on the (active) formation process of these young (massive) stars.

  18. [Comparative study of effects of Escherichia coli M-17 exometabolites and fructooligosaccharides on the growth and antagonistic activity of Lactobacilli].

    PubMed

    Vakkhitov, T Ia; Dobrolezh, O V; Petrov, L N; Verbitskaia, N B; Zadaura, E Iu

    2001-01-01

    Facts concerning the evaluation of the influence of E. coli M17 exometabolites and fructooligosaccharides (FOS) on the growth and antagonistic activity of lactobacilli are presented. As revealed by these facts, preparation "Aktoflor" accelerates the growth of lactobacillary cultures, increases the final yield of biomass and antagonistic activity. E. coli M17 exometabolites contained in "Aktoflor" have been shown to be more active in comparison with FOS. The character of their influence on lactobacilli is discussed and the conclusion is made that the restoration and maintenance of eubiosis is greatly determined by the pool of metabolites excreted by the bacteria. PMID:11550569

  19. Near-Infrared and CO (J=1-0) Observations of Photodissociation Regions in M17

    NASA Astrophysics Data System (ADS)

    Ando, Minoru; Nagata, Tetsuya; Sato, Shuji; Mizuno, Norikazu; Mizuno, Akira; Kawai, Toshihide; Nakaya, Hidehiko; Glass, Ian S.

    2002-07-01

    We have carried out near-infrared mapping observations of photodissociation regions in M17 with the Wide Field Cryogenic Telescope and CO (J=1-0) observations in three isotope lines with the ``NANTEN'' telescope. The observations covered an area of 20'×20' with a spatial resolution of 5.6" for near-infrared wavelengths and with a half-power beamwidth of 2.7‧ for millimeter wavelengths. We detected 38 sources brighter than 7 mag at 3.67 μm (Ln band), five of which show signs of young stellar objects. We have detected two emission bars (the N bar and the S bar) in all four near-infrared bands (J, K, Ln, and 3.3 μm). Their spatial distributions differ considerably from band to band, and we have compared them with the radio continuum, the mid-infrared data, and the CO molecular line emission. The different brightness and spectral energy distributions at near-infrared wavelengths can be well explained by emission from hot dust and ionized gas together with obscuration by local cold dust with a steep gradient from north to south. In the N bar, the free-free emission from ionized gas dominates at shorter wavelengths (J and K) and there is little extinction, whereas in the S bar, the free-free emission is attenuated at shorter wavelengths by the heavy local extinction. In both the N and S bars, the thermal emission from hot dust at around 1000 K dominates in the Ln band. The 3.3 μm unidentified infrared (UIR) emission delineates photodissociation regions between the H II regions and the surrounding molecular clouds. The UIR intensity decreases exponentially from the UIR peak toward the molecular clouds, with scale lengths of 88" and 100", or 0.9 and 1.0 pc, at the N and the S bars, respectively. Far-ultraviolet photons, which excite UIR emission, penetrate into the molecular clouds for ~1 pc, in the nearly edge-on geometry. The 12CO contours are elongated in the direction northwest-southeast, while the C18O contours are round. Far-ultraviolet photons erode the

  20. Characterization of an N-glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris.

    PubMed

    Xi, Hongxing; Tian, Yaping; Zhou, Nandi; Zhou, Zhemin; Shen, Wei

    2015-02-01

    Aminopeptidase is an important flavorsome especially in protein hydrolysate debittering by removing hydrophobic amino acid residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, leucine aminopeptidase from Bacillus subtilis was cloned and expressed in Pichia pastoris, a widely used heterologous protein expression host. Then it was purified and characterized. After methanol induction for 96 h, the aminopeptidase activity in culture supernatant reached 28.4 U ml(À1) , which was 7.1 times that of wild strain B. subtilis Zj016. The optimal temperature and pH of the purified recombinant enzyme were 60 °C and 8.5, respectively. The purified aminopeptidase was stable within 30-60 °C and pH 8.0-9.0. It was intensively inhibited by Ni(2β) , Ca(2β) , DL-dithiothreitol (DTT) and ethylene diamine tetraacetic acid (EDTA), but activated by Co(2β) . The Km toward leucine-p-nitroanilines (Leu-pNA) of the enzyme was 0.97 mM. The sequence analysis of aminopeptidase indicated three potential N-glycosylation sites and it was further verified via MALDI-TOF-MS analysis. Consequently, the N-glycosylated aminopeptidase exhibited higher thermostability and catalytic efficiency. The purified enzyme exhibited two bands through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while a single band can be identified when the enzyme was deglycosylated. Circular dichroism spectroscopy indicated that the secondary structure of recombinant aminopeptidase was similar to the wild-type. PMID:25389014

  1. Submillimeter and far infrared line observations of M17 SW: A clumpy molecular cloud penetrated by UV radiation

    NASA Technical Reports Server (NTRS)

    Stutzki, J.; Stacey, G. J.; Genzel, R.; Harris, A. I.; Jaffe, d. T.; Lugten, J. B.

    1987-01-01

    Millimeter, submillimeter, and far infrared spectroscopic observations of the M17 SW star formation region are discussed. The results require the molecular cloud near the interface to be clumpy or filamentary. As a consequence, far ultraviolet radiation from the central OB stellar cluster can penetrate into the dense molecular cloud to a depth of several pc, thus creating bright and extended (CII) emission from the photodissociated surfaces of dense atomic and molecular clumps or sheets. The extended (CII) emission throughout the molecular cloud SW of the M17 complex has a level 20 times higher than expected from a single molecular cloud interface exposed to an ultraviolet radiation field typical of the solar neighborhood. This suggests that the molecular cloud as a whole is penetrated by ultraviolet radiation and has a clumpy or filamentary structure. The number of B stars expected to be embedded in the M17 molecular cloud probably can provide the UV radiation necessary for the extended (CII) emission. Alternatively, the UV radiation could be external, if the interstellar radiation in the vicinity of M17 is higher than in the solar neighborhood.

  2. Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms.

    PubMed

    Cellier, Marie; James, Arthur L; Orenga, Sylvain; Perry, John D; Rasul, Ari K; Robinson, Shaun N; Stanforth, Stephen P

    2014-10-01

    A series of amino acid derivatives 8-10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8-10 and blue coloured colonies were formed by the substrates 42 and 43. The L-alanyl aminopeptidase substrates 8 targeted L-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted β-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for L-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined. PMID:25172150

  3. Characterisation of the aminopeptidase from non-germinated winter rape (Brassica napus L.) seeds.

    PubMed

    Kania, Joanna; Gillner, Danuta M

    2016-09-15

    Rapeseed plays a crucial role in food and fuel industry. Since aminopeptidases take part in many physiological processes in all organisms, it is important to learn their role and characteristics in economically relevant plants. Extracts of non-germinated winter rape seeds were screened for aminopeptidase activity. Substrate specificity, the influence of pH and temperature, as well as effect of protease inhibitors and chosen metal ions on the aminopeptidase activity were determined. The approximate molecular weight estimated by NATIVE-PAGE and SDS-PAGE electrophoresis was ∼60 kDa. The partially purified enzyme as well as the aminopeptidases present in crude extract cleaved preferentially Phe-pNA. The activity profiles toward several substrates were also determined. Maximum activity was observed at pH 6.5 and temperature of 40 °C for Phe-pNA as a substrate. Two visible picks in the pH profile toward Phe-pNA, together with other results (IEF) suggest the presence of more than one aminopeptidase, having similar molecular mass. Much lower activity and broad pH profiles were observed for Leu- and Ala-pNA as substrates. PMID:27080895

  4. CHARACTERIZATION OF AMINOPEPTIDASE IN THE FREE-LIVING NEMATODE PANAGRELLUS REDIVIVUS: SUBCELLULAR DISTRIBUTION AND POSSIBLE ROLE IN NEUROPEPTIDE METABOLISM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide (Ala-4-NA). Subcellular distribution of the enzyme was unequal, with approximately 80 percent of total aminopeptidase in the soluble fraction and the rem...

  5. Partial purification and characterization of an aminopeptidase from Eimeria tenella.

    PubMed

    Fetterer, R H; Miska, K B; Barfield, R C

    2005-12-01

    Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an Mr of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted Mr is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts. PMID:16539006

  6. Debittering of Protein Hydrolysates by Lactobacillus LBL-4 Aminopeptidase

    PubMed Central

    Tchorbanov, Bozhidar; Marinova, Margarita; Grozeva, Lydia

    2011-01-01

    Yoghurt strain Lactobacillus LBL-4 cultivated for 8–10 h at pH ~6.0 was investigated as a considerable food-grade source of intracellular aminopeptidase. Cell-free extract manifesting >200 AP U/l was obtained from cells harvested from 1 L culture media. Subtilisin-induced hydrolysates of casein, soybean isolate, and Scenedesmus cell protein with degree of hydrolysis 20–22% incubated at 45°C for 10 h by 10 AP U/g peptides caused an enlarging of DH up to 40–42%, 46–48%, and 38–40% respectively. The DH increased rapidly during the first 4 h, but gel chromatography studies on BioGel P-2 showed significant changes occurred during 4–10 h of enzyme action when the DH increased gradually. After the digestion, the remained AP activity can be recovered by ultrafiltration (yield 40–50%). Scenedesmus protein hydrolysate with DH 20% was inoculated by Lactobacillus LBL-4 cells, and after 72 h cultivation the DH reached 32%. The protein hydrolysates (DH above 40%) obtained from casein and soybean isolate (high Q value) demonstrated a negligible bitterness while Scenedesmus protein hydrolysates (low Q value) after both treatments were free of bitterness. PMID:21876793

  7. Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidases by Bengamide Derivatives

    SciTech Connect

    Lu, Jing-Ping; Yuan, Xiu-Hua; Yuan, Hai; Wang, Wen-Long; Wan, Baojie; Franzblau, Scott G.; Ye, Qi-Zhuang

    2012-05-29

    Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.

  8. Characterization and subcellular localization of aminopeptidases in senescing barley leaves

    NASA Technical Reports Server (NTRS)

    Thayer, S. S.; Choe, H. T.; Rausser, S.; Huffaker, R. C.

    1988-01-01

    Four aminopeptidases (APs) were separated using native polyacrylamide gel electrophoresis of cell-free extracts and the stromal fractions of isolated chloroplasts prepared from primary barley (Hordeum vulgare L., var Numar) leaves. Activities were identified using a series of aminoacyl-beta-naphthylamide derivatives as substrates. AP1, 2, and 3 were found in the stromal fraction of isolated chloroplasts with respective molecular masses of 66.7, 56.5, and 54.6 kilodaltons. AP4 was found only in the cytoplasmic fraction. No AP activity was found in vacuoles of these leaves. It was found that 50% of the L-Leu-beta-naphthylamide and 25% of the L-Arg-beta-naphthylamide activities were localized in the chloroplasts. Several AP activities were associated with the membranes of the thylakoid fraction of isolated chloroplasts. AP1, 2, and 4 reacted against a broad range of substrates, whereas AP3 hydrolyzed only L-Arg-beta-naphthylamide. Only AP2 hydrolyzed L-Val-beta-naphthylamide. Since AP2 and AP3 were the only ones reacting against Val-beta-naphthylamide and Arg-beta-naphthylamide, respectively, several protease inhibitors were tested against these substrates using a stromal fraction from isolated chloroplasts as the source of the two APs. Both APs were sensitive to both metallo and sulfhydryl type inhibitors. Although AP activity decreased as leaves senesced, no new APs appeared on gels during senescence and none disappeared.

  9. An Aminopeptidase in the Drosophila Testicular Niche Acts in Germline Stem Cell Maintenance and Spermatogonial Dedifferentiation.

    PubMed

    Lim, Cindy; Gandhi, Shiv; Biniossek, Martin L; Feng, Lijuan; Schilling, Oliver; Urban, Siniša; Chen, Xin

    2015-10-13

    Extrinsic cues from the niche are known to regulate adult stem cell self-renewal versus differentiation. Here, we report that an aminopeptidase Slamdance (Sda) acts in the Drosophila testicular niche to maintain germline stem cells (GSCs) and regulate progenitor germ cell dedifferentiation. Mutations in sda lead to dramatic testicular niche deterioration and stem cell loss. Recombinant Sda has specific aminopeptidase activity in vitro, and the in vivo function of Sda requires an intact aminopeptidase domain. Sda is required for accumulation of mature DE-cadherin, and overexpression of DE-cadherin rescues most sda mutant phenotypes, suggesting that DE-cadherin is an important target of Sda. Finally, Sda is both necessary and sufficient to promote dedifferentiation during aging and recovery from genetically manipulated depletion of GSCs. Together, our results suggest that a niche factor promotes both stem cell maintenance and progenitor cell dedifferentiation. PMID:26440886

  10. The Origin of Hard X-rays in M17's Remarkable O4-O4 Binary

    NASA Astrophysics Data System (ADS)

    Gagne, Marc

    2008-09-01

    At 2 kpc M17 is the nearest giant HII region, with 10,000 stars younger than 1 Myr and a prominent X-ray champagne flow. At the heart of M17 are CEN 1A and 1B, a visual pair of O4 stars separated by only 1.8". A long series of ACIS observations show that each O4 star is a very hard time variable X-ray source. We suggest that one or both O4 stars are themselves colliding wind shock binaries or, alternatively, that one or both have strong magnetic fields. We propose to obtain time-resolved, spatially resolved HEG/MEG spectra of both stars to measure He-like line ratios and line profiles to directly test these two models. This roll-constrained 150-ks GO proposal is tied to a 25-ks GTO proposal (PI: Garmire) for the same target.

  11. Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

    PubMed Central

    2013-01-01

    Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

  12. Antigenic peptide trimming by ER aminopeptidases--insights from structural studies.

    PubMed

    Stratikos, Efstratios; Stern, Lawrence J

    2013-10-01

    Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes. PMID:23545452

  13. Plasmodium falciparum: attenuation by irradiation

    SciTech Connect

    Waki, S.; Yonome, I.; Suzuki, M.

    1983-12-01

    The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.

  14. Exchange Transfusion in Severe Falciparum Malaria

    PubMed Central

    Khatib, Khalid Ismail

    2016-01-01

    Malaria is endemic in India with the incidence of P. falciparum Malaria increasing gradually over the last decade. Severe malaria is an acute disease, caused by P. falciparum, but increasingly also by P. vivax with major signs of organ dysfunction and/or high levels of parasitaemia (>10%) in blood smear. Use of exchange transfusion with antimalarial drug therapy as an additional modality of treatment in severe Falciparum malaria is controversial and is unclear. We report a case of severe malaria complicated by multiorgan failure and ARDS. Patient responded well to manual exchange transfusion with standard artesunate-based chemotherapy. PMID:27042503

  15. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  16. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions. PMID:27221740

  17. Rapid Circumstellar Disk Evolution and an Accelerating Star Formation Rate in the Infrared Dark Cloud M17 SWex

    NASA Astrophysics Data System (ADS)

    Povich, Matthew S.; Townsley, Leisa K.; Robitaille, Thomas P.; Broos, Patrick S.; Orbin, Wesley T.; King, Robert R.; Naylor, Tim; Whitney, Barbara A.

    2016-07-01

    We present a catalog of 840 X-ray sources and first results from a 100 ks Chandra X-ray Observatory imaging study of the filamentary infrared (IR) dark cloud G014.225–00.506, which forms the central regions of a larger cloud complex known as the M17 southwest extension (M17 SWex). In addition to the rich population of protostars and young stellar objects with dusty circumstellar disks revealed by archival data from the Spitzer Space Telescope, we discover a population of X-ray-emitting, intermediate-mass pre-main-sequence stars that lack IR excess emission from circumstellar disks. We model the IR spectral energy distributions of this source population to measure its mass function and place new constraints on the destruction timescales for the inner dust disk for 2–8 M ⊙ stars. We also place a lower limit on the star formation rate (SFR) and find that it is quite high (\\dot{M}≥slant 0.007 M ⊙ yr‑1), equivalent to several Orion Nebula Clusters in G14.225–0.506 alone, and likely accelerating. The cloud complex has not produced a population of massive, O-type stars commensurate with its SFR. This absence of very massive (≳20 M ⊙) stars suggests that either (1) M17 SWex is an example of a distributed mode of star formation that will produce a large OB association dominated by intermediate-mass stars but relatively few massive clusters, or (2) the massive cores are still in the process of accreting sufficient mass to form massive clusters hosting O stars.

  18. Phage displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by po...

  19. Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

    PubMed Central

    Zervoudi, Efthalia; Papakyriakou, Athanasios; Georgiadou, Dimitra; Evnouchidou, Irini; Gajda, Anna; Poreba, Marcin; Salvesen, Guy S.; Drag, Marcin; Hattori, Akira; Swevers, Luc; Vourloumis, Dionisios; Stratikos, Efstratios

    2014-01-01

    Synopsis ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and Insulin Regulated aminopeptidase (IRAP) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding onto MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorogenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the primary specificity (S1) pocket. Molecular modeling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP however, does not achieve this dual specificity by simply combining structural features of ERAP1 and 2, but rather by a unique amino acid change at position 541. Our results provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes. PMID:21314638

  20. COMPARISON OF ALANINE AMINOPEPTIDASE ACTIVITIES IN HETERODERA GLYCINES AND CAENORHABDITIS ELEGANS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aminopeptidase activities in the cytosolic fraction of whole body homogenates of Caenorhabditis elegans and Heterodera glycines were examined. Activities were detected using a colorimetric assay based upon hydrolysis of aminoacyl p-nitroanilides (Xxx-pNA). Properties including substrate preference...

  1. Detectability of Plasmodium falciparum clones

    PubMed Central

    2010-01-01

    Background In areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed. Methods A longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion. Results The distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted. Conclusions A simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week

  2. Cordycepin induces apoptosis and autophagy in human neuroblastoma SK-N-SH and BE(2)-M17 cells

    PubMed Central

    LI, YIFAN; LI, RONG; ZHU, SHENGLANG; ZHOU, RUYUN; WANG, LEI; DU, JIHUI; WANG, YONG; ZHOU, BEI; MAI, LIWEN

    2015-01-01

    Cordycepin, also termed 3′-deoxyadenosine, is a derivative of the nucleoside adenosine that represents a potential novel class of anticancer drugs targeting the 3′ untranslated region of RNAs. Cordycepin has been reported to induce apoptosis in certain cancer cell lines, but the effects of cordycepin on human neuroblastoma cells have not been studied. In the present study, an MTT assay revealed that cordycepin inhibits the viability of neuroblastoma SK-N-SH and BE(2)-M17 cells in a dose-dependent manner. In addition, cordycepin increases the early-apoptotic cell population of SK-N-SH cells, as determined by fluorescence-activated cell sorting analysis. The induction of apoptosis in neuroblastoma cells by cordycepin was further confirmed by western blotting, which revealed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase 1 in the SK-N-SH and BE(2)-M17 cells. Cordycepin also induced the formation of a punctate pattern of light-chain 3 (LC3)-associated green fluorescence in the SK-N-SH cells transfected with a pEGFP-LC3 vector. Furthermore, western blotting revealed cleavage of LC3 A/B in cordycepin-treated neuroblastoma SK-N-SH cells. Taken together, the results indicate that cordycepin significantly increases apoptosis and autophagy in neuroblastoma cells, and may therefore be a drug candidate for neuroblastoma therapy, but requires additional evaluation. PMID:26137103

  3. Interstellar Weather Vanes: GLIMPSE Mid-Infrared Stellar Wind Bow Shocks in M17 and RCW 49

    NASA Astrophysics Data System (ADS)

    Povich, Matthew S.; Benjamin, Robert A.; Whitney, Barbara A.; Babler, Brian L.; Indebetouw, Rémy; Meade, Marilyn R.; Churchwell, Ed

    2008-12-01

    We report the discovery of six infrared stellar wind bow shocks in the Galactic massive star formation regions M17 and RCW 49 from Spitzer GLIMPSE (Galactic Legacy Infrared Mid-Plane Survey Extraordinaire) images. The Infrared Array Camera (IRAC) on the Spitzer Space Telescope clearly resolves the arc-shaped emission produced by the bow shocks. We combine Two Micron All-Sky Survey (2MASS), Spitzer, MSX, and IRAS observations to obtain the spectral energy distributions (SEDs) of the bow shocks and their individual driving stars. We use the stellar SEDs to estimate the spectral types of the three newly identified O stars in RCW 49 and one previously undiscovered O star in M17. One of the bow shocks in RCW 49 reveals the presence of a large-scale flow of gas escaping the H II region at a few 102 km s-1. Radiation transfer modeling of the steep rise in the SED of this bow shock toward longer mid-infrared wavelengths indicates that the emission is coming principally from dust heated by the star driving the shock. The other five bow shocks occur where the stellar winds of O stars sweep up dust in the expanding H II regions.

  4. Epigenetic regulation of the Plasmodium falciparum genome.

    PubMed

    Duffy, Michael F; Selvarajah, Shamista A; Josling, Gabrielle A; Petter, Michaela

    2014-05-01

    Recent research has highlighted some unique aspects of chromatin biology in the malaria parasite Plasmodium falciparum. During its erythrocytic lifecycle P. falciparum maintains its genome primarily as unstructured euchromatin. Indeed there is no clear role for chromatin-mediated silencing of the majority of the developmentally expressed genes in P. falciparum. However discontinuous stretches of heterochromatin are critical for variegated expression of contingency genes that mediate key pathogenic processes in malaria. These range from invasion of erythrocytes and antigenic variation to solute transport and growth adaptation in response to environmental changes. Despite lack of structure within euchromatin the nucleus maintains functional compartments that regulate expression of many genes at the nuclear periphery, particularly genes with clonally variant expression. The typical components of the chromatin regulatory machinery are present in P. falciparum; however, some of these appear to have evolved novel species-specific functions, e.g. the dynamic regulation of histone variants at virulence gene promoters. The parasite also appears to have repeatedly acquired chromatin regulatory proteins through lateral transfer from endosymbionts and from the host. P. falciparum chromatin regulators have been successfully targeted with multiple drugs in laboratory studies; hopefully their functional divergence from human counterparts will allow the development of parasite-specific inhibitors. PMID:24326119

  5. VLT near- to mid-IR imaging and spectroscopy of the M 17 UC1 - IRS5 region

    NASA Astrophysics Data System (ADS)

    Chen, Zhiwei; Nürnberger, Dieter E. A.; Chini, Rolf; Jiang, Zhibo; Fang, Min

    2015-06-01

    Aims: We investigate the surroundings of the hypercompact H ii region M 17 UC1 to probe the physical properties of the associated young stellar objects and the environment of massive star formation. Methods: We use diffraction-limited near-IR (VLT/NACO) and mid-IR (VLT/VISIR) images to reveal the different morphologies at various wavelengths. Likewise, we investigate the stellar and nebular content of the region with VLT/SINFONI integral field spectroscopy with a resolution R ˜ 1500 at H + K bands. Results: Five of the seven point sources in this region show L-band excess emission. A geometric match is found between the H2 emission and near-IR polarized light in the vicinity of IRS5A, and between the diffuse mid-IR emission and near-IR polarization north of UC1. The H2 emission is typical for dense photodissociation regions (PDRs), which are initially far-ultraviolet pumped and repopulated by collisional de-excitation. The spectral types of IRS5A and B273A are B3-B7 V/III and G4-G5 III, respectively. The observed infrared luminosity LIR in the range 1-20 μm is derived for three objects; we obtain 2.0 × 103 L⊙ for IRS5A, 13 L⊙ for IRS5C, and 10 L⊙ for B273A. Conclusions: IRS5 might be a young quadruple system. Its primary star IRS5A is confirmed to be a high-mass protostellar object (˜9 M⊙, ˜1 × 105 yrs); it might have terminated accretion due to the feedback from stellar activities (radiation pressure, outflow) and the expanding H ii region of M 17. The object UC1 might also have terminated accretion because of the expanding hypercompact H ii region, which it ionizes. The disk clearing process of the low-mass young stellar objects in this region might be accelerated by the expanding H ii region. The outflows driven by UC1 are running south-north with its northeastern side suppressed by the expanding ionization front of M 17; the blue-shifted outflow lobe of IRS5A is seen in two types of tracers along the same line of sight in the form of H2 emission

  6. Detection of a large fraction of atomic gas not associated with star-forming material in M17 SW⋆

    NASA Astrophysics Data System (ADS)

    Pérez-Beaupuits, J. P.; Stutzki, J.; Ossenkopf, V.; Spaans, M.; Güsten, R.; Wiesemeyer, H.

    2015-03-01

    Context. The [C II] 158 μm line is one of the dominant coolants of the ISM, and an important probe with which to study the star formation process. Recent Herschel/HIFI and SOFIA/GREAT observations showed that assuming the total velocity-integrated intensity of this line is directly associated with the star-forming material is inadequate. Aims: We probe the column densities and masses traced by the ionized and neutral atomic carbon with spectrally resolved maps, and compare them to the diffuse and dense molecular gas traced by [C I] and low-J CO lines toward the star-forming region M17 SW. Methods: We mapped a 4.1 pc × 4.7 pc region in the [C I] 609 μm line using the APEX telescope, as well as the CO isotopologues with the IRAM 30 m telescope. Because of the velocity-resolved spectra, we analyze the data based on velocity channel maps that are 1 km s-1 wide. We correlate their spatial distribution with that of the [C II] map obtained with SOFIA/GREAT. Optically thin approximations were used to estimate the column densities of [C I] and [C II] in each velocity channel. Results: The distribution of the emission from the isotopologues 13CO, C17O, and C18O resembles more closely that of the [C I] emission than that of the 12CO emission. The spatial distribution of the [C I] and all CO isotopologues emission was found to be associated with that of [C II] in about 20%-80% of the mapped region, with the high correlation found in the central (15-23 km s-1) velocity channels. Conclusions: The excitation temperature of [C I] ranges between 40 K and 100 K in the inner molecular region of M17 SW. Excitation temperatures up to 200 K are found along the ridge. Column densities in 1 km s-1 channels between ~1015 cm-2 and ~1017 cm-2 were found for [C I]. Just ~20 % of the velocity range (~40 km s-1) that the [C II] line spans is associated with the star-forming material traced by [C I] and CO. The total (integrated over the 0-40 km s-1 velocity range) gas mass estimated from the

  7. Anaemia of Plasmodium falciparum malaria.

    PubMed

    Phillips, R E; Pasvol, G

    1992-04-01

    The pathophysiology of the anaemia of falciparum malaria is both complex and multifactorial, and results in a condition which is a major cause of mortality and morbidity in patients, especially children and pregnant women, living in malarial endemic areas. The importance of anaemia as a cause of death in malaria may well be underestimated because of difficulty in diagnosis, especially where parasitaemia may be low and the clinical picture may be confused with other causes of anaemia. Two clinical presentations predominate: severe acute malaria in which anaemia supervenes, and severe anaemia in patients in whom there have been repeated attacks of malaria. The major mechanisms are those of red cell destruction and decreased red cell production. Potential causes of haemolysis include loss of infected cells by rupture or phagocytosis, removal of uninfected cells due to antibody sensitization or other physicochemical membrane changes, and increased reticuloendothelial activity, particularly in organs such as the spleen. Decreased production results from marrow hypoplasia seen in acute infections, and dyserythropoiesis, a morphological appearance, which in functional terms results in ineffective erythropoiesis. The role of parvovirus B19 as a possible cause of bone marrow aplasia in a few cases is postulated. Finally, there is now evidence which points to genetic factors, HLA associated, which may protect against the development of malarial anaemia and which has become common in areas endemic for malaria. PMID:1511178

  8. In Vitro Generation of Plasmodium falciparum Ookinetes

    PubMed Central

    Bounkeua, Viengngeun; Li, Fengwu; Vinetz, Joseph M.

    2010-01-01

    Plasmodium transmission from the human host to the mosquito depends on the ability of gametocytes to differentiate into ookinetes, the invasive form of the parasite that invades and establishes infection in the mosquito midgut. The biology of P. falciparum ookinetes is poorly understood, because sufficient quantities of this stage of this parasite species have not been obtained for detailed study. This report details methods to optimize production of P. falciparum sexual stage parasites, including ookinetes. Flow cytometric sorting was used to separate diploid/tetraploid zygotes and ookinetes from haploid gametetocytes and unfertilized gametes based on DNA content. Consistent production of 106–107 P. falciparum ookinetes per 10 mL culture was observed, with ookinete transformation present in 10–40% of all parasite forms. Transmission electron micrographs of cultured parasites confirmed ookinete development. PMID:21118920

  9. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    PubMed Central

    Kusch, Peter; Deininger, Susanne; Specht, Sabine; Maniako, Rudeka; Haubrich, Stefanie; Pommerening, Tanja; Lin, Paul Kong Thoo; Hoerauf, Achim; Kaiser, Annette

    2011-01-01

    Balanites aegyptiaca (Balanitaceae) is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 μg/μL ± 3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H)-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a Ki of 2.35 μg/μL and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a Ki of 4.8 μg/μL. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 μM ± 3 and 47.82 μM ± 2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed. PMID:21687598

  10. OF4949, new inhibitors of aminopeptidase B. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Sano, S; Ikai, K; Kuroda, H; Nakamura, T; Obayashi, A; Ezure, Y; Enomoto, H

    1986-12-01

    New aminopeptidase B inhibitors that we named OF4949-I, II, III and IV were isolated from the culture broth of a fungus, Penicillium rugulosum OF4949. The molecular formula of I was C23H26N4O8 and that of II, C22H24O8, judging from elemental analysis and secondary ion mass spectrometry. The concentrations of I, II, III and IV required for 50% inhibition of aminopeptidase, using Ehrlich ascites carcinoma cells as the source of the enzyme, were 0.0054, 0.0048, 3.4 and 1.7 micrograms/ml, respectively. Components I and II augmented delayed-type hypersensitivity in mice to sheep red blood cells. PMID:3818441

  11. Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides

    PubMed Central

    Hermans, Stefan J; Ascher, David B; Hancock, Nancy C; Holien, Jessica K; Michell, Belinda J; Yeen Chai, Siew; Morton, Craig J; Parker, Michael W

    2015-01-01

    Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure–activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease. PMID:25408552

  12. Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides.

    PubMed

    Hermans, Stefan J; Ascher, David B; Hancock, Nancy C; Holien, Jessica K; Michell, Belinda J; Chai, Siew Yeen; Morton, Craig J; Parker, Michael W

    2015-02-01

    Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease. PMID:25408552

  13. Purification and amino acid sequence of aminopeptidase P from pig kidney.

    PubMed

    Vergas Romero, C; Neudorfer, I; Mann, K; Schäfer, W

    1995-04-01

    Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. PMID:7744038

  14. The complement of family M1 aminopeptidases of Haemonchus contortus--Biotechnological implications.

    PubMed

    Mohandas, Namitha; Young, Neil D; Jabbar, Abdul; Korhonen, Pasi K; Koehler, Anson V; Hall, Ross S; Hu, Min; Hofmann, Andreas; Gasser, Robin B

    2016-01-01

    Although substantial research has been focused on the 'hidden antigen' H11 of Haemonchus contortus as a vaccine against haemonchosis in small ruminants, little is know about this and related aminopeptidases. In the present article, we reviewed genomic and transcriptomic data sets to define, for the first time, the complement of aminopeptidases (designated Hc-AP-1 to Hc-AP-13) of the family M1 with homologues in Caenorhabditis elegans, characterised by zinc-binding (HEXXH) and exo-peptidase (GAMEN) motifs. The three previously published H11 isoforms (accession nos. X94187, FJ481146 and AJ249941) had most sequence similarity to Hc-AP-2 and Hc-AP-8, whereas unpublished isoforms (accession nos. AJ249942 and AJ311316) were both most similar to Hc-AP-3. The aminopeptidases characterised here had homologues in C. elegans. Hc-AP-1 to Hc-AP-8 were most similar in amino acid sequence (28-41%) to C. elegans T07F10.1; Hc-AP-9 and Hc-AP-10 to C. elegans PAM-1 (isoform b) (53-54% similar); Hc-AP-11 and Hc-AP-12 to C. elegans AC3.5 and Y67D8C.9 (26% and 50% similar, respectively); and Hc-AP-13 to C. elegans C42C1.11 and ZC416.6 (50-58% similar). Comparative analysis suggested that Hc-AP-1 to Hc-AP-8 play roles in digestion, metabolite excretion, neuropeptide processing and/or osmotic regulation, with Hc-AP-4 and Hc-AP-7 having male-specific functional roles. The analysis also indicated that Hc-AP-9 and Hc-AP-10 might be involved in the degradation of cyclin (B3) and required to complete meiosis. Hc-AP-11 represents a leucyl/cystinyl aminopeptidase, predicted to have metallopeptidase and zinc ion binding activity, whereas Hc-AP-12 likely encodes an aminopeptidase Q homologue also with these activities and a possible role in gonad function. Finally, Hc-AP-13 is predicted to encode an aminopeptidase AP-1 homologue of C. elegans with hydrolase activity, suggested to operate, possibly synergistically with a PEPT-1 ortholog, as an oligopeptide transporter in the gut for protein uptake

  15. The Role of Insulin-Regulated Aminopeptidase in MHC Class I Antigen Presentation

    PubMed Central

    Saveanu, Loredana; van Endert, Peter

    2012-01-01

    Production of MHC-I ligands from antigenic proteins generally requires multiple proteolytic events. While the proteolytic steps required for antigen processing in the endogenous pathway are clearly established, persisting gaps of knowledge regarding putative cross-presentation compartments have made it difficult to map the precise proteolytic events required for generation of cross-presented antigens. It is only in the past decade that the importance of aminoterminal trimming as the final step in the endogenous presentation pathway has been recognized and that the corresponding enzymes have been described. This review focuses on the aminoterminal trimming of exogenous cross-presented peptides, with particular emphasis on the identification of insulin responsive aminopeptidase (IRAP) as the principal trimming aminopeptidase in endosomes and phagosomes. PMID:22566938

  16. Bioluminogenic Imaging of AminopeptidaseN In Vitro and In Vivo.

    PubMed

    Wu, Wenxiao; Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

    2016-01-01

    Bioluminescence is a process that converts biochemical energy to visible light. Bioluminescence-based imaging technology has been widely applicable in the imaging of process envisioned in life sciences. As one of the most popular bioluminescence system, the firefly luciferin-luciferase system is exceptionally suitable for deep tissue imaging in living animals owing to its long wavelength emission light. Herein, we report the experimental detail of bioluminogenic imaging of aminopeptidase N activity both in vitro and in vivo. PMID:27424897

  17. A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A.

    PubMed

    Vazeux, G; Iturrioz, X; Corvol, P; Llorens-Cortes, C

    1998-09-01

    Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound aminopeptidase that contains the consensus sequence HEXXH (385-389) found in the zinc metalloprotease family, the zincins. Sequence alignment of the mouse APA with other monozinc-aminopeptidases indicates the presence of a highly conserved glutamate residue (Glu352 in APA) found in the conserved motif GAMEN (349-353). In monozinc-aminopeptidases, the negative charge of the glutamate side-chain carboxylate may constitute the anionic binding site involved in the recognition of the free amino group of substrates or inhibitors. The functional role of Glu352 in APA was investigated by substituting this residue with an aspartate (Asp352), a glycine (Gly352), a glutamine (Gln352) or an arginine (Arg352) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of the mutant enzymes were unaffected, whereas kcat values were decreased 10-250-fold, resulting in a 10-, 30- 260- and 400-fold reduction in cleavage efficiencies for the mutants Asp352, Gly352, Gln352 and Arg352 respectively. The inhibitory potency of two different classes of inhibitors, a thiol and a phosphonate compound, was significantly (P<0.05) decreased by 10- and 4-fold respectively in the mutated enzymes. Moreover, the inhibitory potency of angiotensin I, used as a competitor of the synthetic substrate alpha-l-glutamyl beta-naphthylamide, displayed a 4-fold reduction (P<0.01) in the mutated enzymes, whereas the Ki values of its N-acetyl derivative were unchanged. These data strongly suggest that Glu352 is involved in the catalytic process of APA and contributes to the exopeptidase activity of this enzyme through interaction with the N-terminal part of substrates or inhibitors. PMID:9716499

  18. Inactivation of Caenorhabditis elegans aminopeptidase DNPP-1 restores endocytic sorting and recycling in tat-1 mutants

    PubMed Central

    Li, Xin; Chen, Baohui; Yoshina, Sawako; Cai, Tanxi; Yang, Fuquan; Mitani, Shohei; Wang, Xiaochen

    2013-01-01

    In Caenorhabditis elegans, the P4-ATPase TAT-1 and its chaperone, the Cdc50 family protein CHAT-1, maintain membrane phosphatidylserine (PS) asymmetry, which is required for membrane tubulation during endocytic sorting and recycling. Loss of tat-1 and chat-1 disrupts endocytic sorting, leading to defects in both cargo recycling and degradation. In this study, we identified the C. elegans aspartyl aminopeptidase DNPP-1, loss of which suppresses the sorting and recycling defects in tat-1 mutants without reversing the PS asymmetry defect. We found that tubular membrane structures containing recycling cargoes were restored in dnpp-1 tat-1 double mutants and that these tubules overlap with RME-1–positive recycling endosomes. The restoration of the tubular structures in dnpp-1 tat-1 mutants requires normal functions of RAB-5, RAB-10, and RME-1. In tat-1 mutants, we observed alterations in membrane surface charge and targeting of positively charged proteins that were reversed by loss of dnpp-1. DNPP-1 displays a specific aspartyl aminopeptidase activity in vitro, and its enzymatic activity is required for its function in vivo. Our data reveal the involvement of an aminopeptidase in regulating endocytic sorting and recycling and suggest possible roles of peptide signaling and/or protein metabolism in these processes. PMID:23427264

  19. Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

    SciTech Connect

    Danielsen, E.M. )

    1990-01-09

    The pig intestinal brush border enzymes aminopeptidase and lactase-phlorizin hydrolase are present in the microvilla membrane as homodimers. Dimethyl adipimidate was used to cross-link the two ({sup 35}S)methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.

  20. MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae.

    PubMed

    Jarocki, Veronica M; Santos, Jerran; Tacchi, Jessica L; Raymond, Benjamin B A; Deutscher, Ania T; Jenkins, Cheryl; Padula, Matthew P; Djordjevic, Steven P

    2015-01-01

    Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae. PMID:25589579

  1. MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

    PubMed Central

    Jarocki, Veronica M.; Santos, Jerran; Tacchi, Jessica L.; Raymond, Benjamin B. A.; Deutscher, Ania T.; Jenkins, Cheryl; Padula, Matthew P.; Djordjevic, Steven P.

    2015-01-01

    Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae. PMID:25589579

  2. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase*

    PubMed Central

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

    2012-01-01

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases. PMID:22356908

  3. Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

    PubMed

    Müller, A; Benoist, P; Diem, H G; Schwencke, J

    1991-12-01

    To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest. PMID:15101385

  4. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase

    SciTech Connect

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

    2012-07-11

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.

  5. Genetic distance in housekeeping genes between Plasmodium falciparum and Plasmodium reichenowi and within P. falciparum.

    PubMed

    Tanabe, Kazuyuki; Sakihama, Naoko; Hattori, Tetsuya; Ranford-Cartwright, Lisa; Goldman, Ira; Escalante, Ananias A; Lal, Altaf A

    2004-11-01

    The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca2+ -ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672 +/- 0.0088 and 0.0011 +/- 0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30 +/- 5.40) x 10(4) and (11.62 +/- 7.56) x 10(4) years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively. PMID:15693624

  6. Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

    PubMed Central

    Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

    2015-01-01

    Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field. PMID:26317353

  7. Optical effects of the operation of the onboard engine of the Progress M-17M spacecraft at thermospheric heights

    NASA Astrophysics Data System (ADS)

    Mikhalev, A. V.; Khakhinov, V. V.; Beletskii, A. B.; Lebedev, V. P.

    2016-03-01

    This paper presents the results of optical observations in the active space experiment "Radar-Progress" on April 17, 2013, after switching on the approach-correction engine of the Progress M-17M cargo spacecraft at thermospheric heights (412 km), are presented in this paper. During engine operation, a region of enhanced emission intensity has been recorded. It was presumably related to the scatter of twilight solar emission at the engine exhausts in the cargo spacecraft orbit and, probably to the occurrence of an additional emission in the atomic oxygen line [OI] 630 nm. The maximum observed dimensions of the emission region were ~350 and ~250 km along the orbit and across it, respectively. The velocity of the expansion of the emission region at the first moments after the initiation of engine operation was ~7 and ~3.5 km/s along the orbit and across it, respectively. The maximum intensity of the disturbed region is estimated to be a value equivalent to ~40-60 R within the spectral band of 2 nm. No optical manifestation, which would exceed the natural variations in brightness of the night airglow and which would be related to possible large-scale modification of the ionosphere, was detected in the natural emission lines [O] 557.7 and 630.0 nm in a zone remote from the place of injection of engine exhausts.

  8. Submillimeter and far-infrared line observations of M17 SW - A clumpy molecular cloud penetrated by ultraviolet radiation

    NASA Technical Reports Server (NTRS)

    Stutzki, J.; Genzel, R.; Harris, A. I.; Stacey, G. J.; Jaffe, D. T.

    1988-01-01

    Millimeter, submillimeter, and far-IR spectroscopic observations of the M17 SW star formation region are reported. Strong forbidden C II 158 micron and CO J = 7 - 6 line emission arises in an H II region/molecular cloud interface of several pc thickness. Weaker forbidden C II emission appears to be extended over 15 pc throughout the molecular cloud. CO J = 14 - 13 and forbidden O I 145 micron spectra indicate high temperatures and densities for both molecular and atomic gas in the interface. The results require the molecular cloud near the interface to be clumpy or filamentary. The extended forbidden C II emission throughout the molecular cloud has a level around 20 times higher than expected from a single molecular cloud interface exposed to an ultraviolet radiation field typical of the solar neighborhood. The high gas temperature of molecular material in the UV-illuminated interface region suggests that CO self-shielding and heating of CO by photoelectrons are important.

  9. In silico screening of novel inhibitors of M17 Leucine Amino Peptidase (LAP) of Plasmodium vivax as therapeutic candidate.

    PubMed

    Rout, Subhashree; Mahapatra, Rajani Kanta

    2016-08-01

    M17 LAP (Leucine Amino Peptidase) plays an important role in the hydrolysis of amino acids essential for growth and development of Plasmodium vivax (Pv), the pathogen causing malaria. In this paper a homology model of PvLAP was generated using MODELLER v9.15. From different in-silico methods such as structure based, ligand based and de novo drug designing a total of 90 compounds were selected for docking studies. A final list of 10 compounds was prepared. The study reported the identification of 2-[(3-azaniumyl-2-hydroxy-4-phenylbutanoyl) amino]-4-methylpentanoate as the best inhibitor in terms of docking score and pharmacophoric features. The reliability of the binding mode of the inhibitor is confirmed by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20ns in water environment. Finally, in silico ADMET analysis of the inhibitors using MedChem Designer v3 evaluated the drug likeness of the best hits to be considered for industrial pharmaceutical research. PMID:27470355

  10. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

    PubMed

    Bzik, D J; Fox, B A; Gonyer, K

    1993-05-01

    A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH. PMID:8515777

  11. Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins.

    PubMed Central

    Rajagopal, R; Agrawal, Neema; Selvapandiyan, Angamuthu; Sivakumar, S; Ahmad, Suhail; Bhatnagar, Raj K

    2003-01-01

    Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect. PMID:12441000

  12. Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing.

    PubMed

    Mpakali, Anastasia; Saridakis, Emmanuel; Harlos, Karl; Zhao, Yuguang; Papakyriakou, Athanasios; Kokkala, Paraskevi; Georgiadis, Dimitris; Stratikos, Efstratios

    2015-09-15

    Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes. PMID:26259583

  13. Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket.

    PubMed

    Gajda, Anna D; Pawełczak, Małgorzata; Drag, Marcin

    2012-05-01

    Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized. PMID:22366636

  14. Crystal structure of X-prolyl aminopeptidase from Caenorhabditis elegans: A cytosolic enzyme with a di-nuclear active site.

    PubMed

    Iyer, Shalini; La-Borde, Penelope J; Payne, Karl A P; Parsons, Mark R; Turner, Anthony J; Isaac, R Elwyn; Acharya, K Ravi

    2015-01-01

    Eukaryotic aminopeptidase P1 (APP1), also known as X-prolyl aminopeptidase (XPNPEP1) in human tissues, is a cytosolic exopeptidase that preferentially removes amino acids from the N-terminus of peptides possessing a penultimate N-terminal proline residue. The enzyme has an important role in the catabolism of proline containing peptides since peptide bonds adjacent to the imino acid proline are resistant to cleavage by most peptidases. We show that recombinant and catalytically active Caenorhabditis elegans APP-1 is a dimer that uses dinuclear zinc at the active site and, for the first time, we provide structural information for a eukaryotic APP-1 in complex with the inhibitor, apstatin. Our analysis reveals that C. elegans APP-1 shares similar mode of substrate binding and a common catalytic mechanism with other known X-prolyl aminopeptidases. PMID:25905034

  15. Subchronic haloperidol administration decreases aminopeptidase N activity and [Met5]enkephalin metabolism in rat striatum and cortex.

    PubMed

    Konkoy, C S; Waters, S M; Davis, T P

    1996-02-15

    Previously we have shown that subchronic intraperitoneal (i.p.) administration of haloperidol decreases the degradation of [Met5]enkephalin by regional brain slices (Waters et al., 1995, J. Pharmacol. Exp. Ther. 274, 783). In the present study, subchronic (7-day i.p.) administration of haloperidol (1 mg/kg) decreased the accumulation of aminopeptidase-derived fragments Tyr and Gly-Gly-Phe-Met on cortical and striatal slices. The accumulation of Tyr-Gly-Gly, however, was not altered by haloperidol treatment on slices from either region. Further, aminopeptidase N activity was decreased in P2 membranes isolated from either the cortex or striatum of haloperidol-treated animals. These data suggest that the haloperidol-induced decrease in [Met5]enkephalin metabolism results, at least in part, from a reduction in the activity of aminopeptidase N. PMID:8851165

  16. Crystal structure of X-prolyl aminopeptidase from Caenorhabditis elegans: A cytosolic enzyme with a di-nuclear active site

    PubMed Central

    Iyer, Shalini; La-Borde, Penelope J.; Payne, Karl A.P.; Parsons, Mark R.; Turner, Anthony J.; Isaac, R. Elwyn; Acharya, K. Ravi

    2015-01-01

    Eukaryotic aminopeptidase P1 (APP1), also known as X-prolyl aminopeptidase (XPNPEP1) in human tissues, is a cytosolic exopeptidase that preferentially removes amino acids from the N-terminus of peptides possessing a penultimate N-terminal proline residue. The enzyme has an important role in the catabolism of proline containing peptides since peptide bonds adjacent to the imino acid proline are resistant to cleavage by most peptidases. We show that recombinant and catalytically active Caenorhabditis elegans APP-1 is a dimer that uses dinuclear zinc at the active site and, for the first time, we provide structural information for a eukaryotic APP-1 in complex with the inhibitor, apstatin. Our analysis reveals that C. elegans APP-1 shares similar mode of substrate binding and a common catalytic mechanism with other known X-prolyl aminopeptidases. PMID:25905034

  17. Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen

    PubMed Central

    Dinglasan, Rhoel R.; Kalume, Dario E.; Kanzok, Stefan M.; Ghosh, Anil K.; Muratova, Olga; Pandey, Akhilesh; Jacobs-Lorena, Marcelo

    2007-01-01

    Malaria parasites must undergo development within mosquitoes to be transmitted to a new host. Antivector transmission-blocking vaccines inhibit parasite development by preventing ookinete interaction with mosquito midgut ligands. Therefore, the discovery of novel midgut antigen targets is paramount. Jacalin (a lectin) inhibits ookinete attachment by masking glycan ligands on midgut epithelial surface glycoproteins. However, the identities of these midgut glycoproteins have remained unknown. Here we report on the molecular characterization of an Anopheles gambiae aminopeptidase N (AgAPN1) as the predominant jacalin target on the mosquito midgut luminal surface and provide evidence for its role in ookinete invasion. α-AgAPN1 IgG strongly inhibited both Plasmodium berghei and Plasmodium falciparum development in different mosquito species, implying that AgAPN1 has a conserved role in ookinete invasion of the midgut. Molecules targeting single midgut antigens seldom achieve complete abrogation of parasite development. However, the combined blocking activity of α-AgAPN1 IgG and an unrelated inhibitory peptide, SM1, against P. berghei was incomplete. We also found that SM1 can block only P. berghei, whereas α-AgAPN1 IgG can block both parasite species significantly. Therefore, we hypothesize that ookinetes can evade inhibition by two potent transmission-blocking molecules, presumably through the use of other ligands, and that this process further partitions murine from human parasite midgut invasion models. These results advance our understanding of malaria parasite–mosquito host interactions and guide in the design of transmission-blocking vaccines. PMID:17673553

  18. A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase

    PubMed Central

    Budatha, Madhusudhan; Meur, Gargi; Dutta-Gupta, Aparna

    2007-01-01

    Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX18E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth. PMID:17402938

  19. Plasmodium falciparum: multifaceted resistance to artemisinins.

    PubMed

    Paloque, Lucie; Ramadani, Arba P; Mercereau-Puijalon, Odile; Augereau, Jean-Michel; Benoit-Vical, Françoise

    2016-01-01

    Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance. PMID:26955948

  20. Artemisinin Action and Resistance in Plasmodium falciparum.

    PubMed

    Tilley, Leann; Straimer, Judith; Gnädig, Nina F; Ralph, Stuart A; Fidock, David A

    2016-09-01

    The worldwide use of artemisinin-based combination therapies (ACTs) has contributed in recent years to a substantial reduction in deaths resulting from Plasmodium falciparum malaria. Resistance to artemisinins, however, has emerged in Southeast Asia. Clinically, resistance is defined as a slower rate of parasite clearance in patients treated with an artemisinin derivative or an ACT. These slow clearance rates associate with enhanced survival rates of ring-stage parasites briefly exposed in vitro to dihydroartemisinin. We describe recent progress made in defining the molecular basis of artemisinin resistance, which has identified a primary role for the P. falciparum K13 protein. Using K13 mutations as molecular markers, epidemiological studies are now tracking the emergence and spread of artemisinin resistance. Mechanistic studies suggest potential ways to overcome resistance. PMID:27289273

  1. Acute renal failure due to falciparum malaria.

    PubMed

    Habte, B

    1990-01-01

    Seventy-two patients with severe falciparum malaria are described. Twenty-four (33.3%) were complicated by acute renal failure. Comparing patients with renal failure and those without, statistically significant differences occurred regarding presence of cerebral malaria (83% vs 46%), jaundice (92% vs 33%), and death (54% vs 17%). A significantly higher number of patients with renal failure were nonimmune visitors to malaria endemic regions. Renal failure was oliguric in 45% of cases. Dialysis was indicated in 38%, 29% died in early renal failure, and 33% recovered spontaneously. It is concluded that falciparum malaria is frequently complicated by cerebral malaria and renal failure. As nonimmune individuals are prone to develop serious complications, malaria prophylaxis and vigorous treatment of cases is mandatory. PMID:2236718

  2. The paradoxical population genetics of Plasmodium falciparum.

    PubMed

    Hartl, Daniel L; Volkman, Sarah K; Nielsen, Kaare M; Barry, Alyssa E; Day, Karen P; Wirth, Dyann F; Winzeler, Elizabeth A

    2002-06-01

    Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies. PMID:12036741

  3. Bovine brain pyroglutamyl aminopeptidase (type-1): purification and characterisation of a neuropeptide-inactivating peptidase.

    PubMed

    Cummins, P M; O'Connor, B

    1996-08-01

    Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6% The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3). PMID:8811836

  4. Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

    PubMed

    Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

    2015-10-23

    Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. PMID:26381406

  5. Aminopeptidase N/CD13 targeting fluorescent probes: synthesis and application to tumor cell imaging.

    PubMed

    Zhang, Zhouen; Harada, Hiroshi; Tanabe, Kazuhito; Hatta, Hiroshi; Hiraoka, Masahiro; Nishimoto, Sei-ichi

    2005-11-01

    A family of fluorescein-peptide conjugates (CNP1-3) for aminopeptidase N (APN/CD13) targeting fluorescent probes were designed and synthesized. Among the three conjugates, CNP1 bearing tumor-homing cyclic peptide CNGRC, could selectively label APN/CD13 over-expressing on the surface of tumor cells of HT-1080, as identified by means of fluorescent microscopic cell imaging. CNP1 was shown to be a promising fluorescent probe applicable to tumor-targeting molecular imaging. PMID:15885853

  6. UvrD helicase of Plasmodium falciparum.

    PubMed

    Shankar, Jay; Tuteja, Renu

    2008-03-15

    Malaria caused by the mosquito-transmitted parasite Plasmodium is the cause of enormous number of deaths every year in the tropical and subtropical areas of the world. Among four species of Plasmodium, Plasmodium falciparum causes most fatal form of malaria. With time, the parasite has developed insecticide and drug resistance. Newer strategies and advent of novel drug targets are required so as to combat the deadly form of malaria. Helicases is one such class of enzymes which has previously been suggested as potential antiviral and anticancer targets. These enzymes play an essential role in nearly all the nucleic acid metabolic processes, catalyzing the transient opening of the duplex nucleic acids in an NTP-dependent manner. DNA helicases from the PcrA/UvrD/Rep subfamily are important for the survival of the various organisms. Members from this subfamily can be targeted and inhibited by a variety of synthetic compounds. UvrD from this subfamily is the only member present in the P. falciparum genome, which shows no homology with UvrD from human and thus can be considered as a strong potential drug target. In this manuscript we provide an overview of UvrD family of helicases and bioinformatics analysis of UvrD from P. falciparum. PMID:18242886

  7. Crystallization of Saccharomyces cerevisiae aminopeptidase 1, the major cargo protein of the Cvt pathway

    SciTech Connect

    Adachi, Wakana; Suzuki, Nobuo N.; Fujioka, Yuko; Suzuki, Kuninori; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2007-03-01

    Aminopeptidase 1, a cargo protein in the cytosol-to-vacuole targeting (Cvt) pathway, was expressed, purified and crystallized in two crystal forms. The vacuole hydrolase aminopeptidase 1 (Ape1) is a cargo protein transported to the vacuole by the cytosol-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ape1 is processed into mature Ape1 (mApe1). mApe1 has been expressed, purified and crystallized in two crystal forms. Form I belongs to space group P2{sub 1}, with unit-cell parameters a = 120.6, b = 219.5, c = 133.1 Å, β = 116.5°. Form II belongs to space group R3, with unit-cell parameters a = 141.2, c = 349.4 Å. Diffraction data were collected from these crystals to a resolution of 2.5 Å for form I and 1.83 Å for form II. Self-rotation functions and the volume-to-weight ratio values suggest that forms I and II contain 12 and four mApe1 molecules per asymmetric unit, respectively, and that mApe1 exists as a tetrahedral dodecamer in both crystal forms.

  8. Exploring ligand dissociation pathways from aminopeptidase N using random acceleration molecular dynamics simulation.

    PubMed

    Liu, Ya; Tu, GuoGang; Lai, XiaoPing; Kuang, BinHai; Li, ShaoHua

    2016-10-01

    Aminopeptidase N (APN) is a zinc-dependent ectopeptidase involved in cell proliferation, secretion, invasion, and angiogenesis, and is widely recognized as an important cancer target. However, the mechanisms whereby ligands leave the active site of APN remain unknown. Investigating ligand dissociation processes is quite difficult, both in classical simulation methods and in experimental approaches. In this study, random acceleration molecular dynamics (RAMD) simulation was used to investigate the potential dissociation pathways of ligand from APN. The results revealed three pathways (channels A, B and C) for ligand release. Channel A, which matches the hypothetical channel region, was the most preferred region for bestatin to dissociate from the enzyme, and is probably the major channel for the inner bound ligand. In addition, two alternative channels (channels B and C) were shown to be possible pathways for ligand egression. Meanwhile, we identified key residues controlling the dynamic features of APN channels. Identification of the dissociation routes will provide further mechanistic insights into APN, which will benefit the development of more promising APN inhibitors. Graphical Abstract The release pathways of bestatin inside active site of aminopeptidase N were simulated using RAMD simulation. PMID:27624165

  9. The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion

    PubMed Central

    Fiddler, Christine A.; Parfrey, Helen; Cowburn, Andrew S.; Luo, Ding; Nash, Gerard B.; Murphy, Gillian; Chilvers, Edwin R.

    2016-01-01

    Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo. PMID:27467268

  10. Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloprotein. Purification and properties of the enzyme.

    PubMed

    Spungin, A; Blumberg, S

    1989-08-01

    A heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel-exclusion and anion-exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12. Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine-4-nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25 degrees C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two-stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions. PMID:2503378

  11. Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.

    PubMed Central

    Wu, Q; Li, L; Cooper, M D; Pierres, M; Gorvel, J P

    1991-01-01

    The predicted amino acid sequence of the cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2]. In the present studies, we examined the enzymatic activity of this antigen. From brush border preparations of the small intestine, a rich source of many endopeptidases and exopeptidases, the BP-1 antibody selectively removed aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7] activity. The APA activity of a panel of cell lines correlated in linear fashion with cell-surface levels of the BP-1/6C3 antigen. APA activity was demonstrated for the BP-1/6C3 antigen immunopurified from the pre-B-cell membrane. This activity was enhanced by alkaline earth metals such as Ca2+ and was abrogated by amastatin and angiotensin, which are known competitive inhibitors of APA. The data indicate that the murine BP-1/6C3 antigen is active APA, an enzyme that catalyzes specifically the removal of unsubstituted, N-terminal glutamic acid and aspartic acid residues from peptides. Images PMID:1988965

  12. MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

    PubMed Central

    Robinson, Mark W.; Buchtmann, Kyle A.; Jenkins, Cheryl; Tacchi, Jessica L.; Raymond, Benjamin B. A.; To, Joyce; Roy Chowdhury, Piklu; Woolley, Lauren K.; Labbate, Maurizio; Turnbull, Lynne; Whitchurch, Cynthia B.; Padula, Matthew P.; Djordjevic, Steven P.

    2013-01-01

    Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis. PMID:23594879

  13. Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

    NASA Astrophysics Data System (ADS)

    Essler, Markus; Ruoslahti, Erkki

    2002-02-01

    In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

  14. Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti

    PubMed Central

    Londono, Berlin L.; Eisele, Thomas P.; Keating, Joseph; Bennett, Adam; Chattopadhyay, Chandon; Heyliger, Gaetan; Mack, Brian; Rawson, Ian; Vely, Jean-Francois; Désinor, Olbeg

    2009-01-01

    Plasmodium falciparum parasites have been endemic to Haiti for >40 years without evidence of chloroquine (CQ) resistance. In 2006 and 2007, we obtained blood smears for rapid diagnostic tests (RDTs) and filter paper blots of blood from 821 persons by passive and active case detection. P. falciparum infections diagnosed for 79 persons by blood smear or RDT were confirmed by PCR for the small subunit rRNA gene of P. falciparum. Amplification of the P. falciparum CQ resistance transporter (pfcrt) gene yielded 10 samples with amplicons resistant to cleavage by ApoI. A total of 5 of 9 samples had threonine at position 76 of pfcrt, which is consistent with CQ resistance (haplotypes at positions 72–76 were CVIET [n = 4] and CVMNT [n = 1]); 4 had only the wild-type haplotype associated with CQ susceptibility (CVMNK). These results indicate that CQ-resistant haplotype P. falciparum malaria parasites are present in Haiti. PMID:19402959

  15. PDR MODEL MAPPING OF OBSCURED H{sub 2} EMISSION AND THE LINE-OF-SIGHT STRUCTURE OF M17-SW

    SciTech Connect

    Sheffer, Y.; Wolfire, M. G.

    2013-09-01

    We observed H{sub 2} line emission with Spitzer-IRS toward M17-SW and modeled the data with our photon-dominated region (PDR) code. Derived gas density values of up to few times 10{sup 7} cm{sup -3} indicate that H{sub 2} emission originates in high-density clumps. We discover that the PDR code can be utilized to map the amount of intervening extinction obscuring the H{sub 2} emission layers, and thus we obtain the radial profile of A{sub V} relative to the central ionizing cluster NGC 6618. The extinction has a positive radial gradient, varying between 15-47 mag over the projected distance of 0.9-2.5 pc from the primary ionizer, CEN 1. These high extinction values are in good agreement with previous studies of A{sub V} toward stellar targets in M17-SW. The ratio of data to PDR model values is used to infer the global line-of-sight structure of the PDR surface, which is revealed to resemble a concave surface relative to NGC 6618. Such a configuration confirms that this PDR can be described as a bowl-shaped boundary of the central H II region in M17. The derived structure and physical conditions are important for interpreting the fine-structure and rotational line emission from the PDR.

  16. Artemisinins target the SERCA of Plasmodium falciparum.

    PubMed

    Eckstein-Ludwig, U; Webb, R J; Van Goethem, I D A; East, J M; Lee, A G; Kimura, M; O'Neill, P M; Bray, P G; Ward, S A; Krishna, S

    2003-08-21

    Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron. PMID:12931192

  17. Induction of gene amplification in Plasmodium falciparum

    SciTech Connect

    Rogers, P.L.

    1985-01-01

    Human erythrocytic in vitro cultures of Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with /sup 35/S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.

  18. High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris.

    PubMed

    Tang, Wei; Li, Zhezhe; Li, Chunhua; Yu, Xianhong; Wang, Fei; Wan, Xin; Wang, Yaping; Ma, Lixin

    2016-06-01

    Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases. PMID:26898926

  19. Acute Pancreatitis in a Patient with Complicated Falciparum Malaria

    PubMed Central

    Bhattacharya, Prasanta Kumar; Lynrah, Kryshan G; Ete, Tony; Issar, Neel Kanth

    2016-01-01

    Malaria is one of the most common protozoan diseases, especially in tropical countries. The clinical manifestation of malaria, especially falciparum malaria varies from mild acute febrile illness to life threatening severe systemic complications involving one or more organ systems. We would like to report a case of complicated falciparum malaria involving cerebral, renal, hepatic system along with acute pancreatitis. The patient was successfully treated with anti malarial and other supportive treatment. To the best of our knowledge there are very few reports of acute pancreatitis due to malaria. Falciparum malaria therefore should be added to the list of infectious agents causing acute pancreatitis especially in areas where malaria is endemic. PMID:26894117

  20. Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

    SciTech Connect

    T Nguyen; S Chang; I Evnouchidou; I York; C Zikos; K Rock; A Goldberg; E Stratikos; L Stern

    2011-12-31

    ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

  1. Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.

    PubMed

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-12-17

    The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro. PMID:10606725

  2. Crystallization and preliminary X-ray characterization of aminopeptidase N from Escherichia coli

    PubMed Central

    Onohara, Yuko; Nakajima, Yoshitaka; Ito, Kiyoshi; Xu, Yue; Nakashima, Kanako; Ito, Takashi; Yoshimoto, Tadashi

    2006-01-01

    A recombinant form of aminopeptidase N (molecular weight 99 kDa) from Escherichia coli was crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitating agent. The crystals belong to the hexagonal space group P3121, with unit-cell parameters a = b = 120.5, c = 171.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V M value of 3.62 Å3 Da−1. Diffraction data were collected to 2.0 Å resolution using Cu Kα radiation from a rotating-anode X-ray generator. PMID:16820698

  3. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

    PubMed Central

    Rinaldi, Gabriel; Morales, Maria E.; Alrefaei, Yousef N.; Cancela, Martín; Castillo, Estela; Dalton, John P.; Tort, José F.; Brindley, Paul J.

    2009-01-01

    Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the Schistosoma mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP 1 and 2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP 2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate L-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by ≥ 80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases. PMID:19463860

  4. Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.

    PubMed

    Munkhjargal, Tserendorj; Ishizaki, Takahiro; Guswanto, Azirwan; Takemae, Hitoshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-05-15

    Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this. PMID:27084466

  5. Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

    PubMed Central

    Mayo, Baltasar; Kok, Jan; Bockelmann, Wilhelm; Haandrikman, Alfred; Leenhouts, Kees J.; Venema, Gerard

    1993-01-01

    The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac- and Prt-, the Lac+ Prt+ determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP+ strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products. Images PMID:16348982

  6. Structural and biochemical characterization of a novel aminopeptidase from human intestine.

    PubMed

    Tykvart, Jan; Bařinka, Cyril; Svoboda, Michal; Navrátil, Václav; Souček, Radko; Hubálek, Martin; Hradilek, Martin; Šácha, Pavel; Lubkowski, Jacek; Konvalinka, Jan

    2015-05-01

    N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP). PMID:25752612

  7. Historical review: does stress provoke Plasmodium falciparum recrudescence?

    PubMed

    Shanks, G Dennis

    2015-06-01

    Plasmodium falciparum, unlike P. vivax, must maintain infection in the blood/bone marrow over many months/years in order to bridge periods between transmission periods. Asymptomatic parasitemia at very low concentrations is now known to be quite common due to molecular detection methods. Old tropical medicine texts commonly list many stressful events stated to provoke recrudescent falciparum parasitemia such as fatigue, heat/chill, trauma/surgery, famine/war, transit between areas and other febrile illness. The older literature is reviewed to discover the factual basis of such varied reports since they have not been recently confirmed. It seems likely that human stress sometimes induces falciparum recrudescence of an otherwise asymptomatic infection. Reproducing such observations today has been radically altered as malaria chemotherapy has evolved from suppressive quinine to curative artemisinin combinations. Host stress-provoked recrudescence may be part of P. falciparum's survival strategy. PMID:25918217

  8. Spatial and temporal distribution of falciparum malaria in China

    PubMed Central

    Lin, Hualiang; Lu, Liang; Tian, Linwei; Zhou, Shuisen; Wu, Haixia; Bi, Yan; Ho, Suzanne C; Liu, Qiyong

    2009-01-01

    Background Falciparum malaria is the most deadly among the four main types of human malaria. Although great success has been achieved since the launch of the National Malaria Control Programme in 1955, malaria remains a serious public health problem in China. This paper aimed to analyse the geographic distribution, demographic patterns and time trends of falciparum malaria in China. Methods The annual numbers of falciparum malaria cases during 1992–2003 and the individual case reports of each clinical falciparum malaria during 2004–2005 were extracted from communicable disease information systems in China Center for Diseases Control and Prevention. The annual number of cases and the annual incidence were mapped by matching them to corresponding province- and county-level administrative units in a geographic information system. The distribution of falciparum malaria by age, gender and origin of infection was analysed. Time-series analysis was conducted to investigate the relationship between the falciparum malaria in the endemic provinces and the imported falciparum malaria in non-endemic provinces. Results Falciparum malaria was endemic in two provinces of China during 2004–05. Imported malaria was reported in 26 non-endemic provinces. Annual incidence of falciparum malaria was mapped at county level in the two endemic provinces of China: Yunnan and Hainan. The sex ratio (male vs. female) for the number of cases in Yunnan was 1.6 in the children of 0–15 years and it reached 5.7 in the adults over 15 years of age. The number of malaria cases in Yunnan was positively correlated with the imported malaria of concurrent months in the non-endemic provinces. Conclusion The endemic area of falciparum malaria in China has remained restricted to two provinces, Yunnan and Hainan. Stable transmission occurs in the bordering region of Yunnan and the hilly-forested south of Hainan. The age and gender distribution in the endemic area is characterized by the predominance

  9. Hemoglobinopathies: slicing the Gordian knot of Plasmodium falciparum malaria pathogenesis.

    PubMed

    Taylor, Steve M; Cerami, Carla; Fairhurst, Rick M

    2013-01-01

    Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits--including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia--are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a "natural experiment" to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the "Gordian knot" of host and parasite

  10. Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114.

    PubMed

    Tsakalidou, E; Kalantzopoulos, G

    1992-03-01

    An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89,000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity. PMID:1568949

  11. S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors.

    PubMed

    Poreba, Marcin; Gajda, Anna; Picha, Jan; Jiracek, Jiri; Marschner, Aline; Klein, Christian D; Salvesen, Guy S; Drag, Marcin

    2012-03-01

    Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors. PMID:22085501

  12. Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

    PubMed Central

    Abdullah, Mohd Amir F; Valaitis, Algimantas P; Dean, Donald H

    2006-01-01

    Background Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. Results A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. Conclusion An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba. PMID:16716213

  13. Plasmodium falciparum RuvB proteins

    PubMed Central

    Ahmad, Moaz; Tuteja, Renu

    2012-01-01

    The urgent requirement of next generation antimalarials has been of recent interest due to the emergence of drug-resistant parasite. The genome-wide analysis of Plasmodium falciparum helicases revealed three RuvB proteins. Due to the presence of helicase motif I and II in PfRuvBs, there is a high probability that they contain ATPase and possibly helicase activity. The Plasmodium database has homologs of several key proteins that interact with RuvBs and are most likely involved in the cell cycle progression, chromatin remodeling, and other cellular activities. Phylogenetically PfRuvBs are closely related to Saccharomyces cerevisiae RuvB, which is essential for cell cycle progression and survival of yeast. Thus PfRuvBs can serve as potential drug target if they show an essential role in the survival of parasite. PMID:23060959

  14. Binding, degradation and pressor activity of angiotensins II and III after aminopeptidase inhibition with amastatin and bestatin

    SciTech Connect

    Abhold, R.H.; Sullivan, M.J.; Wright, J.W.; Harding, J.W.

    1987-09-01

    In the metabolism of angiotensin peptides by tissue angiotensinases, aminopeptidases A, B, M and leucine aminopeptidase have been identified as being particularly effective. Because the inhibitory actions of amastatin (AM) and bestatin (BE) are relatively specific for these aminopeptidases, we have examined the effects of these inhibitors on the binding, degradation and pressor activity of angiotensin II (AII) and angiotensin III (AIII). Within 30 min at 37 degrees C, significant metabolism of /sup 125/I-AII and /sup 125/I-AIII by homogenates of a block of tissue containing hypothalamus, thalamus, septum and anteroventral third ventricle regions of the brain was observed. A majority of /sup 125/I-AIII metabolism was due to soluble peptidases, whereas that of /sup 125/I-AII primarily resulted from membrane-bound peptidases. AM, BE and reduced incubation temperatures significantly decreased the metabolism of /sup 125/I-AII and /sup 125/I-AIII. After appropriate adjustments to reflect the proportion of intact radioligand bound, temperature- or inhibitor-induced decreases in metabolism were matched by corresponding increases in specific binding. Heat-treated bovine serum albumin, as a nonspecific peptidase inhibitor, had no effect on either the metabolism or binding of the ligands used. In accordance with their actions in vitro, i.c.v. administration of AM and BE prolonged the pressor activity of subsequently applied AII and AIII. Unexpectedly, the amplitude of the pressor response to AIII was increased by BE, whereas that to AII was decreased by AM. The results of this study indicate that the metabolism of AII and AIII by aminopeptidases is relatively specific and acts to modulate the actions of these peptides.

  15. The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

    PubMed Central

    Gobbetti, M; Smacchi, E; Corsetti, A

    1996-01-01

    A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively. PMID:8795211

  16. The Dynamics of Natural Plasmodium falciparum Infections

    PubMed Central

    Felger, Ingrid; Maire, Martin; Bretscher, Michael T.; Falk, Nicole; Tiaden, André; Sama, Wilson; Beck, Hans-Peter; Owusu-Agyei, Seth; Smith, Thomas A.

    2012-01-01

    Background Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. Methods An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. Results Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5–9 year old children (average duration 319 days, 95% confidence interval 318;320). Conclusions The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections. PMID:23029082

  17. Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double-positive thymocytes.

    PubMed

    Small, M; Kaiser, M; Tse, W; Heimfeld, S; Blumberg, S

    1996-04-01

    The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40 T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double -positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of the specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes. PMID:8625997

  18. A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

    PubMed Central

    Sträter, Norbert; Sun, Lee; Kantrowitz, E. R.; Lipscomb, William N.

    1999-01-01

    The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when l-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces kcat/Km about 10,000-fold. PMID:10500145

  19. Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection

    PubMed Central

    Cheng, Changyong; Wang, Xiaowen; Dong, Zhimei; Shao, Chunyan; Yang, Yongchun; Fang, Weihuan; Fang, Chun; Wang, Hang; Yang, Menghua; Jiang, Lingli; Zhou, Xiangyang; Song, Houhui

    2015-01-01

    The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe3+, Zn2+ and Mn2+, but strongly stimulated by Co2+, indicating that Lmo1603 is a cobalt-dependent aminopeptidase. Single mutation at any of the Glu216, Glu281, His308, Tyr315, His327, and Asp329 completely abolished the enzymatic activity of Lmo1603. More importantly, we showed that Lmo1603 was mainly involved in Listeria infection, but not required for growth in rich laboratory medium and minimal defined medium. Disruption of Lmo1603 resulted in almost complete attenuation of Listeria virulence in a mouse infection model. In addition, we demonstrated that Lmo1603 was mainly localized in the bacterial cytosol and required for invasion and survival inside human epithelial cells and murine macrophages. We conclude that Lmo1603 encodes a functional aminopeptidase T of M29 family, which acts as a novel intracellular virulence factor essential in the successful establishment of L. monocytogenes infections in a mouse model. PMID:26610705

  20. Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

    PubMed Central

    2014-01-01

    Background Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions

  1. Submicroscopic Plasmodium falciparum infections in pregnancy in Ghana.

    PubMed

    Mockenhaupt, F P; Rong, B; Till, H; Eggelte, T A; Beck, S; Gyasi-Sarpong, C; Thompson, W N; Bienzle, U

    2000-03-01

    Malarial parasitaemia below the threshold of microscopy but detectable by polymerase chain reaction (PCR) assays is common in endemic regions. This study was conducted to examine prevalence, predictors, and effects of submicroscopic Plasmodium falciparum infections in pregnancy. In a cross-sectional study among 530 pregnant women in Ghana, plasmodial infections were assessed by microscopy and PCR assays. Concentrations of haemoglobin and C-reactive protein (CRP) were measured and antimalarial drugs (chloroquine, pyrimethamine) in urine were demonstrated by ELISA dipsticks. By microscopy, 32% of the women were found to harbour malaria parasites. This rate increased to 63% adding the results of the parasite-specific PCR. P. falciparum was present in all but one infection. With increasing gravidity, infection rates and parasite densities decreased and the proportions of submicroscopic parasitaemia among infected women grew. Correspondingly, anaemia, fever and evidence of inflammation (CRP > 0.6 mg/dl) were more frequent in primigravidae than in multigravidae. Antimalarial drugs were detected in 65% of the women and were associated with a reduced prevalence of P. falciparum infections and a raised proportion of submicroscopic parasitaemia. Both gravidity and antimalarial drug use were independent predictors of submicroscopic P. falciparum infections. These infections caused a slight reduction of Hb levels and considerably increased serum concentrations of CRP. Conventional microscopy underestimates the actual extent of malarial infections in pregnancy in endemic regions. Submicroscopic P. falciparum infections are frequent and may contribute to mild anaemia and inflammation in seemingly aparasitaemic pregnant women. PMID:10747278

  2. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  3. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

    PubMed Central

    Malik, A; Egan, J E; Houghten, R A; Sadoff, J C; Hoffman, S L

    1991-01-01

    Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID:1707538

  4. Molecular characterization and dietary regulation of aminopeptidase N (APN) in the grass carp (Ctenopharyngodon idella).

    PubMed

    Tang, Jianzhou; Qu, Fufa; Tang, Xiangbei; Zhao, Qiong; Wang, Yonghong; Zhou, Yi; Feng, Junchang; Lu, Shuangqing; Hou, Dexing; Liu, Zhen

    2016-05-10

    Aminopeptidase N (APN) is a member of the peptidase M1 family and plays an important role in protein digestion. In the present study, an APN gene was cloned from the intestine of Ctenopharyngodon idellus. The full-length cDNA sequence of APN encodes an 892-amino-acid peptide that includes one helix trans-membrane region. Phylogenetic analysis showed that the APN sequence clustered with Danio rerio as its closest neighbor, sharing a sequence similarity of 81.5%. APN mRNA was differentially expressed in different tissues, with a gradient expression from high to low in the tissues of the fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart. APN expression in grass carp had a circadian pattern, showing time-dependent higher expression between 06:00 and 18:00 and lower expression between 18:00 and 06:00. In addition, the protein levels and resource in the diet-regulated APN expression suggested that low crude protein (CP) level and fish meal stimulated APN gene expression. Furthermore, the mRNA expression of APN in the intestine was significantly suppressed by high concentrations of glutamine and glutamine dipeptides, respectively. This study may provide valuable knowledge on the regulation of APN expression in teleost, which has potential applications for improving fish dietary formulations. PMID:26828613

  5. Smac-Derived Aza-Peptide As an Aminopeptidase-Resistant XIAP BIR3 Antagonist.

    PubMed

    Elsawy, Mohamed A; Tikhonova, Irina G; Martin, Lorraine; Walker, Brian

    2015-01-01

    The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smacderived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla- Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics. PMID:26282728

  6. Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470

    SciTech Connect

    Alvarado, J.; Nemkal, A; Sauder, J; Russell, M; Akiyoshi, D; Shi, W; Almo, S; Weiss, L

    2009-01-01

    Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity in vitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

  7. Histochemical demonstration of tripeptidyl aminopeptidase I in the rat carotid body.

    PubMed

    Atanasova, Dimitrinka; Lazarov, Nikolai

    2015-03-01

    Tripeptidyl aminopeptidase I (TPP I) is a lysosomal exopeptidase that is widely distributed throughout the central nervous system (CNS) and internal organs in many mammalian species. The enzyme is involved in the breakdown of collagen and different peptides. The carotid body (CB) is the main peripheral arterial chemoreceptor playing an important role in the control of breathing and the autonomic control of cardiovascular function. In response to hypoxia its neuron-like glomus cells release a variety of peptide transmitters that trigger an action potential through the afferent fibers, thus conveying the chemosensory information to the CNS. In the present study we investigated the histochemical localization of TPP I in the CB of rats. Enzyme histochemistry showed high activity of TPP I in CB glomeruli. In particular, the glomus cells contained many TPP I-positive granules, while the glial-like sustentacular cells displayed a slightly fainter reaction. The interglomerular connective tissue was also weakly stained. The results show that both the parenchymal cells of the rat CB express, albeit with different intensity, TPP I. Taken together with our previous enzyme histochemical investigations on the rat CB, it seems likely that the glomus cells possess enzymatic equipment necessary for the neuropeptide intracellular and collagen extracellular initial degradation. These findings also suggest that TPP I is involved in the general turnover of chemotransmitters between glomus cells and sensory nerve endings which emphasizes its importance for chemoreception under hypoxic conditions. PMID:25636608

  8. Identification, biochemical and structural evaluation of species-specific inhibitors against type I methionine aminopeptidases.

    PubMed

    Kishor, Chandan; Arya, Tarun; Reddi, Ravikumar; Chen, Xiaochun; Saddanapu, Venkateshwarlu; Marapaka, Anil Kumar; Gumpena, Rajesh; Ma, Dawei; Liu, Jun O; Addlagatta, Anthony

    2013-07-11

    Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis , Enterococcus faecalis , and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity. PMID:23767698

  9. Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats.

    PubMed

    Alponti, Rafaela Fadoni; Alves, Patricia Lucio; Silveira, Paulo Flavio

    2016-02-01

    The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals. PMID:26577934

  10. Insulin-regulated aminopeptidase deficiency provides protection against ischemic stroke in mice.

    PubMed

    Pham, Vi; Albiston, Anthony L; Downes, Catherine E; Wong, Connie H Y; Diwakarla, Shanti; Ng, Leelee; Lee, Seyoung; Crack, Peter J; Chai, Siew Yeen

    2012-04-10

    Recent studies have demonstrated that angiotensin IV (Ang IV) provides protection against brain injury caused by cerebral ischemia. Ang IV is a potent inhibitor of insulin-regulated aminopeptidase (IRAP). Therefore, we examined the effect of IRAP gene inactivation on neuroprotection following transient middle cerebral artery occlusion (MCAo) in mice. IRAP knockout mice and wild-type controls were subjected to 2 h of transient MCAo using the intraluminal filament technique. Twenty-four hours after reperfusion, neurological deficits of the stroke-induced mice were assessed and infarct volumes were measured by TTC staining. The cerebral infarct volume was significantly reduced in the IRAP knockout mice compared to wild-type littermates with corresponding improvement in neurological performance at 24 h post-ischemia. An increase in compensatory cerebral blood flow during MCAo was observed in the IRAP knockout animals with no differences in cerebral vascular anatomy detected. The current study demonstrates that deletion of the IRAP gene protects the brain from ischemic damage analogous to the effect of the IRAP inhibitor, Ang IV. This study indicates that IRAP is potentially a new therapeutic target for the development of treatment for ischemic stroke. PMID:21895534

  11. Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening.

    PubMed

    Engen, Karin; Rosenström, Ulrika; Axelsson, Hanna; Konda, Vivek; Dahllund, Leif; Otrocka, Magdalena; Sigmundsson, Kristmundur; Nikolaou, Alexandros; Vauquelin, Georges; Hallberg, Mathias; Jenmalm Jensen, Annika; Lundbäck, Thomas; Larhed, Mats

    2016-04-01

    Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z'-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 μM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low μM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors. PMID:27078680

  12. Activity of leucine aminopeptidase of Telchin licus licus: an important insect pest of sugarcane.

    PubMed

    Valencia, Jorge W Arboleda; de Sá, Maria Fátima Grossi; Jiménez, Arnubio Valencia

    2014-06-01

    The enzymatic activity of leucine aminopeptidase (EC 3.4.11.1) from the intestinal tract of sugarcane giant borer (Telchin licus licus) was assayed by using a simple and sensitive spectrophotometric assay that uses L-leucyl-2- naphthylamide as substrate. In this assay, L-leucyl-2-naphthylamide is hydrolyzed to produce 2-naphthylamine and Lleucine. The product 2-naphthylamine reacts with Fast Black K and can be monitored using a continuous spectrophotometric measurement at 590 nm. The data on the kinetic parameters indicates that the Km for the L-leucyl-2- naphthylamide at pH 7.0 was found to be lower than those found for other LAP substrates. The Km and Vmax for the LAP were determined to be 84.03 µM and 357.14 enzymatic units mg(-1), respectively. A noticeable difference of LAP activity between the two insect orders tested was observed. This method could be used to screen for natural LAP inhibitors. PMID:24410745

  13. Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense.

    PubMed

    Pillay, Davita; Boulangé, Alain F V; Coustou, Virginie; Baltz, Théo; Coetzer, Theresa H T

    2013-12-01

    Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. PMID:24177338

  14. LJNK, an indoline-2,3-dione-based aminopeptidase N inhibitor with promising antitumor potency.

    PubMed

    Hou, Jinning; Jin, Kang; Li, Jin; Jiang, Yuqi; Li, Xiaoyang; Wang, Xuejian; Huang, Yongxue; Zhang, Yingjie; Xu, Wenfang

    2016-07-01

    In our previous study, we found that LJNK showed potent aminopeptidase N (APN)-inhibitory activity. In the current study, we further evaluated the antitumor effects of LJNK both in vitro and in vivo. Enzyme experiments showed that LJNK showed better inhibitory activity than bestatin against APN both from human carcinoma cells' surface and from porcine kidney microsomes. In addition, LJNK could suppress rat aortic ring microvessel growth and HUVEC tubular structure formation, which showed its stronger antiangiogenesis effects than bestatin. [(3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide)] assay and clonogenic assay showed that LJNK suppressed cancer cell growth both in the short and the long term. Mice bearing H22 transplantation tumor proved its antitumor effects in vivo. Annexin V-fluorescein isothiocyanate/propidium iodide assay showed that LJNK could induce 28.1% PLC/PRF/5 cell apoptosis and the apoptotic pathway was probably identified by western blot. The above-mentioned results suggested that LJNK inhibited cell proliferation and angiogenesis, and induced apoptosis by decreasing APN activity. PMID:26872309

  15. Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity.

    PubMed

    Alponti, Rafaela Fadoni; Viana, Luciana Godoy; Yamanouye, Norma; Silveira, Paulo Flavio

    2015-08-01

    Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin. PMID:25999180

  16. Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N.

    PubMed Central

    Look, A T; Ashmun, R A; Shapiro, L H; Peiper, S C

    1989-01-01

    To determine the primary structure of CD13, a 150-kD cell surface glycoprotein originally identified on subsets of normal and malignant human myeloid cells, we isolated the complete sequences encoding the polypeptide in overlapping complementary DNA (cDNA) clones. The authenticity of our cDNA clones was demonstrated by the ability of the coding sequences, subcloned in a retroviral expression vector, to mediate expression of bona fide CD13 molecules at the surface of transfected mouse fibroblasts. The nucleotide sequence predicts a 967 amino acid integral membrane protein with a single, 24 amino acid hydrophobic segment near the amino terminus. Amino-terminal protein sequence analysis of CD13 molecules indicated that the hydrophobic segment is not cleaved, but rather serves as both a signal for membrane insertion and as a stable membrane-spanning segment. The remainder of the molecule consists of a large extracellular carboxyterminal domain, which contains a pentapeptide consensus sequence characteristic of members of the zinc-binding metalloprotease superfamily. Sequence comparisons with known enzymes of this class revealed that CD13 is identical to aminopeptidase N, a membrane-bound glycoprotein thought to be involved in the metabolism of regulatory peptides by diverse cell types, including small intestinal and renal tubular epithelial cells, macrophages, granulocytes, and synaptic membranes prepared from cells of the central nervous system. Images PMID:2564851

  17. TLR-mediated secretion of endoplasmic reticulum aminopeptidase 1 from macrophages.

    PubMed

    Goto, Yoshikuni; Ogawa, Kenji; Nakamura, Takahiro J; Hattori, Akira; Tsujimoto, Masafumi

    2014-05-01

    Macrophages play an important role in host defense under several immunological, inflammatory, and/or infectious conditions. In our previous work, we demonstrated that endoplasmic reticulum aminopeptidase 1 (ERAP1) was secreted from macrophages in response to LPS and IFN-γ, and it enhanced their phagocytic activity. In this study, we analyzed the mechanism of LPS/IFN-γ-induced ERAP1 secretion. LPS/IFN-γ-induced secretion of the enzyme from the murine macrophage cell line RAW264.7 was suppressed by polymyxin B. Several agonists of TLRs, such as Pam3CSK4, FSL-1, and ODN1826, induced its secretion. In contrast, neutralizing Abs to IFN-β and TNF-α receptor type 1 suppressed its secretion. Using murine peritoneal macrophages derived from TNF-α and type 1 IFNR knockout mice, we confirmed the involvement of these two cytokines in ERAP1 secretion. In addition, secretion of ERAP1 from both RAW264.7 cells and murine peritoneal macrophages was induced by A23187 and thapsigargin and inhibited by BAPTA-AM and the calmodulin inhibitor W7. These results suggest that LPS/IFN-γ-induced secretion of ERAP1 is mediated by TLRs via induction of intermediate cytokines such as IFN-β and TNF-α, which in turn lead to enhanced cytosolic Ca(2+) levels and calmodulin activation. PMID:24688025

  18. Endoplasmic reticulum-associated amino-peptidase 1 and rheumatic disease: genetics

    PubMed Central

    Ombrello, Michael J.; Kastner, Daniel L.; Remmers, Elaine F.

    2015-01-01

    Purpose of review This article will review the genetic evidence implicating ERAP1, which encodes the endoplasmic reticulum-associated amino-peptidase 1, in susceptibility to rheumatic disease. Recent findings Genetic variants and haplotypes of ERAP1 are associated with ankylosing spondylitis, psoriasis, and Behçet’s disease in people of varying ancestries. In each of these diseases, disease-associated variants of ERAP1 have been shown to interact with disease-associated class I Human Leukocyte Antigen (HLA) alleles to influence disease risk. Functionally, disease-associated missense variants of ERAP1 concertedly alter ERAP1 enzymatic function, both quantitatively and qualitatively, while other disease-associated variants influence ERAP1 expression. Therefore, ERAP1 haplotypes (or allotypes) should be examined as functional units. Biologically, this amounts to an examination of the gene regulation and function of the protein encoded by each allotype. Genetically, the relationship between disease risk and ERAP1 allotypes should be examined to determine whether allotypes or individual variants produce the most parsimonious risk models. Summary Future investigations of ERAP1 should focus on comprehensively characterizing naturally-occurring ERAP1 allotypes, examining the enzymatic function and gene expression of each allotype, and identifying specific allotypes that influence disease susceptibility. PMID:26002026

  19. Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.

    PubMed

    Albiston, A L; McDowall, S G; Matsacos, D; Sim, P; Clune, E; Mustafa, T; Lee, J; Mendelsohn, F A; Simpson, R J; Connolly, L M; Chai, S Y

    2001-12-28

    Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates. PMID:11707427

  20. Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus.

    PubMed

    Wang, Tzu-Fan; Lin, Min-Guan; Lo, Huei-Fen; Chi, Meng-Chun; Lin, Long-Liu

    2014-01-01

    In the present study, the biophysical properties of His6-tagged Bacillus stearothermophilus aminopeptidase II (His6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5-10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His6-tagged BsAmpII. PMID:24165863

  1. Effect of NSAIDs on the aminopeptidase activity of cultured human osteoblasts.

    PubMed

    Lucena, G; Reyes-Botella, C; García-Martínez, O; Ramos-Torrecillas, J; De Luna Bertos, E; Ruiz, C

    2016-05-01

    Aminopeptidases (APs) are involved in various physiological and pathological processes. In tumor tissues the expression of APs, cyclooxygenase-2 and its metabolites are increased. The objective was to determine the effect of certain NSAIDs on the AP activity of osteoblasts. Primary cultures of osteoblast were treated with different concentrations of indomethacin, meloxicam, naproxen, nimesulide, and piroxicam. The AP activity was fluorimetrically determined using aminoacyl-β-naphthylamides (aa-βNAs) as substrates: Ala-βNA, Arg-βNA, Gly-βNA, Leu-βNA, Lys-βNA, Met-βNA, and Phe-βNA. The five NSAIDs showed an inhibitory effect of AP activity against the study substrates depending on the dose tested. Meloxicam and piroxicam had the highest inhibitory effect on enzymatic activity, with an IC50 of around 70 μM. Our results suggest that the physiological alteration of osteoblasts in the presence of NSAIDs may be a consequence of AP inhibition, suggesting a potential clinical role for these drugs against cancer in combination with chemotherapeutic agents. PMID:26930569

  2. Aminopeptidase activity in rat brain synaptosomes - 2-mercaptoethanol stimulation and Arg-vasopressin degradation

    SciTech Connect

    Simmons, W.H.; Orawski, A.T.

    1986-03-05

    Rat brain synaptic plasma membranes contain an amastatin-inhibited aminopeptidase activity which degrades Arg-vaso-pressin (AVP). The pH optimum for AVP cleavage was found to be 6.8, similar to that reported for oxytocin. The ability of other peptides and arylamides such as oxytocin, Tyr-Phe-Met-Arg-Phe-NH/sub 2/ and Arg-Arg-..beta..NA to inhibit cleavage of (/sup 3/H-Tyr/sup 2/)-AVP suggests that the enzyme may not be specific for AVP. The AVP-cleaving activity has been solubilized and partially characterized. Synaptosomes were lysed with hypotonic buffer, washed, and extracted with 1% Nonidet P-40 detergent. The solubilized protein was chromatographed by gel filtration HPLC on Superose 6. A single peak of activity was found with a M.W. = 117,000 which could hydrolyze 1mM Ala-..beta..NA, Arg-..beta..NA, Arg-Arg-..beta..NA, Phe-Met and Phe-Arg as well as slowly cleave AVP with the ultimate release of /sup 3/H-Tyr. 2-Mercaptoethanol (3.9mM) (ME) stimulated activity 3.6 to 6.6-fold for arylamide and dipeptide substrates, but 35-fold for labelled AVP, possibly owing to reduction of the AVP disulfide bond. All activities in the presence of ME were completely inhibited by 0.2mM amastatin.

  3. Discovery of inhibitors of insulin-regulated aminopeptidase as cognitive enhancers.

    PubMed

    Andersson, Hanna; Hallberg, Mathias

    2012-01-01

    The hexapeptide angiotensin IV (Ang IV) is a metabolite of angiotensin II (Ang II) and plays a central role in the brain. It was reported more than two decades ago that intracerebroventricular injection of Ang IV improved memory and learning in the rat. Several hypotheses have been put forward to explain the positive effects of Ang IV and related analogues on cognition. It has been proposed that the insulin-regulated aminopeptidase (IRAP) is the main target of Ang IV. This paper discusses progress in the discovery of inhibitors of IRAP as potential enhancers of cognitive functions. Very potent inhibitors of the protease have been synthesised, but pharmacokinetic issues (including problems associated with crossing the blood-brain barrier) remain to be solved. The paper also briefly presents an overview of the status in the discovery of inhibitors of ACE and renin, and of AT1R antagonists and AT2R agonists, in order to enable other discovery processes within the RAS system to be compared. The paper focuses on the relationship between binding affinities/inhibition capacity and the structures of the ligands that interact with the target proteins. PMID:23304452

  4. Aminopeptidase N (CD13) Is Involved in Phagocytic Processes in Human Dendritic Cells and Macrophages

    PubMed Central

    Villaseñor-Cardoso, Mónica I.; Frausto-Del-Río, Dulce A.

    2013-01-01

    Aminopeptidase N (APN or CD13) is a membrane ectopeptidase expressed by many cell types, including myelomonocytic lineage cells: monocytes, macrophages, and dendritic cells. CD13 is known to regulate the biological activity of various peptides by proteolysis, and it has been proposed that CD13 also participates in several functions such as angiogenesis, cell adhesion, metastasis, and tumor invasion. We had previously reported that, in human monocytes and macrophages, CD13 modulates the phagocytosis mediated by receptors for the Fc portion of IgG antibodies (FcγRs). In this work, we analyzed the possible interaction of CD13 with other phagocytic receptors. We found out that the cross-linking of CD13 positively modulates the phagocytosis mediated by receptors of the innate immune system, since a significant increase in the phagocytosis of zymosan particles or heat-killed E. coli was observed when CD13 was cross-linked using anti-CD13 antibodies, in both macrophages and dendritic cells. Also, we observed that, during the phagocytosis of zymosan, CD13 redistributes and is internalized into the phagosome. These findings suggest that, besides its known functions, CD13 participates in phagocytic processes in dendritic cells and macrophages. PMID:24063007

  5. The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure, paralysis and neuronal failure.

    PubMed Central

    Zhang, HaiGuang; Tan, Jeff; Reynolds, Elaine; Kuebler, Daniel; Faulhaber, Sally; Tanouye, Mark

    2002-01-01

    We report here the characterization of slamdance (sda), a Drosophila melanogaster "bang-sensitive" (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical "bang" or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations. PMID:12454073

  6. Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity.

    PubMed

    Sterkel, Alana K; Lorenzini, Jenna L; Fites, J Scott; Subramanian Vignesh, Kavitha; Sullivan, Thomas D; Wuthrich, Marcel; Brandhorst, Tristan; Hernandez-Santos, Nydiaris; Deepe, George S; Klein, Bruce S

    2016-03-01

    Systemic fungal infections trigger marked immune-regulatory disturbances, but the mechanisms are poorly understood. We report that the pathogenic yeast of Blastomyces dermatitidis elaborates dipeptidyl-peptidase IVA (DppIVA), a close mimic of the mammalian ectopeptidase CD26, which modulates critical aspects of hematopoiesis. We show that, like the mammalian enzyme, fungal DppIVA cleaved C-C chemokines and GM-CSF. Yeast producing DppIVA crippled the recruitment and differentiation of monocytes and prevented phagocyte activation and ROS production. Silencing fungal DppIVA gene expression curtailed virulence and restored recruitment of CCR2(+) monocytes, generation of TipDC, and phagocyte killing of yeast. Pharmacological blockade of DppIVA restored leukocyte effector functions and stemmed infection, while addition of recombinant DppIVA to gene-silenced yeast enabled them to evade leukocyte defense. Thus, fungal DppIVA mediates immune-regulatory disturbances that underlie invasive fungal disease. These findings reveal a form of molecular piracy by a broadly conserved aminopeptidase during disease pathogenesis. PMID:26922990

  7. A novel N(alpha)-acetyl alanine aminopeptidase from Allomyces arbuscula.

    PubMed

    Beti, Raniera; Cattaneo, Arlette; Gabriel, Jean Marc; Ojha, Mukti

    2002-04-01

    An N(alpha)-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of M(r) 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl(2) and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the M(r) 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly N(alpha)-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discussed. PMID:12106909

  8. Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

    PubMed

    Munkhjargal, Tserendorj; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-09-01

    Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis. PMID:27306898

  9. Plasmodium falciparum Secretome in Erythrocyte and Beyond.

    PubMed

    Soni, Rani; Sharma, Drista; Bhatt, Tarun K

    2016-01-01

    Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies. PMID:26925057

  10. Plasmodium falciparum Secretome in Erythrocyte and Beyond

    PubMed Central

    Soni, Rani; Sharma, Drista; Bhatt, Tarun K.

    2016-01-01

    Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies. PMID:26925057

  11. Multiple independent introductions of Plasmodium falciparum in South America

    PubMed Central

    Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J.; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N.; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J.; Renaud, François; Prugnolle, Franck

    2012-01-01

    The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

  12. Functional genomics of Plasmodium falciparum using metabolic modelling and analysis

    PubMed Central

    Oppenheim, Rebecca D.; Soldati-Favre, Dominique; Hatzimanikatis, Vassily

    2013-01-01

    Plasmodium falciparum is an obligate intracellular parasite and the leading cause of severe malaria responsible for tremendous morbidity and mortality particularly in sub-Saharan Africa. Successful completion of the P. falciparum genome sequencing project in 2002 provided a comprehensive foundation for functional genomic studies on this pathogen in the following decade. Over this period, a large spectrum of experimental approaches has been deployed to improve and expand the scope of functionally annotated genes. Meanwhile, rapidly evolving methods of systems biology have also begun to contribute to a more global understanding of various aspects of the biology and pathogenesis of malaria. Herein we provide an overview on metabolic modelling, which has the capability to integrate information from functional genomics studies in P. falciparum and guide future malaria research efforts towards the identification of novel candidate drug targets. PMID:23793264

  13. Nitric oxide inhibits falcipain, the Plasmodium falciparum trophozoite cysteine protease.

    PubMed

    Venturini, G; Colasanti, M; Salvati, L; Gradoni, L; Ascenzi, P

    2000-01-01

    Nitric oxide (NO) is a pluripotent regulatory molecule possessing, among others, an antiparasitic activity. In the present study, the inhibitory effect of NO on the catalytic activity of falcipain, the papain-like cysteine protease involved in Plasmodium falciparum trophozoite hemoglobin degradation, is reported. In particular, NO donors S-nitrosoglutathione (GSNO), (+/-)-(E)-p6ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenami de (NOR-3), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) inhibit dose-dependently the falcipain activity present in the P. falciparum trophozoite extract, this effect likely attributable to S-nitrosylation of the Cys25 catalytic residue. The results represent a new insight into the modulation mechanism of falcipain activity, thereby being relevant in developing new strategies for inhibition of the P. falciparum life cycle. PMID:10623597

  14. Multiple independent introductions of Plasmodium falciparum in South America.

    PubMed

    Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J; Renaud, François; Prugnolle, Franck

    2012-01-10

    The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

  15. Monkey-derived monoclonal antibodies against Plasmodium falciparum.

    PubMed Central

    Stanley, H A; Reese, R T

    1985-01-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen. Images PMID:3898084

  16. Plasmodium falciparum: growth response to potassium channel blocking compounds.

    PubMed

    Waller, Karena L; Kim, Kami; McDonald, Thomas V

    2008-11-01

    Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds. PMID:18703053

  17. [Research Progress on Artemisinin Resistance in Plasmodium falciparum].

    PubMed

    Zhang, Yi-long; Pan, Wei-qing

    2015-12-01

    Artemisinin (ART) is a novel and effective antimalarial drug discovered in China. As recommended by the World Health Organization, the ART-based combination therapies (ACTs) have become the first-line drugs for the treatment of falciparum malaria. ART and its derivatives have contributed greatly to the effective control of malaria globally, leading to yearly decrease of malaria morbidity and mortality. However, there have recently been several reports on the resistance of Plasmodium falciparum to ART in Southeast Asia. This is deemed a serious threat to the global malaria control programs. In this paper, we reviewed recent research progress on ART resistance to P. falciparum, including new tools for resistance measurement, resistance-associated molecular markers, and the origin and spread of the ART-resistant parasite strains. PMID:27089770

  18. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.

    PubMed

    Goel, Suchi; Palmkvist, Mia; Moll, Kirsten; Joannin, Nicolas; Lara, Patricia; Akhouri, Reetesh R; Moradi, Nasim; Öjemalm, Karin; Westman, Mattias; Angeletti, Davide; Kjellin, Hanna; Lehtiö, Janne; Blixt, Ola; Ideström, Lars; Gahmberg, Carl G; Storry, Jill R; Hult, Annika K; Olsson, Martin L; von Heijne, Gunnar; Nilsson, IngMarie; Wahlgren, Mats

    2015-04-01

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population. PMID:25751816

  19. Structure and expression of the Plasmodium falciparum SERA gene.

    PubMed

    Li, W B; Bzik, D J; Horii, T; Inselburg, J

    1989-02-01

    Plasmodium falciparum, strain FCR3, genomic DNA that encodes the SERA gene of P. falciparum was isolated and sequenced. The SERA gene coding region was interrupted by 3 introns, the largest number observed, so far, in any Plasmodium gene. Two SERA gene alleles, allele I and allele II, were identified in the FCR3 strain, while only allele I was found in the Honduras-1 strain. Allele I mRNA was abundant in vivo during the late trophozoite and schizont stages. Allele II mRNA was either not expressed, or it was labile. PMID:2651911

  20. Characterization of a Vibrio fischeri Aminopeptidase and Evidence for Its Influence on an Early Stage of Squid Colonization

    PubMed Central

    Rader, Bethany A.; Gerling, David G.; Gutierrez, Nestor A.; Watkins, Katherine H.; Frey, Michelle West; Nyholm, Spencer V.; Whistler, Cheryl A.

    2012-01-01

    Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and kcat and Km values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization. PMID:22636772

  1. Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization.

    PubMed

    Fidopiastis, Pat M; Rader, Bethany A; Gerling, David G; Gutierrez, Nestor A; Watkins, Katherine H; Frey, Michelle West; Nyholm, Spencer V; Whistler, Cheryl A

    2012-08-01

    Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization. PMID:22636772

  2. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    PubMed

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. PMID:24248368

  3. Screening and Antiviral Analysis of Phages That Display Peptides with an Affinity to Subunit C of Porcine Aminopeptidase

    PubMed Central

    Guo, Donghua; Zhu, Qinghe; Feng, Li

    2013-01-01

    The purified C subunit of the recombinant porcine aminopeptidase N (rpAPN-C) protein was used as an immobilized target to screen potential ligands against rpAPN-C from a 12-mer phage display random peptide library. After five rounds of biopanning, five phage clones showed specific binding affinities to rpAPN-C. In 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays, the phage clone PM1, which contained the HDAISWTHYHPW peptide sequence, had a protective effect against TGEV infection in swine testis cells. Therefore, the HDAISWTHYHPW peptide sequence has a potential use as a small molecular therapeutic agent against TGEV infection. PMID:24111863

  4. Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling

    PubMed Central

    Pham, Viet-Laï; Cadel, Marie-Sandrine; Gouzy-Darmon, Cécile; Hanquez, Chantal; Beinfeld, Margery C; Nicolas, Pierre; Etchebest, Catherine; Foulon, Thierry

    2007-01-01

    Background Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. Results The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. Conclusion Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1

  5. A cross strain Plasmodium falciparum microarray optimized for the transcriptome analysis of Plasmodium falciparum patient derived isolates.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Middha, Sheetal; Acharya, Jyoti; Rao, Sudha Narayana; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2016-09-01

    Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq. PMID:27489776

  6. Artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Fairhurst, Rick M.; Dondorp, Arjen M.

    2016-01-01

    For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins – the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs) – the first-line treatments for malaria – are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in-vitro, genomics, and transcriptomics studies in SEA have defined in-vivo and in-vitro phenotypes of artemisinin resistance; identified its causal genetic determinant; explored its molecular mechanism; and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's ‘K13’ gene; is associated with an upregulated “unfolded protein response” pathway that may antagonize the pro-oxidant activity of artemisinins; and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent; test whether new combinations of currently-available drugs cure ACT failures; and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to Sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest. PMID:27337450

  7. [Activity of alanine aminopeptidase in blood and in urine of smoking and non-smoking smelters].

    PubMed

    Bizoń, Anna; Stasiak, Karolina; Milnerowicz, Halina

    2010-01-01

    The human body is constantly exposed to xenobiotics. This will include exogenous substances from environmental pollution such as heavy metals and lifestyle such as smoking, which may lead to impaired functioning of many organs. The liver and kidney are the critical organs in the case of a long-term occupational or environmental exposure to heavy metals and tobacco smoke. In diagnostics of liver and kidney damage useful are the methods which determine the activity of enzymes such as alanine aminopeptidase (AAP). AAP is a marker for early detection of acute kidney damage, and presence of AAP derive mainly from proximal tubular brush-border. Activity of AAP in urine allows to assess the damage resulting from the nephrotoxic exposure to heavy metals. In the serum AAP is mainly from hepatic. Activity of AAP may be useful to identify liver cancer. The investigation was shown, that AAP activity in the blood is used to detect hepatic cholestasis and congestive jaundice. The aim of present study was to assess the influence of occupational exposure of copper-foundry workers to heavy metals (arsenic, cadmium, lead) on activity of alanine aminopeptidase in blood and urine. The investigations were performed in blood and urine of 166 subjects: 101 male copper smelters and 65 non-exposed male subjects. The study protocol was approved by Local Bioethics Committee of Wroclaw Medical University (KB No: 469/2008). The data on smoking which had been obtained from a direct personal interview were verified by determination of serum cotinine concentrations. Biological material collected from the control group and smelters was divided into subgroups of nonsmokers and smokers. The concentrations of lead and cadmium were determined in whole blood, whilst the level of arsenic and cadmium were determined in urine using FAAS method (Flame Atomic Absorption Spectrometry) in the acetylate flame on the SOLAAR M6. The activity of AA was determined in blood and in urine. The results showed a 9-fold

  8. Binding to and Inhibition of Insulin-Regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density.

    PubMed

    Diwakarla, Shanti; Nylander, Erik; Grönbladh, Alfhild; Vanga, Sudarsana Reddy; Khan, Yasmin Shamsudin; Gutiérrez-de-Terán, Hugo; Ng, Leelee; Pham, Vi; Sävmarker, Jonas; Lundbäck, Thomas; Jenmalm-Jensen, Annika; Andersson, Hanna; Engen, Karin; Rosenström, Ulrika; Larhed, Mats; Åqvist, Johan; Chai, Siew Yeen; Hallberg, Mathias

    2016-04-01

    Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or γ-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity. PMID:26769413

  9. Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

    PubMed

    Lee, Aven; Slattery, Craig; Nikolic-Paterson, David J; Hryciw, Deanne H; Wilk, Sherwin; Wilk, Elizabeth; Zhang, Yuan; Valova, Valentina A; Robinson, Phillip J; Kelly, Darren J; Poronnik, Philip

    2015-04-01

    ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton. PMID:25587118

  10. A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application

    PubMed Central

    Stressler, Timo; Ewert, Jacob; Merz, Michael; Funk, Joshua; Claaßen, Wolfgang; Lutz-Wahl, Sabine; Schmidt, Herbert; Kuhn, Andreas; Fischer, Lutz

    2016-01-01

    Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition. PMID:27003449

  11. Effects of polymorphic variation on the mechanism of Endoplasmic Reticulum Aminopeptidase 1.

    PubMed

    Stamogiannos, Athanasios; Koumantou, Despoina; Papakyriakou, Athanasios; Stratikos, Efstratios

    2015-10-01

    Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) generates antigenic peptides for loading onto Major Histocompatibility Class I molecules (MHCI) and can regulate adaptive immune responses. During the last few years, many genetic studies have revealed strong associations between coding Single Nucleotide Polymorphisms (SNPs) in ERAP1 and common human diseases ranging from viral infections to cancer and autoimmunity. Functional studies have established that these SNPs affect enzyme activity resulting to changes in antigenic peptide processing, presentation by MHCI and cellular cytotoxic responses. These disease-associated polymorphisms are, however, located away from the enzyme's active site and are interspersed to different structural domains. As a result, the mechanism by which these SNPs can affect function remains largely elusive. ERAP1 utilizes a complex catalytic mechanism that involves a large conformational change between inactive and active forms and has the unique property to trim larger peptides more efficiently than smaller ones. We analyzed two of the most consistently discovered disease-associated polymorphisms, namely K528R and Q730E, for their effect on the ability of the enzyme to select substrates based on length and to undergo conformational changes. By utilizing enzymatic and computational analysis we propose that disease-associated SNPs can affect ERAP1 function by influencing: (i) substrate length selection and (ii) the conformational distribution of the protein ensemble. Our results provide novel insight on the mechanisms by which polymorphic variation distal from the active site of ERAP1 can translate to changes in function and contribute to immune system variability in humans. PMID:26224046

  12. Endoplasmic reticulum aminopeptidase-1 functions regulate key aspects of the innate immune response.

    PubMed

    Aldhamen, Yasser A; Seregin, Sergey S; Rastall, David P W; Aylsworth, Charles F; Pepelyayeva, Yuliya; Busuito, Christopher J; Godbehere-Roosa, Sarah; Kim, Sungjin; Amalfitano, Andrea

    2013-01-01

    Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c(+) DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters. PMID:23894499

  13. Atomic far-IR fine-structure line mapping of L1630, M17, and W3: Comparison of (O I) and (C II) distributions

    NASA Technical Reports Server (NTRS)

    Howe, J. E.; Jaffe, Dan T.; Zhou, Shudong

    1995-01-01

    We mapped the distribution of atomic far-IR line emission from (O I) and (C II) over parsec scales in the Galactic star-forming regions L1630, M17, and W3 using the MPE Far-Infrared Fabry-Perot Imaging spectrometer (FIFI) on board the NASA Kuiper Airborne Observatory. The lines mapped include (O I) 63 microns, (O I) 146 microns, and (C II) 158 microns. Comparison of the intensities and ratios of these lines with models of photodissociation regions (e.g., Tielens & Hollenbach 1985, ApJ, 344, 770) allows us to derive temperatures and densities of the primarily neutral atomic gas layers lying on the surfaces of UV-illuminated molecular gas. In general, the (C II) line arises ubiquitously throughout the molecular clouds while the (O I) lines are mainly confined to warm, dense gas (T is greater than 100 K, n is greater than 10(exp 4)/cu cm) near the sites of O and B stars. The distribution of (C II) in the star-forming clouds implies that the (C II) emission arises on the surfaces of molecular clumps throughout the clouds, rather than only at the boundary layer between molecular gas and H II regions.

  14. A genome-wide map of diversity in Plasmodium falciparum.

    PubMed

    Volkman, Sarah K; Sabeti, Pardis C; DeCaprio, David; Neafsey, Daniel E; Schaffner, Stephen F; Milner, Danny A; Daily, Johanna P; Sarr, Ousmane; Ndiaye, Daouda; Ndir, Omar; Mboup, Soulyemane; Duraisingh, Manoj T; Lukens, Amanda; Derr, Alan; Stange-Thomann, Nicole; Waggoner, Skye; Onofrio, Robert; Ziaugra, Liuda; Mauceli, Evan; Gnerre, Sante; Jaffe, David B; Zainoun, Joanne; Wiegand, Roger C; Birren, Bruce W; Hartl, Daniel L; Galagan, James E; Lander, Eric S; Wirth, Dyann F

    2007-01-01

    Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite. PMID:17159979

  15. In vitro drug sensitivity of Plasmodium falciparum in Acre, Brazil.

    PubMed Central

    Kremsner, P. G.; Zotter, G. M.; Feldmeier, H.; Graninger, W.; Kollaritsch, M.; Wiedermann, G.; Rocha, R. M.; Wernsdorfer, W. H.

    1989-01-01

    In Acre, the westernmost state of Brazil in the Amazon region, the sensitivity of Plasmodium falciparum to chloroquine, amodiaquine, mefloquine, quinine and sulfadoxine/pyrimethamine was determined in vitro by the Rieckmann microtechnique. The study was performed between January and June 1987; the in vitro parasite responses to all antimalarial drugs were determined according to the recommendations of WHO. Of 83 isolates of P. falciparum, all were sensitive to mefloquine and of 87 isolates of P. falciparum, 84 (97%) were sensitive to quinine. The EC50 for mefloquine was 0.27 mumol/l and for quinine 4.60 mumol/l. In contrast, 65 of 89 (73%) and 70 of 83 (84%) isolates were resistant to amodiaquine and chloroquine, respectively; 11 isolates even grew at 6.4 mumol chloroquine/l. The EC50 for amodiaquine was 0.34 mumol/l and for chloroquine 0.73 mumol/l. Sulfadoxine/pyrimethamine resistance was seen in 23 of 25 (92%) cases. These data clearly indicate that in the western part of the Amazon region the 4-aminoquinolines, as well as sulfadoxine/pyrimethamine, can no longer be recommended for the treatment of P. falciparum infections. PMID:2670298

  16. Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

    PubMed Central

    Patzewitz, Eva-Maria; Wong, Eleanor H; Müller, Sylke

    2012-01-01

    Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG. PMID:22151036

  17. The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family.

    PubMed Central

    Fanuel, L; Goffin, C; Cheggour, A; Devreese, B; Van Driessche, G; Joris, B; Van Beeumen, J; Frère, J M

    1999-01-01

    The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases. PMID:10377256

  18. Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria

    PubMed Central

    Imwong, Mallika; Woodrow, Charles J.; Hendriksen, Ilse C. E.; Veenemans, Jacobien; Verhoef, Hans; Faiz, M. Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D.; Day, Nicholas P. J.; Dondorp, Arjen M.; White, Nicholas J.

    2015-01-01

    In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples. PMID:25344520

  19. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

    PubMed

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M R K; Freund, Yvonne R; DeRisi, Joseph; Cusack, Stephen; Rosenthal, Philip J

    2016-08-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS. PMID:27270277

  20. [From malaria parasite point of view--Plasmodium falciparum evolution].

    PubMed

    Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa

    2015-01-01

    Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago) than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies. PMID:27259224

  1. Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crystalline (Cry) toxins from Bacillus thuringiensis (Bt) control insect feeding damage on crop plants via foliar applications or by expression within transgenic plants, but continued Bt use is threatened by the buildup of insect resistance traits. Aminopeptidase N (apn) gene family members encode m...

  2. Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis.

    PubMed

    Delmarcelle, Michaël; Boursoit, Marie-Caroline; Filée, Patrice; Baurin, Stéphane Lucius; Frère, Jean-Marie; Joris, Bernard

    2005-09-01

    The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed. PMID:16131658

  3. Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonas gingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant

    PubMed Central

    Yagishita, Hisao; Kumagai, Yumi; Konishi, Kiyoshi; Takahashi, Yukihiro; Aoba, Takaaki; Yoshikawa, Masanosuke

    2001-01-01

    To elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence of Porphyromonas gingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells. PMID:11598093

  4. Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer diatraea saccharalis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aminopeptidase N (APN) proteins located at the midgut epithelium of some lepidopterous species have been implicated as receptors for insecticidal proteins from Bacillus thuringiensis. cDNAs of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-...

  5. Corticotropin releasing factor up-regulates the expression and function of norepinephrine transporter in SK-N-BE (2) M17 cells.

    PubMed

    Huang, Jingjing; Tufan, Turan; Deng, Maoxian; Wright, Gary; Zhu, Meng-Yang

    2015-10-01

    Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [(3) H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status. PMID:26212818

  6. Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli

    PubMed Central

    Mitra, Sanghamitra; Job, Kathleen M.; Meng, Lu; Bennett, Brian; Holz, Richard C.

    2009-01-01

    An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ~ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d–d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal-binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding. PMID:19019076

  7. Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato1

    PubMed Central

    Tu, Chao-Jung; Park, Sang-Youl; Walling, Linda L.

    2003-01-01

    Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Comparison of LAP-N with other plant LAPs identified 28 signature residues that classified LAP proteins as LAP-N or LAP-A like. Overexpression of a His6-LAP-N fusion protein in Escherichia coli demonstrated distinct differences in His6-LAP-N and His6-LAP-A activities. Similar to LapA, the LapN RNA encoded a precursor protein with a molecular mass of 60 kD. The 5-kD presequence had features similar to plastid transit peptides, and processing of the LAP-N presequence could generate the mature 55-kD LAP-N. Unlike LapA, the LapN transcript contained a second in-frame ATG, and utilization of this potential initiation codon would yield a 55-kD LAP-N protein. The localization of LAP-N could be controlled by the balance of translational initiation site utilization and LAP-N preprotein processing. PMID:12746529

  8. Beta-aminopeptidase-catalyzed biotransformations of beta(2)-dipeptides: kinetic resolution and enzymatic coupling.

    PubMed

    Heck, Tobias; Reimer, Artur; Seebach, Dieter; Gardiner, James; Deniau, Gildas; Lukaszuk, Aneta; Kohler, Hans-Peter E; Geueke, Birgit

    2010-05-17

    We have previously shown that the beta-aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non-natural beta(3)-peptides and beta(3)-amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N-terminal beta(2)-amino acid residues as substrates under aqueous conditions. The suitability of a beta(2)-peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C(alpha) substituent of the N-terminal beta(2)-amino acid. BapA was shown to convert a diastereomeric mixture of the beta(2)-peptide H-beta(2)hPhe-beta(2)hAla-OH, but did not act on diastereomerically pure beta(2),beta(3)-dipeptides containing an N-terminal beta(2)-homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA-catalyzed transformation of the diastereomeric mixture of H-beta(2)hPhe-beta(2)hAla-OH proceeded along two highly S-enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis-to-hydrolysis ratio (v(S)/v(H)) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N-terminal beta-amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of beta(2)-homophenylalanine was detected (ee>99 % for H-(S)-beta(2)-hPhe-OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure beta(2)-amino acids and the enantioselective coupling of N-terminal beta(2)-amino acids to peptides. PMID:20340152

  9. Molecular structure of leucine aminopeptidase at 2.7-A resolution.

    PubMed Central

    Burley, S K; David, P R; Taylor, A; Lipscomb, W N

    1990-01-01

    The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-A resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. The monomers are crystallographically equivalent and each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 A3. The secondary structural composition is 40% alpha-helix and 19% beta-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interactions and does not appear to participate directly in catalysis. The C-terminal domain (327 amino acids) is responsible for catalysis and binds the two zinc ions, which are 2.88 A apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped beta-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-255, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes. Images PMID:2395881

  10. Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate.

    PubMed

    Viudes-de-Castro, M P; Mocé, E; Lavara, R; Marco-Jiménez, F; Vicente, J S

    2014-06-01

    Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses. PMID:24629591

  11. An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand.

    PubMed

    Imwong, Mallika; Jindakhad, Thantip; Kunasol, Chanon; Sutawong, Kreepol; Vejakama, Phisitt; Dondorp, Arjen M

    2015-01-01

    Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak. PMID:26616851

  12. How specific is Plasmodium falciparum adherence to chondroitin 4-sulfate?

    PubMed Central

    Goel, Suchi; Gowda, D. Channe

    2011-01-01

    Plasmodium falciparum infection during pregnancy results in the sequestration of infected red blood cells (IRBCs) in the placenta, contributing to pregnancy associated malaria (PAM). IRBC adherence is mediated by the binding of a variant Plasmodium falciparum erythrocyte binding protein 1 named VAR2CSA to the low sulfated chondroitin 4-sulfate (C4S) proteoglycan (CSPG) present predominantly in the intervillous space of the placenta. IRBC binding is highly specific to the level and distribution of 4-sulfate groups in C4S. Given the strict specificity of IRBC-C4S interactions, it is better to use either placental CSPG or CSPGs bearing structurally similar C4S chains in defining VAR2CSA structural architecture that interact with C4S, evaluating VAR2CSA constructs for vaccine development or studying structure-based inhibitors as therapeutics for PAM. PMID:21507719

  13. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014

    PubMed Central

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar

    2016-01-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August–December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  14. Symmetrical peripheral gangrene due to Plasmodium falciparum malaria

    PubMed Central

    Abdali, Nasar; Malik, Azharuddin Mohammed; Kamal, Athar; Ahmad, Mehtab

    2014-01-01

    A 45-year-old man presented with a 4-day history of high-grade fever with rigours and a 2-day history of painful bluish black discolouration of extremities (acrocyanosis). He was haemodynamically stable and all peripheral pulses palpable, but the extremities were cold with gangrene involving bilateral fingers and toes. Mild splenomegaly was present on abdominal examination but rest of the physical examinations were normal. On investigating he was found to have anaemia, thrombocytopaenia with gametocytes of Plasmodium falciparum on peripheral blood smear. His blood was uncoagulable during performance of prothrombin time with a raised D-dimer. Oxygen saturation was normal and the arterial Doppler test showed reduced blood flow to the extremities. A diagnosis of complicated P. falciparum malaria with disseminated intravascular coagulation (DIC) leading to symmetrical peripheral gangrene was performed. Artemisinin combination therapy was started and heparin was given for DIC. A final line of demarcation of gangrene started forming by 12th day. PMID:24862424

  15. Erythrocyte invasion receptors for Plasmodium falciparum: new and old.

    PubMed

    Satchwell, T J

    2016-04-01

    Understanding the complex process by which the invasive form of the Plasmodium falciparum parasite, the merozoite, attaches to and invades erythrocytes as part of its blood stage life cycle represents a key area of research in the battle to combat malaria. Central to this are efforts to determine the identity of receptors on the host cell surface, their corresponding merozoite-binding proteins and the functional relevance of these binding events as part of the invasion process. This review will provide an updated summary of studies identifying receptor interactions essential for or implicated in P. falciparum merozoite invasion of human erythrocytes, highlighting the recent identification of new receptors using groundbreaking high throughput approaches and with particular focus on the properties and putative involvement of the erythrocyte proteins targeted by these invasion pathways. PMID:26862042

  16. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014.

    PubMed

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar; Pradines, Bruno

    2016-05-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August-December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  17. An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand

    PubMed Central

    Imwong, Mallika; Jindakhad, Thantip; Kunasol, Chanon; Sutawong, Kreepol; Vejakama, Phisitt; Dondorp, Arjen M.

    2015-01-01

    Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak. PMID:26616851

  18. Studies on serum requirements for the cultivation of Plasmodium falciparum

    PubMed Central

    Divo, A. A.; Jensen, J. B.

    1982-01-01

    Previous experiments using RPMI 1640 medium have indicated that the dialysis of human serum removes components of low relative molecular mass (6000-8000 RMM) that are essential for continuous cultivation of Plasmodium falciparum. To determine which low-RMM components are important for parasite development, we compared growth in normal serum to that in dialysed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialysable nutrient required for parasite development. High quality bovine serum requires 3 - 12 × 10-5 mol/litre of hypoxanthine as a supplement to support continuous cultures of P. falciparum. Thus far we have been unable to attain parasite growth in medium containing supplemented bovine serum that is as good as growth in medium containing human serum. PMID:6754122

  19. Squalestatin Is an Inhibitor of Carotenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Gabriel, Heloisa B.; Silva, Marcia F.; Kimura, Emília A.; Wunderlich, Gerhard

    2015-01-01

    The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite. PMID:25779575

  20. Modeling Combinations of Pre-erythrocytic Plasmodium falciparum Malaria Vaccines.

    PubMed

    Walker, Andrew S; Lourenço, José; Hill, Adrian V S; Gupta, Sunetra

    2015-12-01

    Despite substantial progress in the control of Plasmodium falciparum infection due to the widespread deployment of insecticide-treated bed nets and artemisinin combination therapies, malaria remains a prolific killer, with over half a million deaths estimated to have occurred in 2013 alone. Recent evidence of the development of resistance to treatments in both parasites and their mosquito vectors has underscored the need for a vaccine. Here, we use a mathematical model of the within-host dynamics of P. falciparum infection, fit to data from controlled human malaria infection clinical trials, to predict the efficacy of co-administering the two most promising subunit vaccines, RTS,S/AS01 and ChAd63-MVA ME-TRAP. We conclude that currently available technologies could be combined to induce very high levels of sterile efficacy, even in immune-naive individuals. PMID:26503278

  1. Heterologous Protection against Malaria after Immunization with Plasmodium falciparum Sporozoites

    PubMed Central

    van Gemert, Geert-Jan; Graumans, Wouter; van de Vegte-Bolmer, Marga; van Lieshout, Lisette; Haks, Mariëlle C.; Hermsen, Cornelus C.; Scholzen, Anja; Visser, Leo G.; Sauerwein, Robert W.

    2015-01-01

    Background Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI) model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization), requiring only 30–45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains. Methods In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa) in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia) at 14 months after the last immunization (NCT01660854). Results Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0–15.5) versus 8.5 days in 5 malaria-naïve controls (p = 0.0005). Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10. Conclusion This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines. Trial Registration Clinicaltrials.gov NCT01660854 PMID:25933168

  2. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  3. Plasmodium falciparum genetic crosses in a humanized mouse model

    PubMed Central

    Vaughan, Ashley M.; Pinapati, Richard S.; Cheeseman, Ian H.; Camargo, Nelly; Fishbaugher, Matthew; Checkley, Lisa A.; Nair, Shalini; Hutyra, Carolyn A.; Nosten, François H.; Anderson, Timothy J. C.; Ferdig, Michael T.; Kappe, Stefan H. I.

    2015-01-01

    Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here, we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations. PMID:26030447

  4. Correlation between 'H' blood group antigen and Plasmodium falciparum invasion.

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2016-06-01

    The ABO blood group system is the most important blood group system in clinical practice. The relationship between Plasmodium falciparum and ABO blood groups has been studied for many years. This study was undertaken to investigate the abilities of different blood group erythrocytes to support in vitro growth of P. falciparum parasites. P. falciparum parasites of four different strains (3D7, 7G8, Dd2 and RKL9) were co-cultured with erythrocytes of blood group 'A', 'B', 'O' (n = 10 for each) and 'O(h)' (Bombay group) (n = 7) for 5 days. Statistically significant differences were observed on the fourth day among the mean percent parasitemias of 'O', non-'O' ('A' and 'B') and 'O(h)' group cultures. The parasitemias of four strains ranged from 12.23 to 14.66, 11.68 to 13.24, 16.89 to 22.3, and 7.37 to 11.27 % in 'A', 'B', 'O' and Bombay group cultures, respectively. As the expression of H antigen decreased from 'O' blood group to 'A' and 'B' and then to Bombay blood group, parasite invasion (percent parasitemia) also decreased significantly (p < 0.01) and concomitantly, indicating the association of parasite invasion with the amount of H antigen present on the surface of erythrocyte. Thus, the question arises, could H antigen be involved in P. falciparum invasion? To evaluate erythrocyte invasion inhibition, 'O' group erythrocytes were virtually converted to Bombay group-like erythrocytes by the treatment of anti-H lectins extracted from Ulex europaeus seeds. Mean percent parasitemia of lectin-treated cultures on the fourth day was significantly lower (p < 0.05) than that of non-treated cultures and was found to be similar with the mean percent parasitemia demonstrated by the Bombay group erythrocyte cultures, thus further strengthening the hypothesis. PMID:27071756

  5. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    SciTech Connect

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  6. Purification and characterisation of an intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4.

    PubMed

    Tsakalidou, E; Anastasiou, R; Papadimitriou, K; Manolopoulou, E; Kalantzopoulos, G

    1997-01-01

    An intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4, isolated from traditional Greek yoghurt, was purified by anion exchange and gel filtration chromatography. A single band of molecular weight of about 80,000 appeared in SDS-PAGE; by gel filtration it was shown that the native enzyme was dimeric. The peptidase showed optimum activity on glycyl-prolyl 4-nitroanilide at pH 7.0 and at 50 degrees C, with K(m) = 3.1 mM and Vmax = 3500 U mg-1; over 50 degrees C the enzyme activity declined rapidly. It was inactivated by PMSF; sulfhydryl group reagents and metal chelators had little effect on enzyme activity. PMID:9519481

  7. The trigger enzyme PepA (aminopeptidase A) of Escherichia coli, a transcriptional repressor that generates positive supercoiling.

    PubMed

    Nguyen Le Minh, Phu; Nadal, Marc; Charlier, Daniel

    2016-06-01

    Escherichia coli aminopeptidase A (PepA) is a trigger enzyme endowed with catalytic activity and DNA-binding properties prominent in transcriptional regulation and site-specific DNA recombination. The current work demonstrates that PepA is a repressor in its own right, capable of specifically inhibiting transcription initiation at promoter P1 of the carAB operon, encoding carbamoylphosphate synthase. Furthermore, in vitro topology studies performed with DNA minicircles demonstrate that PepA binding constrains a single positive supercoil in the carP1 control region. Such a topological event is understood to constitute an impediment to transcription initiation and may serve as a mechanism to regulate gene expression. PMID:27213286

  8. Pfcrt Gene in Plasmodium falciparum Field Isolates from Muzaffargarh, Pakistan

    PubMed Central

    Sahar, Sumrin; Tanveer, Akhtar; Ali, Akbar; Bilal, Hazrat; Muhammad Saleem, Rana

    2015-01-01

    Background: The aim of the study was to identify the prevalence of different species of Plasmodium and haplotypes of pfcrt in Plasmodium falciparum from the selected area. Methods: Overall, 10,372 blood films of suspected malarial patients were examined microscopically from rural health center Sinawan, district Muzaffargarh, Pakistan from November 2008 to November 2010. P. falciparum positive samples (both whole blood and FTA blood spotted cards) were used for DNA extraction. Nested PCR was used to amplify the pfcrt (codon 72–76) gene fragment. Sequencing was carried out to find the haplotypes in the amplified fragment of pfcrt gene. Result: Over all slide positivity rate (SPR), P. vivax and P. falciparum positivity rate was 21.40 %, 19.37 % and 2.03% respectively. FTA blood spotted cards were equally efficient in the blood storage for PCR and sequencing. Analysis of sequencing results of pfcrt showed only one type of haplotype SagtVMNT (AGTGTAATGAATACA) from codon 72–76 in all samples. Conclusion: The results show high prevalence of CQ resistance and AQ resistant genes. AQ is not recommended to be used as a partner drug in ACT in this locality, so as to ward off future catastrophes. PMID:26623432

  9. Population structure and recent evolution of Plasmodium falciparum

    PubMed Central

    Rich, Stephen M.; Ayala, Francisco J.

    2000-01-01

    Plasmodium falciparum is the agent of malignant malaria, one of mankind's most severe maladies. The parasite exhibits antigenic polymorphisms that have been postulated to be ancient. We have proposed that the extant world populations of P. falciparum have derived from one single parasite, a cenancestor, within the last 5,000–50,000 years. This inference derives from the virtual or complete absence of synonymous nucleotide polymorphisms at genes not involved in immune or drug responses. Seeking to conciliate this claim with extensive antigenic polymorphism, we first note that allele substitutions or polymorphisms can arise very rapidly, even in a single generation, in large populations subject to strong natural selection. Second, new alleles can arise not only by single-nucleotide mutations, but also by duplication/deletion of short simple-repeat DNA sequences, a process several orders of magnitude faster than single-nucleotide mutation. We analyze three antigenic genes known to be extremely polymorphic: Csp, Msp-1, and Msp-2. We identify regions consisting of tandem or proximally repetitive short DNA sequences, including some previously unnoticed. We conclude that the antigenic polymorphisms are consistent with the recent origin of the world populations of P. falciparum inferred from the analysis of nonantigenic genes. PMID:10860962

  10. Intrarectal quinine for treating Plasmodium falciparum malaria: a systematic review

    PubMed Central

    Eisenhut, Michael; Omari, Aika; MacLehose, Harriet G

    2005-01-01

    Background In children with malaria caused by Plasmodium falciparum, quinine administered rectally may be easier to use and less painful than intramuscular or intravenous administration. The objective of this review was to compare the effectiveness of intrarectal with intravenous or intramuscular quinine for treating falciparum malaria. Methods All randomized and quasi-randomized controlled trials comparing intrarectal with intramuscular or intravenous quinine for treating people with falciparum malaria located through the following sources were included: Cochrane Infectious Diseases Group Specialized Register, CENTRAL, MEDLINE, EMBASE, LILACS and CINAHL. Trial quality was assessed and data, including adverse event data, were extracted. Dichotomous data were analysed using odds ratios and continuous data using weighted mean difference. Results Eight randomized controlled trials (1,247 children) fulfilled the inclusion criteria. The same principal investigator led seven of the trials. Five compared intrarectal with intravenous quinine, and six compared intrarectal with intramuscular treatment. No statistically significant difference was detected for death, parasite clearance by 48 hours and seven days, parasite and fever clearance time, coma recovery time, duration of hospitalization and time before drinking began. One trial (898 children) reported that intrarectal was less painful than intramuscular administration. Conclusion No difference in the effect on parasites and clinical illness was detected for the use of intrarectal quinine compared with other routes, but most trials were small. Pain during application may be less with intrarectal quinine. Further larger trials, in patients with severe malaria and in adults, are required before the intrarectal route could be recommended. PMID:15904520

  11. Laboratory detection of artemisinin-resistant Plasmodium falciparum.

    PubMed

    Chotivanich, Kesinee; Tripura, Rupam; Das, Debashish; Yi, Poravuth; Day, Nicholas P J; Pukrittayakamee, Sasithon; Chuor, Char Meng; Socheat, Duong; Dondorp, Arjen M; White, Nicholas J

    2014-06-01

    Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility. PMID:24663013

  12. Efficacy of scopadulcic acid A against Plasmodium falciparum in vitro.

    PubMed

    Riel, Michael A; Kyle, Dennis E; Milhous, Wilbur K

    2002-04-01

    Scoparia dulcis is a perennial herb widely distributed in many tropical countries. It is used as an herbal remedy for gastrointestinal and many other ailments, and in Nicaragua extracts are used to treat malaria. Phytochemical screening has shown that scopadulcic acid A (SDA), scopadulcic acid B (SDB), and semisynthetic analogues are pharmacologically active compounds from S. dulcis. SDB has antiviral activity against Herpes simplex virus type 1, antitumor activity in various human cell lines, and direct inhibitory activity against porcine gastric H(+), K(+)-ATPase. A methyl ester of scopadulcic acid B showed the most potent inhibitory activity against gastric proton pumps of 30 compounds tested in one study. Compounds with antiviral, antifungal, and antitumor activity often show activity against Plasmodium falciparum. In P. falciparum, the plasma membrane and food vacuole have H(+)-ATPases and the acidocalcisome has an H(+)-Ppase. These proton pumps are potential targets for antimalarial therapy and may have their function disrupted by compounds known to inhibit gastric proton pumps. We tested pure SDA and found in vitro activity against P. falciparum with an IC(50) of 27 and 19 microM against the D6 and W2 clones, respectively. The IC(50) against the multidrug-resistant isolate, TM91C235, was 23 microM. PMID:11975516

  13. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  14. TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

    SciTech Connect

    Szawlowska, Urszula; Grabowska, Agnieszka; Zdunek-Zastocka, Edyta; Bielawski, Wieslaw

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant prolyl aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

  15. Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene.

    PubMed

    Samarasinghe, Buddhini; Knox, David P; Britton, Collette

    2011-01-01

    Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of β-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, β-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites. PMID:20699100

  16. Caspar Controls Resistance to Plasmodium falciparum in Diverse Anopheline Species

    PubMed Central

    Garver, Lindsey S.; Dong, Yuemei; Dimopoulos, George

    2009-01-01

    Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway–mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort development of P

  17. Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

    1989-06-01

    The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

  18. Artesunate Misuse and Plasmodium falciparum Malaria in Traveler Returning from Africa

    PubMed Central

    Shahinas, Dea; Lau, Rachel; Khairnar, Krishna; Hancock, David

    2010-01-01

    Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility. PMID:20875291

  19. Artesunate misuse and Plasmodium falciparum malaria in traveler returning from Africa.

    PubMed

    Shahinas, Dea; Lau, Rachel; Khairnar, Krishna; Hancock, David; Pillai, Dylan R

    2010-10-01

    Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility. PMID:20875291

  20. Possible Clinical Failure of Artemether-Lumefantrine in an Italian Traveler with Uncomplicated Falciparum Malaria.

    PubMed Central

    Repetto, Ernestina C.; Traverso, Antonio; Giacomazzi, Claudio G.

    2011-01-01

    Artemisinin-combination therapies (ACTs) are recommended for the treatment of uncomplicated malaria in endemic areas with multidrug resistant Plasmodium falciparum. We report a case of possible artemether-lumefantrine clinical failure in an Italian traveler with uncomplicated P. falciparum malaria imported from Democratic Republic of Congo. PMID:22084655

  1. A Cost-Effectiveness Analysis of Plasmodium falciparum Malaria Elimination in Hainan Province, 2002-2012.

    PubMed

    Sun, Ding-Wei; Du, Jian-Wei; Wang, Guang-Ze; Li, Yu-Chun; He, Chang-Hua; Xue, Rui-De; Wang, Shan-Qing; Hu, Xi-Min

    2015-12-01

    In Hainan Province, China, great achievements in elimination of falciparum malaria have been made since 2010. There have been no locally acquired falciparum malaria cases since that time. The cost-effectiveness of elimination of falciparum malaria has been analyzed in Hainan Province. There were 4,422 falciparum malaria cases reported from 2002 to 2012, more cases occurred in males than in females. From 2002 to 2012, a total of 98.5 disability-adjusted life years (DALYs) were reported because of falciparum malaria. Populations in the age ranges of 15-25 and 30-44 years had higher incidences and DALYs than other age groups. From 2002 to 2012, malaria-related costs for salaries of staff, funds from the provincial government, national government, and the GFATM were US$3.02, US$2.24, US$1.44, and US$5.08 million, respectively. An estimated 9,504 falciparum malaria cases were averted during the period 2003-2012. The estimated cost per falciparum malaria case averted was US$116.5. The falciparum malaria elimination program in Hainan was highly effective and successful. However, funding for maintenance is still needed because of imported cases. PMID:26438030

  2. Barley DNA-binding methionine aminopeptidase, which changes the localization from the nucleus to the cytoplasm by low temperature, is involved in freezing tolerance.

    PubMed

    Jeong, Hee-Jeong; Shin, Jeong Sheop; Ok, Sung Han

    2011-01-01

    The polymerase chain reaction-based Mirror Orientation Selection (MOS) method was used to isolate low temperature-induced genes from cold-treated winter barley (Hordeum vulgare L. cv. Dongbori). MOS screening identified a novel methionine (Met) aminopeptidase (MAP) designated as HvMAP. The deduced HvMAP protein was determined to possess an aminopeptidase domain and a nuclear localization signal. An in vitro enzyme assay using recombinant HvMAP protein demonstrated MAP activity. The expression of this gene was induced by low temperature and abscisic acid treatment, and overexpression of this gene conferred stronger freezing tolerance to Arabidopsis transgenic plants as compared to wild-type plants. Interestingly, low temperature treatment changed the localization of HvMAP from the nucleus to the cytoplasm. These findings suggest that HvMAP is a novel MAP that functions in freezing tolerance by facilitating protein maturation. PMID:21421347

  3. Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

    PubMed

    Węglarz-Tomczak, Ewelina; Vassiliou, Stamatia; Mucha, Artur

    2016-08-15

    A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,β-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases. PMID:27390066

  4. Refrigeration provides a simple means to synchronize in vitro cultures of Plasmodium falciparum.

    PubMed

    Yuan, Lili; Hao, Mingming; Wu, Lanou; Zhao, Zhen; Rosenthal, Benjamin M; Li, Xiaomei; He, Yongshu; Sun, Ling; Feng, Guohua; Xiang, Zheng; Cui, Liwang; Yang, Zhaoqing

    2014-05-01

    Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of P. falciparum are routinely used in malaria research. Here, we describe a simple synchronization procedure for P. falciparum asexual erythrocytic culture, which involves storage at 4°C for 8-24 h followed by routine culture. When cultures with 27-60% of ring stage were synchronized using this procedure, 70-93% ring stages were obtained after 48 h of culture and relative growth synchrony remained for at least two erythrocytic cycles. To test the suitability of this procedure for subsequent work, drug sensitivity assays were performed using four laboratory strains and four freshly adapted clinical P. falciparum isolates. Parasites synchronized by sorbitol treatment or refrigeration showed similar dose-response curves and comparable IC50 values to four antimalarial drugs. The refrigeration synchronization method is simple, inexpensive, time-saving, and should be especially useful when large numbers of P. falciparum culture are handled. PMID:24632190

  5. Efficacy of Chloroquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria in Honduras

    PubMed Central

    Torres, Rosa Elena Mejia; Banegas, Engels Ilich; Mendoza, Meisy; Diaz, Cesar; Bucheli, Sandra Tamara Mancero; Fontecha, Gustavo A.; Alam, Md Tauqeer; Goldman, Ira; Udhayakumar, Venkatachalam; Zambrano, Jose Orlinder Nicolas

    2013-01-01

    Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated using the Pan American Health Organization—World Health Organization protocol with a follow-up of 28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations (pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras. PMID:23458957

  6. Molecular characterization of Trichinella spiralis aminopeptidase and its potential as a novel vaccine candidate antigen against trichinellosis in BALB/c mice

    PubMed Central

    2013-01-01

    Background Trichinella spiralis is an intracellular parasite that can cause a serious threat to human health by causing trichinellosis. The aminopeptidase (AP) was found in the proteins produced by T. spiralis infective larvae after in vitro co-culture with intestinal epithelial cells, but its characteristics and function are unknown. The purpose of this study was to identify the T. spiralis aminopeptidase (TsAP) and to investigate its potential as a vaccine candidate antigen against T. spiralis infection. Methods T. spiralis aminopeptidase (TsAP) gene encoding a 54.7 kDa protein was cloned and expressed in Escherichia coli, and purified recombinant TsAP protein was used to immunize BALB/c mice. The antibodies obtained were used to determine where TsAP was localized in the parasite. Transcription and expression of TsAP in different developmental stages of T. spiralis were observed by RT-PCR and Immunofluorescence test (IFT). The immune protection of recombinant TsAP protein against T. spiralis infection in BALB/c mice was evaluated. Results Anti-TsAP antibodies recognized the native protein migrating at 54.7 kDa by Western blotting of the crude antigens from muscle larvae. Transcription and expression of TsAP gene was observed in different developmental stages (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). TsAP appears to be a cytoplasmic protein located primarily at the cuticle and internal organs of this parasite. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TsAP protein displayed a 38.1% reduction in adult worm burden and 59.1% reduction in muscle larval burden. Conclusions In this study, T. spiralis aminopeptidase (TsAP) was first characterized and will help reveal its potential biological functions. TsAP is a novel potential vaccine candidate antigen that merits further investigation. PMID:23972034

  7. The Limits and Intensity of Plasmodium falciparum Transmission: Implications for Malaria Control and Elimination Worldwide

    PubMed Central

    Guerra, Carlos A; Gikandi, Priscilla W; Tatem, Andrew J; Noor, Abdisalan M; Smith, Dave L; Hay, Simon I; Snow, Robert W

    2008-01-01

    Background The efficient allocation of financial resources for malaria control using appropriate combinations of interventions requires accurate information on the geographic distribution of malaria risk. An evidence-based description of the global range of Plasmodium falciparum malaria and its endemicity has not been assembled in almost 40 y. This paper aims to define the global geographic distribution of P. falciparum malaria in 2007 and to provide a preliminary description of its transmission intensity within this range. Methods and Findings The global spatial distribution of P. falciparum malaria was generated using nationally reported case-incidence data, medical intelligence, and biological rules of transmission exclusion, using temperature and aridity limits informed by the bionomics of dominant Anopheles vector species. A total of 4,278 spatially unique cross-sectional survey estimates of P. falciparum parasite rates were assembled. Extractions from a population surface showed that 2.37 billion people lived in areas at any risk of P. falciparum transmission in 2007. Globally, almost 1 billion people lived under unstable, or extremely low, malaria risk. Almost all P. falciparum parasite rates above 50% were reported in Africa in a latitude band consistent with the distribution of Anopheles gambiae s.s. Conditions of low parasite prevalence were also common in Africa, however. Outside of Africa, P. falciparum malaria prevalence is largely hypoendemic (less than 10%), with the median below 5% in the areas surveyed. Conclusions This new map is a plausible representation of the current extent of P. falciparum risk and the most contemporary summary of the population at risk of P. falciparum malaria within these limits. For 1 billion people at risk of unstable malaria transmission, elimination is epidemiologically feasible, and large areas of Africa are more amenable to control than appreciated previously. The release of this information in the public domain will

  8. Determining the involvement of two aminopeptidase Ns in the resistance of Plutella xylostella to the Bt toxin Cry1Ac: cloning and study of in vitro function.

    PubMed

    Chang, Xiaoli; Wu, Qingjun; Wang, Shaoli; Wang, Ran; Yang, Zhongxia; Chen, Defeng; Jiao, Xiaoguo; Mao, Zhenchuan; Zhang, Youjun

    2012-02-01

    The cloning, expression in vitro, and characterization of two aminopeptidase Ns (APN5s and APN2s) isolated from the midgut of Cry1Ac-resistant (R) and susceptible (S) strains of Plutella xylostella larvae are presented in this paper. The deduced amino acid sequences of APN5s included C-terminal GPI-modification sites, the gluzincin aminopeptidase motif GATEN, and three N-glycosylated sites; those of APN2s had no GPI-modification sites, had gluzincin aminopeptidase motif GAMEN, and had four N-glycosylated sites. O-glycosylated sites were not predicted for either APN. Because APN2R and APN2S cDNAs contained the same nucleotides, only full-length cDNAs encoding APN5R and APN5S were expressed in Trichoplusia ni cells. Far-Western blotting showed that the expressed receptor APN5 bound to the Cry1Ac toxin. An enzyme-specific activity experiment also showed that APN5 genes were expressed in T. ni cells. ELISA revealed no differences in the binding of expression proteins from the resistant and susceptible strain with Cry1Ac. PMID:22371317

  9. Characterization of a salt-tolerant aminopeptidase from marine Bacillus licheniformis SWJS33 that improves hydrolysis and debittering efficiency for soy protein isolate.

    PubMed

    Lei, Fenfen; Zhao, Qiangzhong; Sun-Waterhouse, Dongxiao; Zhao, Mouming

    2017-01-01

    An aminopeptidase was isolated from the marine Bacillus licheniformis SWJS33 (BLAP) and purified. According to the tandem mass spectrometry, the enzyme displayed 11% amino acid identity with the aminopeptidase from Bacillus (gi|496687392). BLAP exhibited maximum activity at 60°C and pH 8.0-8.5 and had a molecular mass of 100kDa. The presence of NaCl enabled 50% improvement of enzyme activity with 10-15% NaCl being the best. The observed inactivation by EDTA and bestatin and activation by Co(2+) and Ag(+) indicated that the obtained enzyme was a metalloaminopeptidase. Such an aminopeptidase could further improve the hydrolysis degree of soy protein isolate hydrolysates catalyzed by papain, Alcalase 2.4L or Flavourzyme 500MG from 8.5%, 9.5% or 14.4-18.8%, 18.7% or 20.1%, respectively, while decreasing the bitter intensity score of the SPI hydrolysates catalyzed by Alcalase 2.4L from 3.6 to 0.4. PMID:27507484

  10. Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities).

    PubMed Central

    Amrani, A. E.; Couee, I.; Carde, J. P.; Gaudillere, J. P.; Raymond, P.

    1994-01-01

    We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation. PMID:12232431

  11. The combination of HDAC and aminopeptidase inhibitors is highly synergistic in myeloma and leads to disruption of the NFκB signalling pathway.

    PubMed

    Smith, Emma M; Zhang, Lei; Walker, Brian A; Davenport, Emma L; Aronson, Lauren I; Krige, David; Hooftman, Leon; Drummond, Alan H; Morgan, Gareth J; Davies, Faith E

    2015-07-10

    There is a growing body of evidence supporting the use of epigenetic therapies in the treatment of multiple myeloma. We show the novel HDAC inhibitor CHR-3996 induces apoptosis in myeloma cells at concentrations in the nanomolar range and with apoptosis mediated by p53 and caspase pathways. In addition, HDAC inhibitors are highly synergistic, both in vitro and in vivo, with the aminopeptidase inhibitor tosedostat (CHR-2797). We demonstrate that the basis for this synergy is a consequence of changes in the levels of NFκB regulators BIRC3/cIAP2, A20, CYLD, and IκB, which were markedly affected by the combination. When co-administered the HDAC and aminopeptidase inhibitors caused rapid nuclear translocation of NFκB family members p65 and p52, following activation of both canonical and non-canonical NFκB signalling pathways. The subsequent up-regulation of inhibitors of NFκB activation (most significantly BIRC3/cIAP2) turned off the cytoprotective effects of the NFκB signalling response in a negative feedback loop. These results provide a rationale for combining HDAC and aminopeptidase inhibitors clinically for the treatment of myeloma patients and support the disruption of the NFκB signalling pathway as a therapeutic strategy. PMID:26015393

  12. Enzymatic properties of the glycine D-alanine [corrected] aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji).

    PubMed

    Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi

    2012-01-01

    The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9. PMID:22005737

  13. A World Malaria Map: Plasmodium falciparum Endemicity in 2007

    PubMed Central

    Hay, Simon I; Guerra, Carlos A; Gething, Peter W; Patil, Anand P; Tatem, Andrew J; Noor, Abdisalan M; Kabaria, Caroline W; Manh, Bui H; Elyazar, Iqbal R. F; Brooker, Simon; Smith, David L; Moyeed, Rana A; Snow, Robert W

    2009-01-01

    Background Efficient allocation of resources to intervene against malaria requires a detailed understanding of the contemporary spatial distribution of malaria risk. It is exactly 40 y since the last global map of malaria endemicity was published. This paper describes the generation of a new world map of Plasmodium falciparum malaria endemicity for the year 2007. Methods and Findings A total of 8,938 P. falciparum parasite rate (PfPR) surveys were identified using a variety of exhaustive search strategies. Of these, 7,953 passed strict data fidelity tests for inclusion into a global database of PfPR data, age-standardized to 2–10 y for endemicity mapping. A model-based geostatistical procedure was used to create a continuous surface of malaria endemicity within previously defined stable spatial limits of P. falciparum transmission. These procedures were implemented within a Bayesian statistical framework so that the uncertainty of these predictions could be evaluated robustly. The uncertainty was expressed as the probability of predicting correctly one of three endemicity classes; previously stratified to be an informative guide for malaria control. Population at risk estimates, adjusted for the transmission modifying effects of urbanization in Africa, were then derived with reference to human population surfaces in 2007. Of the 1.38 billion people at risk of stable P. falciparum malaria, 0.69 billion were found in Central and South East Asia (CSE Asia), 0.66 billion in Africa, Yemen, and Saudi Arabia (Africa+), and 0.04 billion in the Americas. All those exposed to stable risk in the Americas were in the lowest endemicity class (PfPR2−10 ≤ 5%). The vast majority (88%) of those living under stable risk in CSE Asia were also in this low endemicity class; a small remainder (11%) were in the intermediate endemicity class (PfPR2−10 > 5 to < 40%); and the remaining fraction (1%) in high endemicity (PfPR2−10 ≥ 40%) areas. High endemicity was widespread in the

  14. [Plasmodium falciparum malaria: evaluation of three imported cases].

    PubMed

    İnkaya, Ahmet Çağkan; Kaya, Filiz; Yıldız, İrem; Uzun, Ömrüm; Ergüven, Sibel

    2016-04-01

    Among Plasmodium species the causative agent of malaria in Turkey is P.vivax, however the incidence of imported falciparum malaria cases is steadily increasing. P.falciparum may cause severe malaria with the involvement of central nervous system, acute renal failure, severe anemia or acute respiratory distress syndrome. Furhermore most of the casualties due to malaria are related with P.falciparum. There is recently, a considerable increase in malaria infections especially in tropical areas. In this report, three cases, who have admitted to our hospital with three different clinical presentations of falciparum malaria, and all shared common history of travelling to Africa were presented. First case was a 27 years old, male patient who returned from Malawi seven days ago where he stayed for two weeks. He admitted to our hospital with the complaints of sensation of cold, shivering and fever. In physical examination his body temperature was 37.9°C, C-reactive protein level was high, and the other systemic results were normal. The second case was a 25 years old, male patient who returned from Gambia two weeks ago. He was suffering from fever, headache, shivering and unable to maintain his balance. The patient's body temperature was 38°C. Laboratory tests revealed hyperbilirubinemia and thrombocytopenia. Parasitological examination of the Giemsa-stained peripheral blood smear of these two patients demonstrated ring forms compatible with P.falciparum. Treatment was commenced with arthemeter plus lumefantrine, resulting with complete cure. Third case was a 46 years old, male patient who had been working in Uganda, and returned to Turkey two weeks ago. He had sudden onset of fever, headache, nausea and vomiting and impaired consciousness. His peripheral blood smear revealed ring-formed trophozoites and banana-shaped gametocytes of P.falciparum. Arthemeter plus lumefantrine therapy was started, however, he developed severe thrombocytopenia and jaundice under treatment

  15. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  16. pfmdr2 confers heavy metal resistance to Plasmodium falciparum.

    PubMed

    Rosenberg, Elli; Litus, Ilena; Schwarzfuchs, Nurit; Sinay, Rosa; Schlesinger, Pnina; Golenser, Jacob; Baumeister, Stefan; Lingelbach, Klaus; Pollack, Yaakov

    2006-09-15

    Heavy metals are required by all organisms for normal function, but high levels of heavy metals are toxic. Therefore, homeostasis of these metals is crucial. In the human malaria-causing agent Plasmodium falciparum, the mechanisms of heavy metal transport have yet to be characterized. We have developed a P. falciparum line resistant to heavy metals from a wild-type line sensitive to heavy metals. A molecular and biochemical analysis of the involvement of the P. falciparum multidrug resistance 2 (pfmdr2) gene, an ABC-type transporter, in heavy metal homeostasis was studied. Using a novel uptake assay applied on these two strains, it was demonstrated that, when exposed to heavy metals, the sensitive line accumulates metal, whereas no accumulation was observed in the resistant line. The accumulation occurs within the parasite itself and not in the cytoplasm of the red blood cell. This difference in the accumulation pattern is not a result of amplification of the pfmdr2 gene or of a change in the expression pattern of the gene in the two lines. Sequencing of the gene from both lines revealed a major difference; a stop codon is found in the sensitive line upstream of the normal termination, resulting in a truncated protein that lacks 188 amino acids that contain a portion of the essential cytoplasmatic transporter domain, thereby rendering it inactive. In contrast, the resistant line harbors a full-length, active protein. These findings strongly suggest that the PFMDR2 protein acts as an efflux pump of heavy metals. PMID:16849328

  17. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M; Holder, Anthony A

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  18. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  19. Immunoregulatory alterations in Plasmodium falciparum and Plasmodium vivax infections.

    PubMed

    Merino, F; Layrisse, Z; Godoy, G; Volcán, G

    1986-09-01

    Studies on the immune function of patients with acute Plasmodium vivax or P. falciparum infections were performed. All subjects were residing in recent malaria endemic areas of Venezuela. Lymphopenia, reduction of peripheral blood T-lymphocytes positive for monoclonal antibody OKT4 (T helper) a decrease of in vitro mitogenic proliferative response and natural killer cell activity were observed. Serum lymphocytotoxic antibodies reactive at 37 degrees C were detected in both groups of patients as well as serum autoantibodies. The possible role of lymphocytotoxic autoantibodies in the etiology of the T-lymphocyte depletion and acquired immunological perturbations in human malaria is discussed. PMID:2947313

  20. Unraveling the 'DEAD-box' helicases of Plasmodium falciparum.

    PubMed

    Tuteja, Renu; Pradhan, Arun

    2006-07-01

    The causative agent for the most fatal form of malaria, Plasmodium falciparum, has developed insecticide and drug resistance with time. Therefore combating this disease is becoming increasingly difficult and this calls for finding alternate ways to control malaria. One of the feasible ways could be to find out inhibitors/drugs specific for the indispensable enzymes of malaria parasite such as helicases. These helicases, which contain intrinsic nucleic acid-dependent ATPase activity, are capable of enzymatically unwinding energetically stable duplex nucleic acids into single-stranded templates and are required for all the nucleic acid transactions. Most of the helicases contain a set of nine extremely conserved amino acid sequences, which are called 'helicase motifs'. Due to the presence of the DEAD (Asp-Glu-Ala-Asp) in one of the conserved motifs, this family is also known as the 'DEAD-box' family. In this review, using bioinformatic approach, we describe the 'DEAD-box' helicases of malaria parasite P. falciparum. An in depth analysis shows that the parasite contains 22 full-length genes, some of which are homologues of well-characterized helicases of this family from other organisms. Recently we have cloned and characterized the first member of this family, which is a homologue of p68 and is expressed during the schizont stage of the development of the parasite [Pradhan, A., Chauhan, V.S., Tuteja, R., 2005a. A novel 'DEAD-box' DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. 140, 55-60.; Pradhan A., Chauhan V.S., Tuteja R., 2005b. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. 144, 133-141.]. It will be really interesting to clone and characterize other members of the 'DEAD-box' family and understand their role in the replication and transmission of the parasite. These detailed studies may help to identify a parasite

  1. Spread of Artemisinin Resistance in Plasmodium falciparum Malaria

    PubMed Central

    Ashley, E.A.; Dhorda, M.; Fairhurst, R.M.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J.M.; Mao, S.; Sam, B.; Sopha, C.; Chuor, C.M.; Nguon, C.; Sovannaroth, S.; Pukrittayakamee, S.; Jittamala, P.; Chotivanich, K.; Chutasmit, K.; Suchatsoonthorn, C.; Runcharoen, R.; Hien, T.T.; Thuy-Nhien, N.T.; Thanh, N.V.; Phu, N.H.; Htut, Y.; Han, K-T.; Aye, K.H.; Mokuolu, O.A.; Olaosebikan, R.R.; Folaranmi, O.O.; Mayxay, M.; Khanthavong, M.; Hongvanthong, B.; Newton, P.N.; Onyamboko, M.A.; Fanello, C.I.; Tshefu, A.K.; Mishra, N.; Valecha, N.; Phyo, A.P.; Nosten, F.; Yi, P.; Tripura, R.; Borrmann, S.; Bashraheil, M.; Peshu, J.; Faiz, M.A.; Ghose, A.; Hossain, M.A.; Samad, R.; Rahman, M.R.; Hasan, M.M.; Islam, A.; Miotto, O.; Amato, R.; MacInnis, B.; Stalker, J.; Kwiatkowski, D.P.; Bozdech, Z.; Jeeyapant, A.; Cheah, P.Y.; Sakulthaew, T.; Chalk, J.; Intharabut, B.; Silamut, K.; Lee, S.J.; Vihokhern, B.; Kunasol, C.; Imwong, M.; Tarning, J.; Taylor, W.J.; Yeung, S.; Woodrow, C.J.; Flegg, J.A.; Das, D.; Smith, J.; Venkatesan, M.; Plowe, C.V.; Stepniewska, K.; Guerin, P.J.; Dondorp, A.M.; Day, N.P.; White, N.J.

    2014-01-01

    BACKGROUND Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand–Cambodia border. Slowly clearing in fections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the “propeller” region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of

  2. Replication and maintenance of the Plasmodium falciparum apicoplast genome.

    PubMed

    Milton, Morgan E; Nelson, Scott W

    2016-08-01

    Members of the phylum Apicomplexa are responsible for many devastating diseases including malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), babesiosis (Babesia bovis), and cyclosporiasis (Cyclospora cayetanensis). Most Apicomplexans contain a unique and essential organelle called the apicoplast. Derived from an ancient chloroplast, the apicoplast replicates and maintains a 35 kilobase (kb) circular genome. Due to its essential nature within the parasite, drugs targeted to proteins involved in DNA replication and repair of the apicoplast should be potent and specific. This review summarizes the current knowledge surrounding the replication and repair of the Plasmodium falciparum apicoplast genome and identifies several putative proteins involved in replication and repair pathways. PMID:27338018

  3. [Artemisinin resistance in Plasmodium falciparum: global status and basic research].

    PubMed

    Zhao, Shao-min; Wang, Man-yuan

    2014-10-01

    Artemisinin-resistant Plasmodium falciparum has been identified by WHO in the Greater Mekong subregion. While there is no report on artemisinin resistance in Africa and South America by now, related surveillance measures have been taken place. The genes related artemisinin-resistance has been identified and the molecular markers will be used for large-scale surveillance efforts to contain artemisinin resistance. The emergence and spread of artemisinin resistance worldwide is a present danger and needs more attention. This article reviews the progress of artemisininresistance malaria parasites and artemisinin-based combination therapies. PMID:25726605

  4. Discrete-Event Simulation Models of Plasmodium falciparum Malaria

    PubMed Central

    McKenzie, F. Ellis; Wong, Roger C.; Bossert, William H.

    2008-01-01

    We develop discrete-event simulation models using a single “timeline” variable to represent the Plasmodium falciparum lifecycle in individual hosts and vectors within interacting host and vector populations. Where they are comparable our conclusions regarding the relative importance of vector mortality and the durations of host immunity and parasite development are congruent with those of classic differential-equation models of malaria, epidemiology. However, our results also imply that in regions with intense perennial transmission, the influence of mosquito mortality on malaria prevalence in humans may be rivaled by that of the duration of host infectivity. PMID:18668185

  5. Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli.

    PubMed Central

    Hwang, D D; Liu, L F; Kuan, I C; Lin, L Y; Tam, T C; Tam, M F

    1999-01-01

    We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers. PMID:10024508

  6. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

    PubMed Central

    Crabb, B S; Cowman, A F

    1996-01-01

    Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest. Images Fig. 4 Fig. 5 PMID:8692985

  7. Plasmodium falciparum polypeptides released during in vitro cultivation*

    PubMed Central

    Da Silva, L. Rodriguez; Loche, M.; Dayal, R.; Perrin, L. H.

    1983-01-01

    Synchronous cultures of Plasmodium falciparum were successively labelled with (35S)-methionine and both the supernatants and the pellets of infected red blood cells were collected. The release of TCA-precipitable material in the culture supernatants was low during the development of ring forms and trophozoites, increased during schizogony, and was maximum at the time of schizont rupture and merozoite reinvasion. Analysis of the supernatants by SDS — PAGE and autoradiography showed that both polypeptides common to the various developmental stages of the parasite and schizont/merozoite-specific polypeptides were released. Polypeptides of relative molecular mass 140 000, 82 000 and, to a lower degree, 41 000 were present in high amounts in the culture supernatants. These polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the growth of P. falciparum cultures, and may be involved in protective immunity. The released polypeptides may also be used as target antigens in immunodiagnostic tests aiming at the detection of malaria infection. ImagesFig. 2AFig. 2BFig. 3 PMID:6340846

  8. Resistance of Plasmodium falciparum to antimalarial drugs in Equatorial Guinea.

    PubMed

    Roche, J; Benito, A; Ayecaba, S; Amela, C; Molina, R; Alvar, J

    1993-10-01

    One hundred and sixty-six children from Equatorial Guinea, all under 10 years of age and with acute uncomplicated falciparum malaria, were randomly allocated to four groups and treated with one of the following regimens: chloroquine or amodiaquine (25 mg base/kg body weight over 3 days), quinine (8 mg/kg every 8 h for 3 or 5 days), and sulphadoxine-pyrimethamine (25-1.25 mg/kg, in one dose). The parasite clearance rates up to day 14 were 28% with chloroquine, 74% with amodiaquine, and 95% with quinine or sulphadoxine-pyrimethamine. The times required to clear asexual blood forms of Plasmodium falciparum in sensitive cases were 64, 70, 73 and 65 h, respectively. Although quinine and sulphadoxine-pyrimethamine are equally effective, quinine is recommended for treatment of multidrug-resistant malaria in paediatric patients, essentially because of the risk of serious reactions to sulpha drugs. Health providers are, however, encouraged to keep supplies of sulphadoxine-pyrimethamine as an option and to refer patients quickly, if required. PMID:8311568

  9. Delayed Diagnosis of Falciparum Malaria with Acute Kidney Injury.

    PubMed

    Choi, Iee Ho; Hwang, Pyoung Han; Choi, Sam Im; Lee, Dae Yeol; Kim, Min Sun

    2016-09-01

    Prompt malaria diagnosis is crucial so antimalarial drugs and supportive care can then be rapidly initiated. A 15-year-old boy who had traveled to Africa (South Africa, Kenya, and Nigeria between January 3 and 25, 2011) presented with fever persisting over 5 days, headache, diarrhea, and dysuria, approximately 17 days after his return from the journey. Urinalysis showed pyuria and hematuria. Blood examination showed hemolytic anemia, thrombocytopenia, disseminated intravascular coagulation, and hyperbilirubinemia. Plasmapheresis and hemodialysis were performed for 19 hospital days. Falciparum malaria was then confirmed by peripheral blood smear, and antimalarial medications were initiated. The patient's condition and laboratory results were quickly normalized. We report a case of severe acute renal failure associated with delayed diagnosis of falciparum malaria, and primary use of supportive treatment rather than antimalarial medicine. The present case suggests that early diagnosis and treatment is important because untreated tropical malaria can be associated with severe acute renal failure and fatality. Physicians must be alert for correct diagnosis and proper management of imported tropical malaria when patients have travel history of endemic areas. PMID:27510397

  10. Atorvastatin prevents Plasmodium falciparum cytoadherence and endothelial damage

    PubMed Central

    2011-01-01

    Background The adhesion of Plasmodium falciparum parasitized red blood cell (PRBC) to human endothelial cells (EC) induces inflammatory processes, coagulation cascades, oxidative stress and apoptosis. These pathological processes are suspected to be responsible for the blood-brain-barrier and other organs' endothelial dysfunctions observed in fatal cases of malaria. Atorvastatin, a drug that belongs to the lowering cholesterol molecule family of statins, has been shown to ameliorate endothelial functions and is widely used in patients with cardiovascular disorders. Methods The effect of this compound on PRBC induced endothelial impairments was assessed using endothelial co-culture models. Results Atorvastatin pre-treatment of EC was found to reduce the expression of adhesion molecules and P. falciparum cytoadherence, to protect cells against PRBC-induced apoptosis and to enhance endothelial monolayer integrity during co-incubation with parasites. Conclusions These results might suggest a potential interest use of atorvastatin as a protective treatment to interfere with the pathophysiological cascades leading to severe malaria. PMID:21356073

  11. The Molecular Basis of Folate Salvage in Plasmodium falciparum

    PubMed Central

    Salcedo-Sora, J. Enrique; Ochong, Edwin; Beveridge, Susan; Johnson, David; Nzila, Alexis; Biagini, Giancarlo A.; Stocks, Paul A.; O'Neill, Paul M.; Krishna, Sanjeev; Bray, Patrick G.; Ward, Stephen A.

    2011-01-01

    Tetrahydrofolates are essential cofactors for DNA synthesis and methionine metabolism. Malaria parasites are capable both of synthesizing tetrahydrofolates and precursors de novo and of salvaging them from the environment. The biosynthetic route has been studied in some detail over decades, whereas the molecular mechanisms that underpin the salvage pathway lag behind. Here we identify two functional folate transporters (named PfFT1 and PfFT2) and delineate unexpected substrate preferences of the folate salvage pathway in Plasmodium falciparum. Both proteins are localized in the plasma membrane and internal membranes of the parasite intra-erythrocytic stages. Transport substrates include folic acid, folinic acid, the folate precursor p-amino benzoic acid (pABA), and the human folate catabolite pABAGn. Intriguingly, the major circulating plasma folate, 5-methyltetrahydrofolate, was a poor substrate for transport via PfFT2 and was not transported by PfFT1. Transport of all folates studied was inhibited by probenecid and methotrexate. Growth rescue in Escherichia coli and antifolate antagonism experiments in P. falciparum indicate that functional salvage of 5-methyltetrahydrofolate is detectable but trivial. In fact pABA was the only effective salvage substrate at normal physiological levels. Because pABA is neither synthesized nor required by the human host, pABA metabolism may offer opportunities for chemotherapeutic intervention. PMID:21998306

  12. Reduced erythrocyte deformability associated with hypoargininemia during Plasmodium falciparum malaria

    PubMed Central

    Rey, Juliana; Buffet, Pierre A.; Ciceron, Liliane; Milon, Geneviève; Mercereau-Puijalon, Odile; Safeukui, Innocent

    2014-01-01

    The mechanisms underlying reduced red blood cell (RBC) deformability during Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels (r = 0.69; p = 0.01) and weakly with parasitemia (r = −0.38; p = 0.006). In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability, and this effect correlated strongly with plasma L-arginine levels (r = 0.89; p < 0.0001). Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P. falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature. PMID:24441939

  13. Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents.

    PubMed

    Barr, P J; Inselburg, J; Green, K M; Kansopon, J; Hahm, B K; Gibson, H L; Lee-Ng, C T; Bzik, D J; Li, W B; Bathurst, I C

    1991-03-01

    We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine. PMID:2052035

  14. In vitro sensitivity of Plasmodium falciparum to artesunate in Thailand.

    PubMed Central

    Wongsrichanalai, C.; Wimonwattrawatee, T.; Sookto, P.; Laoboonchai, A.; Heppner, D. G.; Kyle, D. E.; Wernsdorfer, W. H.

    1999-01-01

    Reported are the in vitro susceptibilities of Plasmodium falciparum to artesunate, mefloquine, quinine and chloroquine of 86 isolates and to dihydroartemisinin of 45 isolates collected from areas of high resistance to mefloquine within Thailand near the borders with Myanmar and Cambodia, and from southern Thailand where P. falciparum is generally still sensitive to mefloquine. All the isolates were highly sensitive to artesunate, but the geometric mean IC50S were higher in isolates from the Thai-Myanmar and Thai-Cambodian borders than in those from southern Thailand. The IC50S for mefloquine and artesunate were strongly correlated (Pearson r = 0.605; n = 86; P < 0.00001). As expected, the in vitro sensitivities to dihydroartemisinin and artesunate were similar and strongly correlated (at IC50, Pearson r = 0.695; n = 45; P < 0.00002). The correlation between the activity of mefloquine and artesunate requires further investigation in order to determine the potential for development of cross-resistance in nature. Our results suggest that combination with mefloquine is not the ideal way of protecting the usefulness of artemisinin and its derivatives. A search for more suitable partner drugs to these compounds and careful regulation of their use are necessary in the interest of ensuring their long therapeutic life span. PMID:10361756

  15. Characterization of N-myristoyltransferase from Plasmodium falciparum.

    PubMed Central

    Gunaratne, R S; Sajid, M; Ling, I T; Tripathi, R; Pachebat, J A; Holder, A A

    2000-01-01

    The gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) has been cloned from the malaria parasite Plasmodium falciparum. The gene appears to be single copy and mRNA is expressed in asexual blood-stage forms. Comparison of cDNA and genomic sequences identified three small introns. The open reading frame codes for a 410-amino-acid protein and no evidence of forms with an extended N-terminal coding sequence was obtained. Residues important in substrate binding and in the catalytic mechanism in other species are conserved. The protein was expressed from a plasmid in Escherichia coli, partially purified and shown to have enzymic activity using a synthetic peptide substrate. Comparison of the malaria parasite protein with that derived from the human gene showed a different pattern of inhibition by chemical modification. Human NMT activity was inhibited by diethylpyrocarbonate and partially inhibited by iodacetamide, whereas P. falciparum NMT activity was not inhibited by either pre-treatment. Since the enzyme in infectious fungi is a target for potential chemotherapeutic drugs, it should also be investigated in the context of parasitic infections such as that responsible for malaria. PMID:10816442

  16. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

  17. Genome sequence of the human malaria parasite Plasmodium falciparum

    PubMed Central

    Gardner, Malcolm J.; Hall, Neil; Fung, Eula; White, Owen; Berriman, Matthew; Hyman, Richard W.; Carlton, Jane M.; Pain, Arnab; Nelson, Karen E.; Bowman, Sharen; Paulsen, Ian T.; James, Keith; Eisen, Jonathan A.; Rutherford, Kim; Salzberg, Steven L.; Craig, Alister; Kyes, Sue; Chan, Man-Suen; Nene, Vishvanath; Shallom, Shamira J.; Suh, Bernard; Peterson, Jeremy; Angiuoli, Sam; Pertea, Mihaela; Allen, Jonathan; Selengut, Jeremy; Haft, Daniel; Mather, Michael W.; Vaidya, Akhil B.; Martin, David M. A.; Fairlamb, Alan H.; Fraunholz, Martin J.; Roos, David S.; Ralph, Stuart A.; McFadden, Geoffrey I.; Cummings, Leda M.; Subramanian, G. Mani; Mungall, Chris; Venter, J. Craig; Carucci, Daniel J.; Hoffman, Stephen L.; Newbold, Chris; Davis, Ronald W.; Fraser, Claire M.; Barrell, Bart

    2013-01-01

    The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host–parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria. PMID:12368864

  18. Dynamic alteration in splenic function during acute falciparum malaria

    SciTech Connect

    Looareesuwan, S.; Ho, M.; Wattanagoon, Y.; White, N.J.; Warrell, D.A.; Bunnag, D.; Harinasuta, T.; Wyler, D.J.

    1987-09-10

    Plasmodium-infected erythrocytes lose their normal deformability and become susceptible to splenic filtration. In animal models, this is one mechanism of antimalarial defense. To assess the effect of acute falciparum malaria on splenic filtration, we measured the clearance of heated /sup 51/Cr-labeled autologous erythrocytes in 25 patients with acute falciparum malaria and in 10 uninfected controls. Two groups of patients could be distinguished. Sixteen patients had splenomegaly, markedly accelerated clearance of the labeled erythrocytes (clearance half-time, 8.4 +/- 4.4 minutes (mean +/- SD) vs. 62.5 +/- 36.5 minutes in controls; P less than 0.001), and a lower mean hematocrit than did the patients without splenomegaly (P less than 0.001). In the nine patients without splenomegaly, clearance was normal. After institution of antimalarial chemotherapy, however, the clearance in this group accelerated to supernormal rates similar to those in the patients with splenomegaly, but without the development of detectable splenomegaly. Clearance was not significantly altered by treatment in the group with splenomegaly. Six weeks later, normal clearance rates were reestablished in most patients in both groups. We conclude that splenic clearance of labeled erythrocytes is enhanced in patients with malaria if splenomegaly is present and is enhanced only after treatment if splenomegaly is absent. Whether this enhanced splenic function applies to parasite-infected erythrocytes in patients with malaria and has any clinical benefit will require further studies.

  19. Identification of a Plasmodium falciparum Phospholipid Transfer Protein*

    PubMed Central

    van Ooij, Christiaan; Withers-Martinez, Chrislaine; Ringel, Alessa; Cockcroft, Shamshad; Haldar, Kasturi; Blackman, Michael J.

    2013-01-01

    Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte. PMID:24043620

  20. Molecular Aspects of Plasmodium falciparum Infection during Pregnancy

    PubMed Central

    Ndam, Nicaise Tuikue; Deloron, Philippe

    2007-01-01

    Cytoadherence of Plasmodium-falciparum-parasitized red blood cells (PRBCs) to host receptors is the key phenomenon in the pathological process of the malaria disease. Some of these interactions can originate poor outcomes responsible for 1 to 3 million annual deaths mostly occurring among children in sub-Saharan Africa. Pregnancy-associated malaria (PAM) represents an important exception of the disease occurring at adulthood in malaria endemic settings. Consequences of this are shared between the mother (maternal anemia) and the baby (low birth weight and infant mortality). Demonstrating that parasites causing PAM express specific variant surface antigens (VSAPAM), including the P. falciparum erythrocyte membrane protein 1 (P f EMP1) variant VAR2CSA, that are targets for protective immunity has strengthened the possibility for the development of PAM-specific vaccine. In this paper, we review the molecular basis of malaria pathogenesis attributable to the erythrocyte stages of the parasites, and findings supporting potential anti-PAM vaccine components evidenced in PAM. PMID:17641725

  1. Aminoindoles, a novel scaffold with potent activity against Plasmodium falciparum.

    PubMed

    Barker, Robert H; Urgaonkar, Sameer; Mazitschek, Ralph; Celatka, Cassandra; Skerlj, Renato; Cortese, Joseph F; Tyndall, Erin; Liu, Hanlan; Cromwell, Mandy; Sidhu, Amar Bir; Guerrero-Bravo, Jose E; Crespo-Llado, Keila N; Serrano, Adelfa E; Lin, Jing-Wen; Janse, Chris J; Khan, Shahid M; Duraisingh, Manoj; Coleman, Bradley I; Angulo-Barturen, Inigo; Jiménez-Díaz, María Belén; Magán, Noemí; Gomez, Vanesa; Ferrer, Santiago; Martínez, María Santos; Wittlin, Sergio; Papastogiannidis, Petros; O'Shea, Thomas; Klinger, Jeffrey D; Bree, Mark; Lee, Edward; Levine, Mikaela; Wiegand, Roger C; Munoz, Benito; Wirth, Dyann F; Clardy, Jon; Bathurst, Ian; Sybertz, Edmund

    2011-06-01

    This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ~70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50% inhibitory concentrations [IC₅₀s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of > 99% in an adapted version of Peters' 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155-171, 1975). Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC₅₀s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC₅₀s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels

  2. The extravascular compartment of the bone marrow: a niche for Plasmodium falciparum gametocyte maturation?

    PubMed Central

    2012-01-01

    Background Plasmodium falciparum immature gametocytes accumulate in the bone marrow, but their exact location in this tissue remains unclear. Methods The stage and deposition pattern of gametocytes was analysed on histological sections of a bone marrow sample collected in a patient with subacute P. falciparum malaria. Results A majority (89%) of immature stages II to IV gametocytes and a minority (29%) of mature stage V gametocytes were observed in extravascular spaces. Discussion and conclusion These observations represent a valuable step towards understanding sequestration patterns of P. falciparum gametocytes and may ultimately lead to novel transmission-blocking interventions. PMID:22905863

  3. Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP)

    SciTech Connect

    Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish

    2012-03-26

    The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 {angstrom} resolution. Multiwavelength anomalous diffraction (MAD) data were collected utilizing the Zn{sup 2+} ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

  4. In Vitro Activity of Fluorescent Dyes against Asexual Blood Stages of Plasmodium falciparum

    PubMed Central

    Joanny, Fanny; Held, Jana

    2012-01-01

    Many successful antimicrobial drugs originate from synthetic dyes. This paper reports the in vitro activity of 14 fluorescent dyes against Plasmodium falciparum. Five of these dyes (Hoechst 33342, MitoRed, DiOC6, SYTO 9, and rhodamine B) show activity at a low nanomolar concentration against two P. falciparum strains in the histidine-rich protein 2 drug sensitivity assay, while toxicity in HeLa cells is low. These dyes may be a starting point for developing new drugs against P. falciparum. PMID:22850520

  5. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  6. Plasmodium falciparum: analysis of chromosomes separated by contour-clamped homogenous electric fields.

    PubMed

    Gu, H; Inselburg, J W; Bzik, D J; Li, W B

    1990-08-01

    We have established improved conditions for separating the chromosomes of Plasmodium falciparum by pulsed field gradient gel electrophoresis (PFG) using a contour-clamped homogenous electric field (CHEF) apparatus. Thirteen clearly separable chromosomal bands were reproducibly isolated from the strain FCR3 and their sizes have been determined. Evidence that indicates one band may contain two chromosomes is presented. The relationship between the PFG separable DNA and the number of unique chromosomes in P. falciparum is considered. We have established a relationship between the maximum resolvable sizes of the chromosomes and the pulse times. The chromosomal location of twenty-seven P. falciparum DNA probes is also reported. PMID:2197113

  7. Comparison of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase activities for evaluation of microangiopathy in diabetes mellitus.

    PubMed

    Shimojo, N; Kitahashi, S; Naka, K; Fujii, A; Okuda, K; Tanaka, S; Fujii, S

    1987-03-01

    The activities of urinary N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured in 207 diabetic patients and 57 healthy controls, and the relationship of these enzymes to different stages of diabetic microangiopathy was studied. Diabetics with clinical proteinuria had higher urinary NAG and AAP (17.7 +/- 1.9 and 42.8 +/- 4.9 U/g creatinine, mean +/- SE, respectively) than healthy controls (1.8 +/- 0.1 and 10.0 +/- 0.4) or diabetics without proteinuria. Among diabetics without proteinuria, NAG excretion in those with retinopathy was slightly higher than in those without (6.4 +/- 0.5 v 5.4 +/- 0.4), and AAP in those with retinopathy was significantly higher than in those without (23.0 +/- 1.5 v 17.4 +/- 0.8, P less than 0.01). Urinary albumin measured by radioimmunoassay and lysozyme in diabetics with retinopathy but without proteinuria was higher than those without retinopathy (P less than 0.001 and P less than 0.01). The increase in albumin was the greatest in diabetics with long duration of the disease (greater than or equal to 8 years); however, NAG and AAP increased more significantly in those with high hemoglobin A1c than in patients with long duration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2881186

  8. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    PubMed

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis. PMID:25274570

  9. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Zhang, Shaoping; Cheng, Hongmei; Gao, Yulin; Wang, Guirong; Liang, Gemei; Wu, Kongming

    2009-07-01

    A Cry1Ac-resistant strain (Bt-R) of Helicoverpa armigera, with 2971-fold resistance, was derived by selection with Cry1Ac toxin for 75 generations. We used cDNA-amplified fragment length polymorphism analysis to identify those genes differentially expressed in the Cry1Ac-resistant and -susceptible strains, which revealed 212 differentially expressed transcripts among 2000 screened cDNAs. Among these transcript-derived fragments (TDFs), 37 showed some homology to known sequences, including Aminopeptidase N (APN), which is expressed in the midgut epithelium and has been implicated as a Cry1A subfamily receptor in several moths, including H. armigera. We confirmed the TDF by RT-PCR and identified a deletion mutation of apn1 in the Bt-R strain. We expressed the TDF in bacteria. The partial HaAPN1-96S wild-type protein, bound to Cry1Ac on ligand blots, whereas HaAPN1-BtR did not. This suggested that HaAPN1 is a receptor for Bt Cry1Ac and that its deletion mutation is associated with Cry1Ac resistance in H. armigera. The absence of one binding site is responsible for its resistance to Cry1Ac. We developed an allele-specific PCR to monitor whether the apn1 gene in an H. armigera field population produced a similar mutation. No deleted mutants were found in 2250 individuals collected from the field in 2006-2007. PMID:19376227

  10. Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

    PubMed Central

    Mayo, B; Kok, J; Venema, K; Bockelmann, W; Teuber, M; Reinke, H; Venema, G

    1991-01-01

    Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown. Images PMID:1674655

  11. Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

    PubMed Central

    Kochan, Grazyna; Krojer, Tobias; Harvey, David; Fischer, Roman; Chen, Liye; Vollmar, Melanie; von Delft, Frank; Kavanagh, Kathryn L.; Brown, Matthew A.; Bowness, Paul; Wordsworth, Paul; Kessler, Benedikt M.; Oppermann, Udo

    2011-01-01

    Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions. PMID:21508329

  12. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction

    PubMed Central

    Ochiya, Takahiro; Takenaga, Keizo; Asagiri, Masataka; Nakano, Kazumi; Satoh, Hitoshi; Watanabe, Toshiki; Imajoh-Ohmi, Shinobu; Endo, Hideya

    2015-01-01

    The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy. PMID:26029719

  13. Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

    PubMed

    Melkebeek, V; Rasschaert, K; Bellot, P; Tilleman, K; Favoreel, H; Deforce, D; De Geest, B G; Goddeeris, B M; Cox, E

    2012-11-01

    Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea in human and animal. In piglets, ETEC having F4 fimbriae (F4(+) ETEC) induce severe diarrhea, dependent on the presence of receptors for F4 (F4R). In this study, porcine aminopeptidase N (pAPN) was identified as an F4R by comparative proteomic analysis of brush border proteins of F4R(+) and F4R(-) pigs and by adherence/internalization experiments on pAPN-transfected cells. Binding of F4 fimbriae to pAPN depended on sialic acid containing carbohydrate moieties, and resulted in clathrin-mediated endocytosis of the fimbriae. Endocytosis via pAPN was not restricted to F4 fimbriae, but was also observed for anti-pAPN antibodies. Both F4 fimbriae- and pAPN-specific antibodies were taken up in vivo by porcine enterocytes and induced subsequently a rapid immunoglobulin A and G response. In conclusion, we identified pAPN as an endocytotic receptor for F4 fimbriae and highlight the opportunity to target vaccine antigens to this epithelial receptor. PMID:22669578

  14. Discovery of inhibitors of Escherichia coli methionine aminopeptidase with the Fe(II)-form selectivity and antibacterial activity†

    PubMed Central

    Wang, Wen-Long; Chai, Sergio C.; Huang, Min; He, Hong-Zhen; Hurley, Thomas D.; Ye, Qi-Zhuang

    2009-01-01

    Methionine aminopeptidase (MetAP) is a promising target to develop novel antibiotics, because all bacteria express MetAP from a single gene that carries out the essential function of removing N-terminal methionine from nascent proteins. Divalent metal ions play a critical role in the catalysis, and there is an urgent need to define the actual metal used by MetAP in bacterial cells. By high throughput screening, we identified a novel class of catechol-containing MetAP inhibitors that display selectivity for the Fe(II)-form of MetAP. X-ray structure revealed that the inhibitor binds to MetAP at the active site with the catechol coordinating to the metal ions. Importantly, some of the inhibitors showed antibacterial activity at low micromolar concentration on Gram-positive and Gram-negative bacteria. Our data indicate that Fe(II) is the likely metal used by MetAP in the cellular environment, and MetAP inhibitors need to inhibit this metalloform of MetAP effectively to be therapeutically useful. PMID:18785729

  15. A role for naturally occurring alleles of endoplasmic reticulum aminopeptidases in tumor immunity and cancer pre-disposition.

    PubMed

    Stratikos, Efstratios; Stamogiannos, Athanasios; Zervoudi, Efthalia; Fruci, Doriana

    2014-01-01

    Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2) are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs). Coding single nucleotide polymorphisms (SNPs) in these enzymes have been associated with pre-disposition to several major human diseases including inflammatory diseases with autoimmune etiology, viral infections, and virally induced cancer. The function of these enzymes has been demonstrated to affect CTL and natural killer cell responses toward healthy and malignant cells as well as the production of inflammatory cytokines. Recent studies have demonstrated that SNPs in ERAP1 and ERAP2 can affect their ability to generate or destroy antigenic epitopes and define the immunopeptidome. In this review, we examine the potential role of these enzymes and their polymorphic states on the generation of cytotoxic responses toward malignantly transformed cells. Given the current state-of-the-art, it is possible that polymorphic variation in these enzymes may contribute to the individual's pre-disposition to cancer through altered generation or destruction of tumor antigens that can facilitate tumor immune evasion. PMID:25566501

  16. Autoimmune disease-associated variants of extracellular endoplasmic reticulum aminopeptidase 1 induce altered innate immune responses by human immune cells.

    PubMed

    Aldhamen, Yasser A; Pepelyayeva, Yuliya; Rastall, David P W; Seregin, Sergey S; Zervoudi, Efthalia; Koumantou, Despoina; Aylsworth, Charles F; Quiroga, Dionisia; Godbehere, Sarah; Georgiadis, Dimitris; Stratikos, Efstratios; Amalfitano, Andrea

    2015-01-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we demonstrated that ERAP1 regulates key aspects of the innate immune response. Previous studies show ERAP1 to be endoplasmic reticulum-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating the innate immune responses of human peripheral blood mononuclear cells (hPBMCs) using two experimental methods: extracellular exposure of hPBMCs to ERAP1 variants and adenovirus (Ad)-based ERAP1 expression. We found that exposure of hPBMCs to ERAP1 variant proteins as well as ERAP1 overexpression by Ad5 vectors increased inflammatory cytokine and chemokine production, and enhanced immune cell activation. Investigating the molecular mechanisms behind these responses revealed that ERAP1 is able to activate innate immunity via multiple pathways, including the NLRP3 (NOD-like receptor, pyrin domain-containing 3) inflammasome. Importantly, these responses varied if autoimmune disease-associated variants of ERAP1 were examined in the assay systems. Unexpectedly, blocking ERAP1 cellular internalization augmented IL-1β production. To our knowledge, this is the first report identifying ERAP1 as being involved in modulating innate responses of human immune cells, a finding that may explain why ERAP1 has been genetically associated with several autoimmune diseases. PMID:25591727

  17. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

    PubMed

    Ochiya, Takahiro; Takenaga, Keizo; Asagiri, Masataka; Nakano, Kazumi; Satoh, Hitoshi; Watanabe, Toshiki; Imajoh-Ohmi, Shinobu; Endo, Hideya

    2015-01-01

    The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy. PMID:26029719

  18. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    PubMed Central

    Jenkins, Jeremy L; Dean, Donald H

    2001-01-01

    Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

  19. Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment.

    PubMed Central

    Cooper, M A; Carroll, J; Travis, E R; Williams, D H; Ellar, D J

    1998-01-01

    The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles. PMID:9677328

  20. Artemisinin-naphthoquine for treating uncomplicated Plasmodium falciparum malaria

    PubMed Central

    Isba, Rachel; Zani, Babalwa; Gathu, Michael; Sinclair, David

    2015-01-01

    Background The World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) for treating people with Plasmodium falciparum malaria. Five combinations are currently recommended, all administered over three days. Artemisinin-naphthoquine is a new combination developed in China, which is being marketed as a one-day treatment. Although shorter treatment courses may improve adherence, the WHO recommends at least three days of the short-acting artemisinin component to eliminate 90% P. falciparum parasites in the bloodstream, before leaving the longer-acting partner drug to clear the remaining parasites. Objectives To evaluate the efficacy and safety of the artemisinin-naphthoquine combination for treating adults and children with uncomplicated P. falciparum malaria. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register; Cochrane Central Register of Controlled Trials (CENTRAL) published in The Cochrane Library; MEDLINE; EMBASE; and LILACS up to January 2015. We also searched the metaRegister of Controlled Trials (mRCT) using 'malaria' and 'arte* OR dihydroarte*' as search terms. Selection criteria Randomized controlled trials comparing artemisinin-naphthoquine combinations with established WHO-recommended ACTs for the treatment of adults and children with uncomplicated malaria due to P. falciparum. Data collection and analysis Two review authors independently assessed trials for eligibility and risk of bias, and extracted data. We analysed primary outcomes in line with the WHO 'Protocol for assessing and monitoring antimalarial drug efficacy' and compared drugs using risk ratios (RR) and 95% confidence intervals (CI). Secondary outcomes were effects on gametocytes, haemoglobin, and adverse events. We assessed the quality of evidence using the GRADE approach. Main results Four trials, enrolling 740 adults and children, met the inclusion criteria. Artemisinin-naphthoquine was administered as a single dose (two

  1. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species [1-3], providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database [4], and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6-8], proteomics [9-12], literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle

  2. In Vitro Activity and Interaction of Clindamycin Combined with Dihydroartemisinin against Plasmodium falciparum

    PubMed Central

    Ramharter, M.; Noedl, H.; Winkler, H.; Graninger, W.; Wernsdorfer, W. H.; Kremsner, P. G.; Winkler, S.

    2003-01-01

    Combination regimens are considered a valuable tool for the fight against drug-resistant falciparum malaria. This study was conducted to evaluate the antimalarial potential of clindamycin in combination with dihydroartemisinin in continuously cultured and in freshly isolated Plasmodium falciparum parasites, measuring the inhibition of Plasmodium falciparum histidine-rich protein II synthesis. Interaction analysis revealed a synergistic or additive mode of interaction at various concentration ratios in all continuously cultured parasites at the 50% effective concentration (EC50) level. Antagonism was not found for any of the culture-adapted parasites. In fresh P. falciparum isolates, a fixed clindamycin-dihydroartemisinin combination exhibited additive activity at the EC50 and EC90 levels. The drug mixture showed no significant activity correlation to other commonly used antimalarials. The clindamycin-dihydroartemisinin combination appears to be a promising candidate for clinical investigation. PMID:14576107

  3. Impact of Plasmodium falciparum Coinfection on Longitudinal Epstein-Barr Virus Kinetics in Kenyan Children.

    PubMed

    Reynaldi, Arnold; Schlub, Timothy E; Chelimo, Kiprotich; Sumba, Peter Odada; Piriou, Erwan; Ogolla, Sidney; Moormann, Ann M; Rochford, Rosemary; Davenport, Miles P

    2016-03-15

    Endemic Burkitt lymphoma is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfection, although how P. falciparum exposure affects the dynamics of EBV infection is unclear. We have used a modeling approach to study EBV infection kinetics in a longitudinal cohort of children living in regions of high and low malaria transmission in Kenya. Residence in an area of high malaria transmission was associated with a higher rate of EBV expansion during primary EBV infection in infants and during subsequent episodes of EBV DNA detection, as well as with longer episodes of EBV DNA detection and shorter intervals between subsequent episodes of EBV DNA detection. In addition, we found that concurrent P. falciparum parasitemia also increases the likelihood of the first and subsequent peaks of EBV in peripheral blood. This suggests that P. falciparum infection is associated with increased EBV growth and contributes to endemic Burkitt lymphoma pathogenesis. PMID:26531246

  4. Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

    SciTech Connect

    Ting, L; Shi, W; Lewandowicz, A; Singh, V; Mwakingwe, A; Birck, M R; Taylor Ringia, E A; Bench, G; Madrid, D C; Tyler, P C; Evans, G B; Furneaux, R H; Schramm, V L; Kim, K

    2004-05-19

    Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials.

  5. The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

    PubMed Central

    Molina-Cruz, Alvaro; Barillas-Mury, Carolina

    2014-01-01

    Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas. PMID:25185006

  6. The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes.

    PubMed

    Molina-Cruz, Alvaro; Barillas-Mury, Carolina

    2014-08-01

    Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas. PMID:25185006

  7. Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens.

    PubMed

    Juliano, Jonathan J; Parobek, Christian M; Brazeau, Nicholas F; Ngasala, Billy; Randrianarivelojosia, Milijaona; Lon, Chanthap; Mwandagalirwa, Kashamuka; Tshefu, Antoinette; Dhar, Ravi; Das, Bidyut K; Hoffman, Irving; Martinson, Francis; Mårtensson, Andreas; Saunders, David L; Kumar, Nirbhay; Meshnick, Steven R

    2016-01-01

    Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens. PMID:26503281

  8. Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors

    PubMed Central

    Ponder, Elizabeth L.; Albrow, Victoria E.; Leader, Brittany A.; Békés, Miklós; Mikolajczyk, Jowita; Fonović, Urša Pečar; Shen, Aimee; Drag, Marcin; Xiao, Junpeng; Deu, Edgar; Campbell, Amy J.; Powers, James C.; Salvesen, Guy S.; Bogyo, Matthew

    2011-01-01

    SUMMARY Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite lifecycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a novel class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum. PMID:21700207

  9. Population genetics of Plasmodium falciparum and Plasmodium vivax and asymptomatic malaria in Temotu Province, Solomon Islands

    PubMed Central

    2013-01-01

    Background Temotu Province, Solomon Islands is progressing toward malaria elimination. A baseline survey conducted in 2008 showed that most Plasmodium infections in the province were of low parasite density and asymptomatic infections. To better understand mechanisms underlying these malaria transmission characteristics genetic diversity and relationships among Plasmodium falciparum and Plasmodium vivax populations in the province were examined. Methods Forty-five P. falciparum and 67 P. vivax samples collected in the 2008 baseline survey were successfully genotyped using eight P. falciparum and seven P. vivax microsatellite markers. Genetic diversity, relationships and distribution of both P. falciparum and P. vivax populations were analysed. Results Plasmodium falciparum population exhibited low diversity with 19 haplotypes identified and had closely related clusters indicating clonal expansion. Interestingly, a dominant haplotype was significantly associated with fever and high parasite density. In contrast, the P. vivax population was highly diverse with 58 haplotypes identified that were not closely related. Parasite populations between different islands in the province showed low genetic differentiation. Conclusion The low diversity and clonal population of P. falciparum population may partially account for clinical immunity developed against illness. However, it is possible that importation of a new P. falciparum strain was the major cause of illness. High diversity in P. vivax population and low relatedness between strains suggested clinical immunity to P. vivax may be maintained by different mechanisms. The genetic diversity, population structure and distribution of strains indicate that transmission of P. falciparum was low, but that of P. vivax was still high in 2008. These data will be useful for assessing changes in malaria transmission resulting from interventions. PMID:24261646

  10. An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling, inhibitory studies and molecular modeling.

    PubMed

    Węglarz-Tomczak, Ewelina; Poręba, Marcin; Byzia, Anna; Berlicki, Łukasz; Nocek, Bogusław; Mulligan, Rory; Joachimiak, Andrzej; Drąg, Marcin; Mucha, Artur

    2013-02-01

    Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria. To explore the possibility of NmAPN being a potential drug target for inhibition and development of novel therapeutic agents, the specificity of the S1 and S1' binding sites was explored using an integrated approach. Initially, an extensive library consisting of almost 100 fluorogenic substrates derived from both natural and unnatural amino acids, were used to obtain a detailed substrate fingerprint of the S1 pocket of NmAPN. A broad substrate tolerance of NmAPN was revealed, with bulky basic and hydrophobic ligands being the most favored substrates. Additionally, the potency of a set of organophosphorus inhibitors of neutral aminopeptidases, amino acid and dipeptide analogs was determined. Inhibition constants in the nanomolar range, determined for phosphinic dipeptides, proves the positive increase in inhibition impact of the P1' ligand elongation. The results were further verified via molecular modeling and docking of canonical aminopeptidase phosphinic dipeptide inhibitors in the NmAPN active site. These studies present comprehensive characterization of interactions responsible for specific ligand binding. This knowledge provides invaluable insight into understanding of the enzyme and development of novel NmAPN inhibitors. PMID:23131591

  11. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Specificity and immobilization of aminopeptidase.

    PubMed

    Vosbeck, K D; Greenberg, B D; Awad, W M

    1975-05-25

    We recently described the purification of two aminopeptidases from Streptomyces griseus (Vosbeck, K.D., Chow, K.-F., and Awad, W.M., Jr. (1973) J. Biol. Chem. 248, 6329-6034). An analysis of the amino acid composition reveals very little differences in the two proteins. Each protein has alanine as the NH2-terminal residue. The aminopeptidases were treated separately with acetic anhydride; as noted in the past, the presence of glycerol is required to achieve excellent yields of acetylated active derivatives (Siegel, S., and Awad, W.M., Jr. (1973 J. Biol. Chem. 248, 3233-3240). However, in the present case much higher concentrations of glycerol (50%) are needed during acetylation to obtain derivatives with completely reacted NH2-terminal residues. The epsilon-amino groups were not completely acetylated. In contrast to the native enzymes, the acetylated derivatives show an affinity for DEAE-cellulose, a property consonant with the changes in net charge. The kinetic constants for each enzyme against L-leucine-p-nitroanilide do not change significantly after acetylation. The specificities of the two aminopeptidases were examined extensively on a semiquantitative basis. The activities are not restricted by the length of substrate chains. Each enzyme shows a preference for hydrophobic residues at the ultimate and penultimate positions. Charge residues are released a slower rates. No prolidase activity is demonstrable even at high enzyme to substrate ratios; however, NH2-terminal proline residues are released readily. D-Amino acid residues at the ultimate or penultimate position substantially reduce the rate of hydrolysis; D-leucyl-D-leucine is not hydrolyzed... PMID:805135

  12. Anti-Plasmodium falciparum activity of quinoline-sulfonamide hybrids.

    PubMed

    Pinheiro, Luiz C S; Boechat, Núbia; Ferreira, Maria de Lourdes G; Júnior, Carlos C S; Jesus, Antônio M L; Leite, Milene M M; Souza, Nicolli B; Krettli, Antoniana U

    2015-09-01

    Fifteen quinoline-sulfonamide hybrids, with a 7-chloroquinoline moiety connected by a linker group to arylsulfonamide moieties with different substituents in the 4-position were synthesized and assayed against Plasmodium falciparum. The compounds displayed high schizonticidal blood activity in vitro, with IC50 values ranging from 0.05 to 1.63 μM, in the anti-HPR2 assay against clone W2-chloroquine-resistant; ten of them showed an IC50 (ranging from 0.05 to 0.40 μM) lower than that of chloroquine and sulfadoxine. Among them, two compounds inhibited Plasmodium berghei parasitemia by 47% and 49% on day 5 after mice inoculation. The most active, in vivo, hybrid 13 is considered to be a new prototype for the development of an antimalarial drug against chloroquine-resistant parasites. PMID:26190461

  13. Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties.

    PubMed

    Sanyal, Sohini; Egée, Stéphane; Bouyer, Guillaume; Perrot, Sylvie; Safeukui, Innocent; Bischoff, Emmanuel; Buffet, Pierre; Deitsch, Kirk W; Mercereau-Puijalon, Odile; David, Peter H; Templeton, Thomas J; Lavazec, Catherine

    2012-01-12

    Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature. PMID:22106347

  14. Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum.

    PubMed Central

    Brockelman, C R; Wongsattayanont, B; Tan-ariya, P; Fucharoen, S

    1987-01-01

    Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixture designated REM consisted of 9 volumes of minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) and 1 volume of RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated human serum. Parasite multiplication in erythrocytes containing normal hemoglobin cultured in RPMI or REM was similar. Significant reduction in parasite multiplication rates was observed in erythrocytes containing abnormal hemoglobin when these were cultured in REM. The degree of reduction in five types of thalassemic erythrocytes was in the following descending order: hemoglobin H disease with Hb Constant Spring, classical hemoglobin H disease, beta(0)-thalassemia-hemoglobin E in which blood harbored a high percentage of hemoglobin F-containing cells, beta (0)-thalassemia-hemoglobin E in which blood harbored few hemoglobin F-containing cells, and beta-thalassemia heterozygous variant. PMID:3539999

  15. Plasmodium falciparum Rab1A Localizes to Rhoptries in Schizonts

    PubMed Central

    Morse, David; Webster, Wesley; Kalanon, Ming; Langsley, Gordon; McFadden, Geoffrey I.

    2016-01-01

    Over-expression of a GFP-PfRab1A fusion protein in Plasmodium falciparum schizonts produces a punctate pattern of fluorescence typical of rhoptries, secretory organelles involved in host cell invasion. The GFP-positive bodies were purified by a combination of differential and density gradient centrifugation and their protein content determined by MS/MS sequencing. Consistent with the GFP rhoptry-like pattern of transgenic parasites, four of the 19 proteins identified have been previously described to be rhoptry-associated and another four are ER or ER-associated proteins. Confirmation that GFP-PfRab1A decorates rhoptries was obtained by its co-localization with Rap1 and Ron4 in late phase schizonts. We conclude that PfRab1A potentially regulates vesicular traffic from the endoplasmic reticulum to the rhoptries in Apicomplexa parasites. PMID:27348424

  16. Economic access to effective drugs for falciparum malaria.

    PubMed

    Panosian, Claire B

    2005-03-01

    The increasing death toll from drug-resistant falciparum malaria is cause for international concern. In 2002, the US Agency for International Development commissioned the Institute of Medicine (IOM) to recommend global actions to ensure the broadest possible access to new, effective antimalarial treatments. In a report issued in 2004, the IOM Committee on Economics of Antimalarial Drugs recommended a global subsidy of 300 million dollars to 500 million dollars per year to replace increasingly ineffective drugs with coformulated artemisinin combination treatments to be distributed through public and private channels in affected areas. This approach allows the existing market to support the switch to new drugs and keeps treatment costs for consumers at levels similar to the current price of chloroquine. The leverage of an international subsidy of combination therapy can also discourage the distribution of monotherapies (such as solo artemisinins), the use of which might foster increasing resistance to antimalarial drugs in the future. PMID:15714418

  17. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia

    PubMed Central

    Miotto, Olivo; Almagro-Garcia, Jacob; Manske, Magnus; MacInnis, Bronwyn; Campino, Susana; Rockett, Kirk A; Amaratunga, Chanaki; Lim, Pharath; Suon, Seila; Sreng, Sokunthea; Anderson, Jennifer M; Duong, Socheat; Nguon, Chea; Chuor, Char Meng; Saunders, David; Se, Youry; Lon, Chantap; Fukuda, Mark M; Amenga-Etego, Lucas; Hodgson, Abraham VO; Asoala, Victor; Imwong, Mallika; Takala-Harrison, Shannon; Nosten, Francois; Su, Xin-zhuan; Ringwald, Pascal; Ariey, Frédéric; Dolecek, Christiane; Hien, Tran Tinh; Boni, Maciej F; Thai, Cao Quang; Amambua-Ngwa, Alfred; Conway, David J; Djimdé, Abdoulaye A; Doumbo, Ogobara K; Zongo, Issaka; Ouedraogo, Jean-Bosco; Alcock, Daniel; Drury, Eleanor; Auburn, Sarah; Koch, Oliver; Sanders, Mandy; Hubbart, Christina; Maslen, Gareth; Ruano-Rubio, Valentin; Jyothi, Dushyanth; Miles, Alistair; O’Brien, John; Gamble, Chris; Oyola, Samuel O; Rayner, Julian C; Newbold, Chris I; Berriman, Matthew; Spencer, Chris CA; McVean, Gilean; Day, Nicholas P; White, Nicholas J; Bethell, Delia; Dondorp, Arjen M; Plowe, Christopher V; Fairhurst, Rick M; Kwiatkowski, Dominic P

    2013-01-01

    We describe an analysis of genome variation in 825 Plasmodium falciparum samples from Asia and Africa that reveals an unusual pattern of parasite population structure at the epicentre of artemisinin resistance in western Cambodia. Within this relatively small geographical area we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and remarkably high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalogue of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in various transporter proteins and DNA mismatch repair proteins. These data provide a population genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist its elimination. PMID:23624527

  18. Localization of heme biosynthesis pathway enzymes in Plasmodium falciparum.

    PubMed

    Rao, Aditya; Yeleswarapu, Sri Jyothsna; Srinivasan, Rajgopal; Bulusu, Gopalakrishnan

    2008-12-01

    Protein trafficking in the malarial parasite Plasmodium falciparum is dictated by a complex life-cycle that involves a variety of intra-cellular and host cell destinations, such as the mitochondrion, apicoplast, rhoptries and micronemes. Of these, the apicoplast and mitochondrion are believed to account for more than 10% of this traffic. Studies have shown that mechanisms for mitochondrion and apicoplast targeting are distinct, despite their close physical proximity. The heme biosynthesis pathway spans both these organelles, making trafficking studies crucial for the spatial demarcation of the constituent interactions. This minireview highlights the challenges in identifying the possible sub-cellular destinations of the heme pathway enzymes using gleanings from literature survey as well as focussed bioinformatic analysis. PMID:19239121

  19. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.

    PubMed

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C Y; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S W; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  20. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

    PubMed Central

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C. Y.; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S. W.; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  1. Diagnosis and management of the neurological complications of falciparum malaria

    PubMed Central

    Mishra, Saroj K.; Newton, Charles R. J. C.

    2010-01-01

    Malaria is a major public health problem in the developing world owing to its high rates of morbidity and mortality. Of all the malarial parasites that infect humans, Plasmodium falciparum is most commonly associated with neurological complications, which manifest as agitation, psychosis, seizures, impaired consciousness and coma (cerebral malaria). Cerebral malaria is the most severe neurological complication; the condition is associated with mortality of 15–20%, and a substantial proportion of individuals with this condition develop neurocognitive sequelae. In this Review, we describe the various neurological complications encountered in malaria, discuss the underlying pathogenesis, and outline current management strategies for these complications. Furthermore, we discuss the role of adjunctive therapies in improving outcome. PMID:19347024

  2. Subcellular localization of adenylate kinases in Plasmodium falciparum.

    PubMed

    Ma, Jipeng; Rahlfs, Stefan; Jortzik, Esther; Schirmer, R Heiner; Przyborski, Jude M; Becker, Katja

    2012-09-21

    Adenylate kinases (AK) play a key role in nucleotide signaling processes and energy metabolism by catalyzing the reversible conversion of ATP and AMP to 2 ADP. In the malaria parasite Plasmodium falciparum this reaction is mediated by AK1, AK2, and a GTP:AMP phosphotransferase (GAK). Here, we describe two additional adenylate kinase-like proteins: PfAKLP1, which is homologous to human AK6, and PfAKLP2. Using GFP-fusion proteins and life cell imaging, we demonstrate a cytosolic localization for PfAK1, PfAKLP1, and PfAKLP2, whereas PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane, and this localization is driven by N-myristoylation. PMID:22819813

  3. J-dot targeting of an exported HSP40 in Plasmodium falciparum-infected erythrocytes.

    PubMed

    Petersen, Wiebke; Külzer, Simone; Engels, Sonja; Zhang, Qi; Ingmundson, Alyssa; Rug, Melanie; Maier, Alexander G; Przyborski, Jude M

    2016-07-01

    Plasmodium falciparum exports a large number of proteins to its host cell, the mature human erythrocyte, where they are involved in host cell modification. Amongst the proteins trafficked to the host cell, many are heat shock protein (HSP)40 homologues. We previously demonstrated that at least two exported PfHSP40s (referred to as PFE55 and PFA660) localise to mobile structures in the P. falciparum-infected erythrocyte (Kulzer et al., 2010), termed J-dots. The complete molecular content of these structures has not yet been completely resolved, however it is known that they also contain an exported HSP70, PfHSP70x, and are potentially involved in transport of the major cytoadherance ligand, PfEMP1, through the host cell. To understand more about the nature of the association of exported HSP40s with J-dots, here we have studied the signal requirements for recruitment of the proteins to these structures. By expressing various exported GFP chimeras, we can demonstrate that the predicted substrate binding domain is necessary and sufficient for J-dot targeting. This targeting only occurs in human erythrocytes infected with P. falciparum, as it is not conserved when expressing a P. falciparum HSP40 in Plasmodium berghei-infected murine red blood cells, suggesting that J-dots are P. falciparum-specific. This data reveals a new mechanism for targeting of exported proteins to intracellular structures in the P. falciparum-infected erythrocyte. PMID:27063072

  4. Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA

    PubMed Central

    Nagano, Soshichiro; Seki, Eiko; Lin, Ting-Yu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Heddle, Jonathan G.

    2015-01-01

    Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping. PMID:26566222

  5. Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

    PubMed

    Serrán-Aguilera, Lucía; Denton, Helen; Rubio-Ruiz, Belén; López-Gutiérrez, Borja; Entrena, Antonio; Izquierdo, Luis; Smith, Terry K; Conejo-García, Ana; Hurtado-Guerrero, Ramon

    2016-01-01

    Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase. PMID:27616047

  6. Targeting the gyrase of Plasmodium falciparum with topoisomerase poisons.

    PubMed

    Tang Girdwood, Sonya C; Nenortas, Elizabeth; Shapiro, Theresa A

    2015-06-15

    Drug-resistant malaria poses a major public health problem throughout the world and the need for new antimalarial drugs is growing. The apicoplast, a chloroplast-like organelle essential for malaria parasite survival and with no counterpart in humans, offers an attractive target for selectively toxic new therapies. The apicoplast genome (plDNA) is a 35 kb circular DNA that is served by gyrase, a prokaryotic type II topoisomerase. Gyrase is poisoned by fluoroquinolone antibacterials that stabilize a catalytically inert ternary complex of enzyme, its plDNA substrate, and inhibitor. We used fluoroquinolones to study the gyrase and plDNA of Plasmodium falciparum. New methods for isolating and separating plDNA reveal four topologically different forms and permit a quantitative exam of perturbations that result from gyrase poisoning. In keeping with its role in DNA replication, gyrase is most abundant in late stages of the parasite lifecycle, but several lines of evidence indicate that even in these cells the enzyme is present in relatively low abundance: about 1 enzyme for every two plDNAs or a ratio of 1 gyrase: 70 kb DNA. For a spectrum of quinolones, correlation was generally good between antimalarial activity and gyrase poisoning, the putative molecular mechanism of drug action. However, in P. falciparum there is evidence for off-target toxicity, particularly for ciprofloxacin. These studies highlight the utility of the new methods and of fluoroquinolones as a tool for studying the in situ workings of gyrase and its plDNA substrate. PMID:25881748

  7. Characterization of native PfABCG protein in Plasmodium falciparum.

    PubMed

    Edaye, Sonia; Georges, Elias

    2015-09-15

    The Plasmodium falciparum genome encodes 16 members of ABC proteins, with one member of the ABCG subfamily (PfABCG). Analysis of PfABCG amino acid sequence shows equal sequence identity to hsABCG1 and G2. Using N-terminal directed antibody against a recombinant fragment of PfABCG, we show that PfABCG migrates with an apparent molecular mass of 65KDa polypeptide on SDS-PAGE. PfABCG is expressed in all four stages of the parasite erythrocytic life cycle, with lower and higher expression in ring and late trophozoite stages, respectively. The protein localizes to the plasma membrane and a novel spherical structure beneath the cell membrane. Similar localization is also observed in gametocytes where PfABCG is highly expressed. Analysis of PfABCG genomic sequences for polymorphisms and changes in protein expression between different strains of P. falciparum revealed identical nucleotide sequence among the different strains, but variable protein expression. PfABCG expression is least in HB3 chloroquine sensitive strain, while higher expression levels are seen in other chloroquine-sensitive and -resistant strains, with highest levels of expression in 7G8. The differential expression of PfABCG in three chloroquine-sensitive strains (e.g., 3D7, HB3 and D10) predicts the sensitivity of the different strains to ketotifen, an anti-histaminic drug, whereby low expression is associated with decreased sensitivity to ketotifen. Taken together, the results in this report provide the first description of native PfABCG expression and subcellular localization in asexual stages of the parasite and its localization in gametocytes. It remains to be determined if PfABCG is functionally equivalent to mammalian ABCG1, ABCG2 or both. PMID:26239803

  8. Fucosylated Chondroitin Sulfate Inhibits Plasmodium falciparum Cytoadhesion and Merozoite Invasion

    PubMed Central

    Bastos, Marcele F.; Albrecht, Letusa; Kozlowski, Eliene O.; Lopes, Stefanie C. P.; Blanco, Yara C.; Carlos, Bianca C.; Castiñeiras, Catarina; Vicente, Cristina P.; Werneck, Claudio C.; Wunderlich, Gerhard; Ferreira, Marcelo U.; Marinho, Claudio R. F.; Mourão, Paulo A. S.; Pavão, Mauro S. G.

    2014-01-01

    Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria. PMID:24395239

  9. Identification of inhibitors of Plasmodium falciparum gametocyte development

    PubMed Central

    2013-01-01

    Background Plasmodium falciparum gametocytes, specifically mature stages, are the only stage in man transmissible to the mosquito vector responsible for malaria transmission. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria. The comprehensive profiling of in vitro activity of anti-malarial compounds against both early (I-III) and late (IV-V) stage P. falciparum gametocytes, along with the high throughput screening (HTS) outcomes from the MMV malaria box are described. Method Two anti-gametocyte HTS assays based on confocal fluorescence microscopy, utilizing both a gametocyte specific protein (pfs16-Luc-GFP) and a viability marker (MitoTracker Red CM-H2XRos) (MTR), were used for the measurement of anti-gametocytocidal activity. This combination provided a direct observation of gametocyte number per assay well, whilst defining the viability of each gametocyte imaged. Results IC50 values were obtained for 36 current anti-malarial compounds for activities against asexual, early and late stage gametocytes. The MMV malaria box was screened and actives progressed for IC50 evaluation. Seven % of the “drug-like” and 21% of the “probe-like” compounds from the MMV malaria box demonstrated equivalent activity against both asexual and late stage gametocytes. Conclusions The assays described were shown to selectively identify compounds with gametocytocidal activity and have been demonstrated suitable for HTS with the capability of screening in the order of 20,000 compounds per screening campaign, two to three times per seven-day week. PMID:24206914

  10. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria

    PubMed Central

    Mbengue, Alassane; Bhattacharjee, Souvik; Pandharkar, Trupti; Liu, Haining; Estiu, Guillermina; Stahelin, Robert V.; Rizk, Shahir; Njimoh, Dieudonne L.; Ryan, Yana; Chotivanich, Kesinee; Nguon, Chea; Ghorbal, Mehdi; Lopez-Rubio, Jose-Juan; Pfrender, Michael; Emrich, Scott; Mohandas, Narla; Dondorp, Arjen M.; Wiest, Olaf; Haldar, Kasturi

    2015-01-01

    Artemisinins are the corner stone of anti-malarial drugs1. Emergence and spread of resistance to them2–4 raises risk of wiping out recent gains achieved in reducing world-wide malaria burden and threatens future malaria control and elimination on a global level. Genome wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance5–10. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase as well as its lipid product phosphatidylinositol 3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signaling, where transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination. PMID:25874676

  11. Plasmodium falciparum var gene expression is modified by host immunity

    PubMed Central

    Warimwe, George M.; Keane, Thomas M.; Fegan, Gregory; Musyoki, Jennifer N.; Newton, Charles R. J. C.; Pain, Arnab; Berriman, Matthew; Marsh, Kevin; Bull, Peter C.

    2009-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, which play a central role in the host–parasite interaction by binding to various host molecules. They are encoded by a diverse family of genes called var, of which there are ≈60 copies in each parasite genome. In sub-Saharan Africa, although P. falciparum infection occurs throughout life, severe malarial disease tends to occur only in childhood. This could potentially be explained if (i) PfEMP1 variants differ in their capacity to support pathogenesis of severe malaria and (ii) this capacity is linked to the likelihood of each molecule being recognized and cleared by naturally acquired antibodies. Here, in a study of 217 Kenyan children with malaria, we show that expression of a group of var genes “cys2,” containing a distinct pattern of cysteine residues, is associated with low host immunity. Expression of cys2 genes was associated with parasites from young children, those with severe malaria, and those with a poorly developed antibody response to parasite-infected erythrocyte surface antigens. Cys-2 var genes form a minor component of all genomic var repertoires analyzed to date. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organized such that PfEMP1 molecules that confer the most virulence to the parasite tend also to be those that are most susceptible to the development of host immunity. This may help the parasite to adapt effectively to the development of host antibodies through modification of the host–parasite relationship. PMID:20018734

  12. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii.

    PubMed

    Mahon, Cathal S; O'Donoghue, Anthony J; Goetz, David H; Murray, Patrick G; Craik, Charles S; Tuohy, Maria G

    2009-11-01

    Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k(cat)/K(m)=2.1 x 10(6) M(-1) s(-1)) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 degrees C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an alpha/beta hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are

  13. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

    PubMed Central

    Mahon, Cathal S.; O'Donoghue, Anthony J.; Goetz, David H.; Murray, Patrick G.; Craik, Charles S.; Tuohy, Maria G.

    2009-01-01

    Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro–X (kcat/Km=2.1×106 M−1 s−1) compared with Ala–X or Val–X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 °C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an α/β hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are

  14. In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector

    PubMed Central

    Linz, Lucas B.; Liu, Sijun; Chougule, Nanasaheb P.

    2015-01-01

    ABSTRACT Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and surface plasmon resonance were used to confirm and characterize CP-APN interaction. PEMV virions and a peptide comprised of PEMV CP fused to a proline-rich hinge (-P-) and green fluorescent protein (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles, binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W. O. Sparks, W. A. Miller, and B. C. Bonning, Virology 401:107–116, 2010, http://dx.doi.org/10.1016/j.virol.2010.02.009) support the identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV-APN interaction designed to block plant virus transmission and to suppress aphid populations. IMPORTANCE A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers

  15. Molecular Determinants of Species Specificity in the Coronavirus Receptor Aminopeptidase N (CD13): Influence of N-Linked Glycosylation

    PubMed Central

    Wentworth, David E.; Holmes, Kathryn V.

    2001-01-01

    Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection. PMID:11559807

  16. Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

    SciTech Connect

    Appolaire, Alexandre; Girard, Eric; Colombo, Matteo; Durá, M. Asunción; Moulin, Martine; Härtlein, Michael; Franzetti, Bruno; Gabel, Frank

    2014-11-01

    The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme. The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

  17. Human immunodeficiency virus 1 Tat binds to dipeptidyl aminopeptidase IV (CD26): a possible mechanism for Tat's immunosuppressive activity.

    PubMed

    Gutheil, W G; Subramanyam, M; Flentke, G R; Sanford, D G; Munoz, E; Huber, B T; Bachovchin, W W

    1994-07-01

    The human immunodeficiency virus 1 (HIV-1) Tat protein suppresses antigen-induced, but not mitogen-induced, activation of human T cells when added to T-cell cultures [Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. (1989) Science 246, 1606-1608]. This activity is potentially pertinent to the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Here we report that Tat binds with high affinity to the T-cell activation molecule dipeptidyl aminopeptidase IV (DP IV), also known as CD26. This molecule occurs on the surface of CD4+ cells responsible for the recall antigen response and appears to play an essential role in this response. Tat binds to both the cell surface and soluble forms of DP IV at physiological salt concentrations without inhibiting the protease activity of DP IV against small chromogenic substrates used to assay activity, but Tat markedly inhibits the activity of DP IV at lower salt concentrations. The kinetics of inhibition indicate the affinity of Tat for DP IV varies from 20 pM to 11 nM, and the activity of the Tat-DP IV complex varies from 13% to 100%, as the NaCl concentration varies from 0 to 140 mM. Cytofluorometry experiments demonstrate that Tat competes with anti-Ta1, a monoclonal antibody (mAb) specific for DP IV, for binding to cell surface DP IV, thus indicating that Tat binds DP IV at or near the Ta1 epitope. Moreover, the anti-Ta1 mAb blocks the immunosuppressive activity of Tat. The high affinity of Tat for DP IV, previous evidence implicating DP IV in antigen-specific T-cell activation events, and the ability of anti-Ta1 mAb to block the immunosuppressive effect of Tat make DP IV a plausible receptor for Tat's immunosuppressive activity. PMID:7912830

  18. Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481

    PubMed Central

    2012-01-01

    The aminopeptidase P (PepP, EC 3.4.11.9) gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA. PMID:22853547

  19. Aminopeptidase p mediated detoxification of organophosphonate analogues of sarin: mechanistic and stereochemical study at the phosphorus atom of the substrate.

    PubMed

    Huang, Li-Fang; Su, Benson; Jao, Shu-Chuan; Liu, Kwang-Ting; Li, Wen-Shan

    2006-03-01

    The activity of the aminopeptidase P from Escherichia coli in hydrolyzing a series of organophosphonate sarin analogues (1-6) was evaluated. The enzymatic rates of hydrolysis for methylphosphonate 1 with a methoxy group attached to the phosphorus center were 7- to 15-fold higher than those for the corresponding analogues 2-6. Double mutant R153W/R370L was able to hydrolyze the S(p) enantiomer of racemic 1 at a considerable rate. This mutant allowed the preparation of the R(p) isomer of the sarin analogue 1. All the mutants, R370L, R153A, W88L, R153L/R370L, and R153W/R370L, preferred the formation of (S(p))-8 to that of the corresponding (R(p))-8 enantiomer and displayed a better enantiomeric excess of products, by 1.4- to 2-fold as compared to the wild-type enzyme. Enzymatic hydrolysis of O,O-diisopropyl-p-nitrophenyl phosphate (9) in H(2) (18)O led to the formation of the (18)O-labeled O,O-diisopropyl phosphate product and confirmed that the catalytic reaction starts with cleavage of the P--O bond. From chemical and kinetic studies, the utilization of an optically pure S(p) enantiomer of O-methyl-p-nitrophenyl methylphosphonothioate (S(p))-MNMPT, 7) has demonstrated that the enzymatic reaction proceeds through a displacement mechanism and generates a chiral product in situ with an inversion of stereochemical configuration at the phosphorus atom. The results also lead to the conclusion that alteration of the active site through site-directed mutagenesis can result in a preference for (S(p))-MNMPT (7) rather than the R(p) isomer. PMID:16470765

  20. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    PubMed

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  1. Effect of thyroid hormone-nitric oxide interaction on tumor growth, angiogenesis, and aminopeptidase activity in mice.

    PubMed

    Carmona-Cortés, Javier; Rodríguez-Gómez, Isabel; Wangensteen, Rosemary; Banegas, Inmaculada; García-Lora, Ángel M; Quesada, Andrés; Osuna, Antonio; Vargas, Félix

    2014-06-01

    This study evaluated the effects of thyroid hormone-NO interaction on tumor development, vascularization, vascular endothelial growth factor (VEGF), and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Experiments were performed in male CBA-C57 mice. Animals were untreated (controls) or treated with: T4, the antithyroid drug methimazole, the NO inhibitor L-NAME, T4+L-NAME, methimazole+NAME, the αvß3 integrin antagonist tetrac, T4+tetrac, the iNOS inhibitor aminoguanidine (AG), and T4 + AG; all treatments were for 6 weeks except for tetrac, administered for the last 11 days. Mice were subcutaneously inoculated with 1 × 10(6) exponentially growing Lewis carcinoma 3LL cells into the dorsum. Study variables 9 days later were tumor weight (TW), Hb content, an index of tumor vascularization, VEGF, and AP activity. T4 produced parallel increases in TW and angiogenesis. L-NAME reduced TW and angiogenesis in control, hyperthyroid, and hypothyroid mice, whereas AG had no effect on these variables. Tetrac arrested TW in normal and T4-treated mice but did not decrease angiogenesis in T4-treated animals. Negative correlations were found between TW and AP activity in tumors from control hyper- and hypothyroid groups and an inverse relationship was observed between TW and AP activities in tetrac-treated mice. T4 enhances TW and angiogenesis, in which NO participates, but requires activation of integrin αvß3 to promote carcinogenesis. NO blockade reduces TW, regardless of the thyroid status. Thyroid hormone negatively modulates AP activity in the tumor. Accordingly, blockade of the membrane TH receptor αvß3 integrin reduces TW associated with an increase in AP activity. PMID:24549786

  2. Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis1

    PubMed Central

    Murphy, Angus S.; Hoogner, Karen R.; Peer, Wendy Ann; Taiz, Lincoln

    2002-01-01

    Polar transport of the plant hormone auxin is regulated at the cellular level by inhibition of efflux from a plasma membrane (PM) carrier. Binding of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to a regulatory site associated with the carrier has been characterized, but the NPA-binding protein(s) have not been identified. Experimental disparities between levels of high-affinity NPA binding and auxin transport inhibition can be explained by the presence of a low-affinity binding site and in vivo hydrolysis of NPA. In Arabidopsis, colocalization of NPA amidase and aminopeptidase (AP) activities, inhibition of auxin transport by artificial β-naphthylamide substrates, and saturable displacement of NPA by the AP inhibitor bestatin suggest that PM APs may be involved in both low-affinity NPA binding and hydrolysis. We report the purification and molecular cloning of NPA-binding PM APs and associated proteins from Arabidopsis. This is the first report of PM APs in plants. PM proteins were purified by gel permeation, anion exchange, and NPA affinity chromatography monitored for tyrosine-AP activity. Lower affinity fractions contained two orthologs of mammalian APs involved in signal transduction and cell surface-extracellular matrix interactions. AtAPM1 and ATAPP1 have substrate specificities and inhibitor sensitivities similar to their mammalian orthologs, and have temporal and spatial expression patterns consistent with previous in planta histochemical data. Copurifying proteins suggest that the APs interact with secreted cell surface and cell wall proline-rich proteins. AtAPM1 and AtAPP1 are encoded by single genes. In vitro translation products of ATAPM1 and AtAPP1 have enzymatic activities similar to those of native proteins. PMID:11891249

  3. A common SNP in ER aminopeptidase 2 induces a specificity switch that leads to altered antigen processing

    PubMed Central

    Evnouchidou, Irini; Birtley, James; Seregin, Sergey; Papakyriakou, Athanasios; Zervoudi, Efthalia; Samiotaki, Martina; Panayotou, George; Giastas, Petros; Petrakis, Olivia; Georgiadis, Dimitris; Amalfitano, Andrea; Saridakis, Emmanuel; Mavridis, Irene M.; Stratikos, Efstratios

    2012-01-01

    ER aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered antigen processing by these enzymes is a causal link to disease etiology but the molecular mechanisms are obscure. We report here that the common ERAP2 SNP rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity-switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans. PMID:22837489

  4. Microarray Analysis of Tomato’s Early and Late Wound Response Reveals New Regulatory Targets for Leucine Aminopeptidase A

    PubMed Central

    Scranton, Melissa A.; Fowler, Jonathan H.; Girke, Thomas; Walling, Linda L.

    2013-01-01

    Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A’s role in modulation of wound responses extends beyond the well described late-wound gene core. PMID:24205013

  5. Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

    PubMed

    Ghale, Garima; Kuhnert, Nikolai; Nau, Werner M

    2012-03-01

    A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases. PMID:22545408

  6. Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

    PubMed Central

    2013-01-01

    With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection. PMID:24289031

  7. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.

    PubMed

    Atsumi, Shogo; Mizuno, Eri; Hara, Hirotaka; Nakanishi, Kazuko; Kitami, Madoka; Miura, Nami; Tabunoki, Hiroko; Watanabe, Ayako; Sato, Ryoichi

    2005-07-01

    We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding. PMID:16000811

  8. Location of the Bombyx mori Aminopeptidase N Type 1 Binding Site on Bacillus thuringiensis Cry1Aa Toxin

    PubMed Central

    Atsumi, Shogo; Mizuno, Eri; Hara, Hirotaka; Nakanishi, Kazuko; Kitami, Madoka; Miura, Nami; Tabunoki, Hiroko; Watanabe, Ayako; Sato, Ryoichi

    2005-01-01

    We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding. PMID:16000811

  9. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

    PubMed Central

    Herrero, Salvador; Gechev, Tsanko; Bakker, Petra L; Moar, William J; de Maagd, Ruud A

    2005-01-01

    Background Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. Results Suppression Subtractive Hybridization (SSH) was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4). A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1) were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3), the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. Conclusion We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony. PMID:15978131

  10. Primaquine or other 8-aminoquinoline for reducing Plasmodium falciparum transmission

    PubMed Central

    Graves, Patricia M; Gelband, Hellen; Garner, Paul

    2015-01-01

    Background Mosquitoes become infected with Plasmodium when they ingest gametocyte-stage parasites from an infected person's blood. Plasmodium falciparum gametocytes are sensitive to the drug primaquine (PQ) and other 8-aminoquinolines (8AQ); these drugs could prevent parasite transmission from infected people to mosquitoes, and consequently reduce the incidence of malaria. However, PQ will not directly benefit the individual, and could be harmful to those with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In 2010, The World Health Organization (WHO) recommended a single dose of PQ at 0.75 mg/kg, alongside treatment for P. falciparum malaria to reduce transmission in areas approaching malaria elimination. In 2013 the WHO revised this to 0.25 mg/kg due to concerns about safety. Objectives To assess whether giving PQ or an alternative 8AQ alongside treatment for P. falciparum malaria reduces malaria transmission, and to estimate the frequency of severe or haematological adverse events when PQ is given for this purpose. Search methods We searched the following databases up to 10 Feb 2014 for trials: the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL), published in The Cochrane Library; MEDLINE; EMBASE; LILACS; metaRegister of Controlled Trials (mRCT); and the WHO trials search portal using 'malaria*', 'falciparum', and 'primaquine' as search terms. In addition, we searched conference proceedings and reference lists of included studies, and contacted researchers and organizations. Selection criteria Randomized controlled trials (RCTs) or quasi-RCTs comparing PQ (or alternative 8AQ) given as a single dose or short course alongside treatment for P. falciparum malaria with malaria treatment given without PQ/8AQ in adults or children. Data collection and analysis Two authors independently screened all abstracts, applied inclusion criteria, and extracted data. We sought evidence of an impact on

  11. Primaquine or other 8-aminoquinoline for reducing P. falciparum transmission

    PubMed Central

    Graves, Patricia M; Gelband, Hellen; Garner, Paul

    2014-01-01

    Background Mosquitoes become infected with Plasmodium when they ingest gametocyte-stage parasites from an infected person's blood. Plasmodium falciparum gametocytes are sensitive to the drug primaquine (PQ) and other 8-aminoquinolines (8AQ); these drugs could prevent parasite transmission from infected people to mosquitoes, and consequently reduce the incidence of malaria. However, PQ will not directly benefit the individual, and could be harmful to those with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In 2010, The World Health Organization (WHO) recommended a single dose of PQ at 0.75 mg/kg, alongside treatment for P. falciparum malaria to reduce transmission in areas approaching malaria elimination. In 2013 the WHO revised this to 0.25 mg/kg due to concerns about safety. Objectives To assess whether giving PQ or an alternative 8AQ alongside treatment for P. falciparum malaria reduces malaria transmission, and to estimate the frequency of severe or haematological adverse events when PQ is given for this purpose. Search methods We searched the following databases up to 10 Feb 2014 for trials: the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL), published in The Cochrane Library; MEDLINE; EMBASE; LILACS; metaRegister of Controlled Trials (mRCT); and the WHO trials search portal using 'malaria*', 'falciparum', and 'primaquine' as search terms. In addition, we searched conference proceedings and reference lists of included studies, and contacted researchers and organizations. Selection criteria Randomized controlled trials (RCTs) or quasi-RCTs comparing PQ (or alternative 8AQ) given as a single dose or short course alongside treatment for P. falciparum malaria with malaria treatment given without PQ/8AQ in adults or children. Data collection and analysis Two authors independently screened all abstracts, applied inclusion criteria, and extracted data. We sought evidence of an impact on

  12. Structure-Guided, Single-Point Modifications in the Phosphinic Dipeptide Structure Yield Highly Potent and Selective Inhibitors of Neutral Aminopeptidases

    SciTech Connect

    Vassiliou, Stamatia; Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Mulligan, Rory; Joachimiak, Andrzej; Mucha, Artur

    2014-10-09

    Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor–enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. In conclusion, another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π–π stacking interaction between a pyridine ring and Tyr372.

  13. 3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of M1 aminopeptidases with immune-regulating properties.

    PubMed

    Papakyriakou, Athanasios; Zervoudi, Efthalia; Tsoukalidou, Sofia; Mauvais, Francois-Xavier; Sfyroera, Georgia; Mastellos, Dimitrios C; van Endert, Peter; Theodorakis, Emmanuel A; Vourloumis, Dionisios; Stratikos, Efstratios

    2015-02-12

    Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study, we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 = 105 nM). Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of immune responses. PMID:25635706

  14. Structure-guided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases.

    PubMed

    Vassiliou, Stamatia; Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Mulligan, Rory; Joachimiak, Andrzej; Mucha, Artur

    2014-10-01

    Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π-π stacking interaction between a pyridine ring and Tyr372. PMID:25192493

  15. Plasmodium vivax Populations Are More Genetically Diverse and Less Structured than Sympatric Plasmodium falciparum Populations

    PubMed Central

    Jennison, Charlie; Arnott, Alicia; Tessier, Natacha; Tavul, Livingstone; Koepfli, Cristian; Felger, Ingrid; Siba, Peter M.; Reeder, John C.; Bahlo, Melanie; Mueller, Ivo; Barry, Alyssa E.

    2015-01-01

    Introduction The human malaria parasite, Plasmodium vivax, is proving more difficult to control and eliminate than Plasmodium falciparum in areas of co-transmission. Comparisons of the genetic structure of sympatric parasite populations may provide insight into the mechanisms underlying the resilience of P. vivax and can help guide malaria control programs. Methodology/Principle findings P. vivax isolates representing the parasite populations of four areas on the north coast of Papua New Guinea (PNG) were genotyped using microsatellite markers and compared with previously published microsatellite data from sympatric P. falciparum isolates. The genetic diversity of P. vivax (He = 0.83–0.85) was higher than that of P. falciparum (He = 0.64–0.77) in all four populations. Moderate levels of genetic differentiation were found between P. falciparum populations, even over relatively short distances (less than 50 km), with 21–28% private alleles and clear geospatial genetic clustering. Conversely, very low population differentiation was found between P. vivax catchments, with less than 5% private alleles and no genetic clustering observed. In addition, the effective population size of P. vivax (30353; 13043–69142) was larger than that of P. falciparum (18871; 8109–42986). Conclusions/Significance Despite comparably high prevalence, P. vivax had higher diversity and a panmictic population structure compared to sympatric P. falciparum populations, which were fragmented into subpopulations. The results suggest that in comparison to P. falciparum, P. vivax has had a long-term large effective population size, consistent with more intense and stable transmission, and limited impact of past control and elimination efforts. This underlines suggestions that more intensive and sustained interventions will be needed to control and eventually eliminate P. vivax. This research clearly demonstrates how population genetic analyses can reveal deeper insight into transmission

  16. Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

    PubMed Central

    Moonah, Shannon; Sanders, Natalie G.; Persichetti, Jason K.

    2014-01-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  17. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    PubMed Central

    2012-01-01

    Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

  18. Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan

    PubMed Central

    2010-01-01

    Background Plasmodium vivax and Plasmodium falciparum are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of P. vivax and P. falciparum in this understudied region. Methods The genetic diversities of P. vivax and P. falciparum populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (msp) genes by PCR-RFLP. Specifically, the Pvmsp-3α and Pvmsp-3β genes of P. vivax and the Pfmsp-1 and Pfmsp-2 genes of P. falciparum were analysed. Results In P. vivax, genotyping of Pvmsp-3α and Pvmsp-3β genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for Pvmsp-3α, type A being the most prevalent (82%). Conversely, amplification of the P. vivax msp-3β locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of Pvmsp-3α and Pvmsp-3β yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In P. falciparum, all three known genotypes of Pfmsp-1 and two of Pfmsp-2 were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% P. falciparum samples exhibited mixed-strain infections. Conclusion These results indicate that both P. vivax and P. falciparum populations in Pakistan are highly diverse. PMID:20416089

  19. Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

    PubMed

    Moonah, Shannon; Sanders, Natalie G; Persichetti, Jason K; Sullivan, David J

    2014-10-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  20. The mRNA expression of amino acid transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1 in the intestine and liver of post-hatch broiler chicks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acid transporter (AAT) proteins are responsible for the movement of amino acids (AA) in and out of cells. Aminopeptidase (APN) cleaves AAs from the N terminus of polypeptides making them available for transport, while PepT1 is a di- and tri- peptide transporter. In the intestine, these prote...

  1. Identification of a novel aminopeptidase p-like gene (OnAPP) and it's possible involvement in Bt toxicity and resistance in a major corn pest(Ostrinia nubilalis)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study we identified and characterized 10 aminopeptidase-like genes in relation t...

  2. The mRNA expression of amino acid transporters, aminopeptidase N, and the di- and tri- peptide transporter PepT1 in the embryo of the domesticated chicken (Gallus gallus) shows developmental regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mRNA expression profile for ten amino acid transporters (AAT), the di-and tri- peptide transporter (Pept1), and aminopeptidase N (APN) during chick embryogenesis was determined. Fertilized eggs were sampled at days 9, 11, 15, 17, 19, and 20, post fertilization. Three to four embryos were sampl...

  3. Segregation of European corn borer, Ostrinia nubilalis, aminopeptidase 1, cadherin, and bre5-like alleles, from a colony resistant to Bacillus thuringiensis Cry1Ab toxins, are not associated ...fed a diet containing Cry1Ab

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peptide receptors may be required for activated Bacillus thuringiensis (Bt) Cry toxins to bind midgut epithelium prior to pore formation. Single nucleotide polymorphism (SNP) markers from two Ostrinia nubilalis midgut peptide receptors, cadherin (OnCad) and aminopeptidase N 1 (OnAPN1), and OnBre5 (...

  4. Artesunate plus pyronaridine for treating uncomplicated Plasmodium falciparum malaria

    PubMed Central

    Bukirwa, Hasifa; Unnikrishnan, B; Kramer, Christine V; Sinclair, David; Nair, Suma; Tharyan, Prathap

    2014-01-01

    Background The World Health Organization (WHO) recommends that people with uncomplicated Plasmodium falciparum malaria are treated using Artemisinin-based Combination Therapy (ACT). ACT combines three-days of a short-acting artemisinin derivative with a longer-acting antimalarial which has a different mode of action. Pyronaridine has been reported as an effective antimalarial over two decades of use in parts of Asia, and is currently being evaluated as a partner drug for artesunate. Objectives To evaluate the efficacy and safety of artesunate-pyronaridine compared to alternative ACTs for treating people with uncomplicated P. falciparum malaria. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register; Cochrane Central Register of Controlled Trials (CENTRAL), published in The Cochrane Library; MEDLINE; EMBASE; LILACS; ClinicalTrials.gov; the metaRegister of Controlled Trials (mRCT); and the WHO International Clinical Trials Search Portal up to 16 January 2014. We searched reference lists and conference abstracts, and contacted experts for information about ongoing and unpublished trials. Selection criteria Randomized controlled trials of artesunate-pyronaridine versus other ACTs in adults and children with uncomplicated P. falciparum malaria. For the safety analysis, we also included adverse events data from trials comparing any treatment regimen containing pyronaridine with regimens not containing pyronaridine. Data collection and analysis Two authors independently assessed trial eligibility and risk of bias, and extracted data. We combined dichotomous data using risk ratios (RR) and continuous data using mean differences (MD), and presented all results with a 95% confidence interval (CI). We used the GRADE approach to assess the quality of evidence. Main results We included six randomized controlled trials enrolling 3718 children and adults. Artesunate-pyronaridine versus artemether-lumefantrine In two multicentre trials, enrolling

  5. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  6. Trend and manifestations of falciparum malaria in a tertiary care hospital of India

    PubMed Central

    Saya, Rama Prakasha; Saya, Ganesh Kumar; Debabrata, Goswami

    2016-01-01

    Background: The recent focus is on the increase in the burden of falciparum cases with a varied spectrum of presentation and outcome, especially in developing countries like India. This study was undertaken to analyze the trend and manifestations of falciparum malaria in a tertiary care hospital. Materials and Methods: This descriptive study was carried out at the Gauhati Government Medical College and Hospital from June 2006 to May 2007. The data were collected on demographic and time characteristics, clinical and laboratory findings, the outcome of disease and expressed in proportion or percentages. Results: Out of the 100 cases, around 2nd/3rd (63%) of cases were in the age group of 15–30 years and the mean age was found to be 29.51 years. About 66% of them were males. Clinical presentations included pain abdomen (42, 42%), nausea and vomiting (35, 35%), jaundice (34, 34%), oliguria (24, 24%), altered sensorium (24, 24%), breathing difficulty (10, 10%), and seizures (5, 5%). Number of cases and mortality were more with a peak in the month of May and September. Manifestations of severe falciparum malaria included hepatopathy (38%), renal failure (28%), shock (9%), acute respiratory distress syndrome (7%), hypoglycemia (3%), and severe anemia (1%). Eighty-two cases (82%) recovered and 18 cases (18%) expired. Conclusion: Falciparum malaria is more among younger adult age group and males. Complications and mortality are also more due to falciparum malaria. PMID:27563638

  7. 3-Iodo-4-aminoquinoline derivative sensitises resistant strains of Plasmodium falciparum to chloroquine.

    PubMed

    Edaye, Sonia; Tazoo, Dagobert; Bohle, D Scott; Georges, Elias

    2016-06-01

    Chloroquine (CQ), the first cost-effective synthetic antimalarial, is rendered ineffective in malaria-endemic regions owing to the rise and spread of CQ-resistant Plasmodium falciparum. In this report, we show that a halogen derivative of CQ, namely 3-iodo-CQ, inhibits the proliferation of CQ-sensitive and -resistant P. falciparum in a verapamil-insensitive manner. Similar to CQ, the antimalarial activity of 3-iodo-CQ is likely due to its inhibition of β-haematin formation. Interestingly, the presence of non-inhibitory concentrations of 3-iodo-CQ potentiated the antiproliferative activity of CQ against CQ-resistant strains or P. falciparum transfectants expressing wild-type or mutant P. falciparum CQ resistance transporter (PfCRT) (C2(GC03) or C4(Dd2), respectively). These findings demonstrate that halogenation of the third position of 4-aminoquinoline, with a simple one-step reaction from CQ, generates a novel derivative that is active against CQ-sensitive and -resistant P. falciparum, possibly by inhibiting the activity of mutant PfCRT. PMID:27211211

  8. Functional interpretation of a non-gut hemocoelic tissue aminopeptidase N (APN) in a lepidopteran insect pest Achaea janata.

    PubMed

    Ningshen, Thuirei Jacob; Aparoy, Polamarasetty; Ventaku, Venkat Rao; Dutta-Gupta, Aparna

    2013-01-01

    Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn(++) dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation. PMID:24244508

  9. Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

    PubMed Central

    Ningshen, Thuirei Jacob; Aparoy, Polamarasetty; Ventaku, Venkat Rao; Dutta-Gupta, Aparna

    2013-01-01

    Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn++ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation. PMID:24244508

  10. Genetic architecture of artemisinin-resistant Plasmodium falciparum.

    PubMed

    Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P

    2015-03-01

    We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. PMID:25599401

  11. Analysis of Breath Specimens for Biomarkers of Plasmodium falciparum Infection

    PubMed Central

    Berna, Amalia Z.; McCarthy, James S.; Wang, Rosalind X.; Saliba, Kevin J.; Bravo, Florence G.; Cassells, Julie; Padovan, Benjamin; Trowell, Stephen C.

    2015-01-01

    Currently, the majority of diagnoses of malaria rely on a combination of the patient's clinical presentation and the visualization of parasites on a stained blood film. Breath offers an attractive alternative to blood as the basis for simple, noninvasive diagnosis of infectious diseases. In this study, breath samples were collected from individuals during controlled malaria to determine whether specific malaria-associated volatiles could be detected in breath. We identified 9 compounds whose concentrations varied significantly over the course of malaria: carbon dioxide, isoprene, acetone, benzene, cyclohexanone, and 4 thioethers. The latter group, consisting of allyl methyl sulfide, 1-methylthio-propane, (Z)-1-methylthio-1-propene, and (E)-1-methylthio-1-propene, had not previously been associated with any disease or condition. Before the availability of antimalarial drug treatment, there was evidence of concurrent 48-hour cyclical changes in the levels of both thioethers and parasitemia. When thioether concentrations were subjected to a phase shift of 24 hours, a direct correlation between the parasitemia and volatile levels was revealed. Volatile levels declined monotonically approximately 6.5 hours after initial drug treatment, correlating with clearance of parasitemia. No thioethers were detected in in vitro cultures of Plasmodium falciparum. The metabolic origin of the thioethers is not known, but results suggest that interplay between host and parasite metabolic pathways is involved in the production of these thioethers. PMID:25810441

  12. Plasmodium falciparum Merozoite Surface Protein 6 Is a Dimorphic Antigen

    PubMed Central

    Pearce, J. Andrew; Triglia, Tony; Hodder, Anthony N.; Jackson, David C.; Cowman, Alan F.; Anders, Robin F.

    2004-01-01

    Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition. PMID:15039357

  13. Human cerebral malaria and Plasmodium falciparum genotypes in Malawi

    PubMed Central

    2012-01-01

    Background Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. Methods The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. Results Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. Conclusions Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity. PMID:22314206

  14. Serological Evidence of Discrete Spatial Clusters of Plasmodium falciparum Parasites

    PubMed Central

    Bejon, Philip; Turner, Louise; Lavstsen, Thomas; Cham, Gerald; Olotu, Ally; Drakeley, Chris J.; Lievens, Marc; Vekemans, Johan; Savarese, Barbara; Lusingu, John; von Seidlein, Lorenz; Bull, Peter C.; Marsh, Kevin; Theander, Thor G.

    2011-01-01

    Background Malaria transmission may be considered to be homogenous with well-mixed parasite populations (as in the classic Ross/Macdonald models). Marked fine-scale heterogeneity of transmission has been observed in the field (i.e., over a few kilometres), but there are relatively few data on the degree of mixing. Since the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is highly polymorphic, the host's serological responses may be used to infer exposure to parasite sub-populations. Methods and Findings We measured the antibody responses to 46 individual PfEMP1 domains at four time points among 450 children in Kenya, and identified distinct spatial clusters of antibody responses to individual domains. 35 domains showed strongly significant sero-clusters at p = 0.001. Individuals within the high transmission hotspot showed the greatest diversity of anti-PfEMP1 responses. Individuals outside the hotspot had a less diverse range of responses, even if as individuals they were at relatively intense exposure. Conclusions We infer that antigenically distinct sub-populations of parasites exist on a fine spatial scale in a study area of rural Kenya. Further studies should examine antigenic variation over longer periods of time and in different study areas. PMID:21747921

  15. Genetic architecture of artemisinin-resistant Plasmodium falciparum

    PubMed Central

    Miotto, Olivo; Amato, Roberto; Ashley, Elizabeth A; MacInnis, Bronwyn; Almagro-Garcia, Jacob; Amaratunga, Chanaki; Lim, Pharath; Mead, Daniel; Oyola, Samuel O; Dhorda, Mehul; Imwong, Mallika; Woodrow, Charles; Manske, Magnus; Stalker, Jim; Drury, Eleanor; Campino, Susana; Amenga-Etego, Lucas; Thanh, Thuy-Nhien Nguyen; Tran, Hien Tinh; Ringwald, Pascal; Bethell, Delia; Nosten, Francois; Phyo, Aung Pyae; Pukrittayakamee, Sasithon; Chotivanich, Kesinee; Chuor, Char Meng; Nguon, Chea; Suon, Seila; Sreng, Sokunthea; Newton, Paul N; Mayxay, Mayfong; Khanthavong, Maniphone; Hongvanthong, Bouasy; Htut, Ye; Han, Kay Thwe; Kyaw, Myat Phone; Faiz, Md Abul; Fanello, Caterina I; Onyamboko, Marie; Mokuolu, Olugbenga A; Jacob, Christopher G; Takala-Harrison, Shannon; Plowe, Christopher V; Day, Nicholas P; Dondorp, Arjen M; Spencer, Chris C A; McVean, Gilean; Fairhurst, Rick M; White, Nicholas J; Kwiatkowski, Dominic P

    2015-01-01

    We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. PMID:25599401

  16. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    NASA Astrophysics Data System (ADS)

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2014-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (`K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

  17. Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

    SciTech Connect

    Shams-Eldin, Hosam Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.

    2008-06-06

    Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.

  18. Malaria vaccines: identifying Plasmodium falciparum liver-stage targets

    PubMed Central

    Longley, Rhea J.; Hill, Adrian V. S.; Spencer, Alexandra J.

    2015-01-01

    The development of a highly efficacious and durable vaccine for malaria remains a top priority for global health researchers. Despite the huge rise in recognition of malaria as a global health problem and the concurrent rise in funding over the past 10–15 years, malaria continues to remain a widespread burden. The evidence of increasing resistance to anti-malarial drugs and insecticides is a growing concern. Hence, an efficacious and durable preventative vaccine for malaria is urgently needed. Vaccines are one of the most cost-effective tools and have successfully been used in the prevention and control of many diseases, however, the development of a vaccine for the Plasmodium parasite has proved difficult. Given the early success of whole sporozoite mosquito-bite delivered vaccination strategies, we know that a vaccine for malaria is an achievable goal, with sub-unit vaccines holding great promise as they are simple and cheap to both manufacture and deploy. However a major difficulty in development of sub-unit vaccines lies within choosing the appropriate antigenic target from the 5000 or so genes expressed by the parasite. Given the liver-stage of malaria represents a bottle-neck in the parasite’s life cycle, there is widespread agreement that a multi-component sub-unit malaria vaccine should preferably contain a liver-stage target. In this article we review progress in identifying and screening Plasmodium falciparum liver-stage targets for use in a malaria vaccine. PMID:26441899

  19. The gene encoding topoisomerase II from Plasmodium falciparum.

    PubMed Central

    Cheesman, S; McAleese, S; Goman, M; Johnson, D; Horrocks, P; Ridley, R G; Kilbey, B J

    1994-01-01

    The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite. Images PMID:8041616

  20. Polycyclic amines as chloroquine resistance modulating agents in Plasmodium falciparum.

    PubMed

    Joubert, Jacques; Kapp, Erika; Taylor, Dale; Smith, Peter J; Malan, Sarel F

    2016-02-15

    Pentacycloundecylamines (PCUs) and adamantane amines, such as NGP1-01 (1) and amantadine, have shown significant channel blocking activities. They are postulated to act as chemosensitizers and circumvent the resistance of the plasmodia parasite against chloroquine (CQ) by inhibiting the p-glycoprotein efflux pump and enabling the accumulation of CQ inside the parasite digestive vacuole. Twelve polycyclic amines containing either a PCU or adamantane amine moiety conjugated to different aromatic functionalities through various tethered linkers were selected based on their channel blocking abilities and evaluated as potential chemosensitizers. Compounds 2, 4, 5 and 10 showed significant voltage-gated calcium channel (VGCC) blocking ability (IC50=0.27-35 μM) and were able to alter the CQ IC50 in differing degrees (45-81%) in the multidrug resistant Plasmodium falciparum Dd2 isolate. Among them, the PCU-dansyl amine compound (4) displayed the best potential to act as a chemosensitizer against the Dd2 strain at a 1 μM concentration (RMI=0.19) while displaying moderate antiplasmodial activity (Dd2 IC50=6.25 μM) and low in vitro cytotoxicity against a mammalian cell line (CHO, IC50=119 μM). Compounds 2 and 10 also showed some promising chemosensitizing abilities (RMI=0.36 and 0.35 respectively). A direct correlation was found between the VGCC blocking ability of these polycyclic amines and their capacity to act as CQ resistance modulating agents. PMID:26832222

  1. Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum

    PubMed Central

    Yamagishi, Junya; Natori, Anna; Tolba, Mohammed E.M.; Mongan, Arthur E.; Sugimoto, Chihiro; Katayama, Toshiaki; Kawashima, Shuichi; Makalowski, Wojciech; Maeda, Ryuichiro; Eshita, Yuki; Tuda, Josef

    2014-01-01

    To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions. PMID:25091627

  2. Platelets Potentiate Brain Endothelial Alterations Induced by Plasmodium falciparum

    PubMed Central

    Wassmer, Samuel C.; Combes, Valéry; Candal, Francisco J.; Juhan-Vague, Irène; Grau, Georges E.

    2006-01-01

    Brain lesions of cerebral malaria (CM) are characterized by a sequestration of Plasmodium falciparum-parasitized red blood cells (PRBC) and platelets within brain microvessels, as well as by blood-brain barrier (BBB) disruption. In the present study, we evaluated the possibility that PRBC and platelets induce functional alterations in brain endothelium. In a human brain endothelial cell line, named HBEC-5i, exhibiting most of the features demanded for a pathophysiological study of BBB, tumor necrosis factor (TNF) or lymphotoxin α (LT-α) reduced transendothelial electrical resistance (TEER), enhanced the permeability to 70-kDa dextran, and increased the release of microparticles, a recently described indicator of disease severity in CM patients. In vitro cocultures showed that platelets or PRBC can have a direct cytotoxic effect on activated, but not on resting, HBEC-5i cells. Platelet binding was required, as platelet supernatant had no effect. Furthermore, platelets potentiated the cytotoxicity of PRBC for TNF- or LT-α-activated HBEC-5i cells when they were added prior to these cells on the endothelial monolayers. This effect was not observed when platelets were added after PRBC. Both permeability and TEER were strongly affected, and the apoptosis rate of HBEC-5i cells was dramatically increased. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. PMID:16369021

  3. The periodicity of Plasmodium vivax and Plasmodium falciparum in Venezuela.

    PubMed

    Grillet, María-Eugenia; El Souki, Mayida; Laguna, Francisco; León, José Rafael

    2014-01-01

    We investigated the periodicity of Plasmodium vivax and P. falciparum incidence in time-series of malaria data (1990-2010) from three endemic regions in Venezuela. In particular, we determined whether disease epidemics were related to local climate variability and regional climate anomalies such as the El Niño Southern Oscillation (ENSO). Malaria periodicity was found to exhibit unique features in each studied region. Significant multi-annual cycles of 2- to about 6-year periods were identified. The inter-annual variability of malaria cases was coherent with that of SSTs (ENSO), mainly at temporal scales within the 3-6 year periods. Additionally, malaria cases were intensified approximately 1 year after an El Niño event, a pattern that highlights the role of climate inter-annual variability in the epidemic patterns. Rainfall mediated the effect of ENSO on malaria locally. Particularly, rains from the last phase of the season had a critical role in the temporal dynamics of Plasmodium. The malaria-climate relationship was complex and transient, varying in strength with the region and species. By identifying temporal cycles of malaria we have made a first step in predicting high-risk years in Venezuela. Our findings emphasize the importance of analyzing high-resolution spatial-temporal data to better understand malaria transmission dynamics. PMID:24149288

  4. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

    Smythe, J.A.; Coppel, R.L.; Brown, G.V.; Ramasamy, R.; Kemp, D.J.; Anders, R.F. )

    1988-07-01

    The authors describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage {lambda}gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a M{sub r} 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a M{sub r} 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with ({sup 3}H)glucosamine and ({sup 3}H)myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the M{sub r} 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

  5. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2016-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. PMID:24352242

  6. Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

    PubMed

    Roy, Sourav; Nagappa, Lakshmeesha K; Prahladarao, Vasudeva S; Balaram, Hemalatha

    2015-12-01

    Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) exhibits a kinetic mechanism that differs from that of the human homolog. Human HGPRT follows a steady-state ordered mechanism, wherein PRPP binding precedes the binding of hypoxanthine/guanine and release of product IMP/GMP is the rate limiting step. In the current study, initial velocity kinetics with PfHGXPRT indicates a steady-state ordered mechanism, wherein xanthine binding is conditional to the binding of PRPP. The value of the rate constant for IMP dissociation is greater by 183-fold than the kcat for hypoxanthine phosphoribosylation and this results in the absence of burst in progress curves from pre-steady-state kinetics. Further, IMP binding is 1000 times faster (4s(-1) at 0.5μM IMP) when compared to the kcat (3.9±0.2×10(-3)s(-1)) for the reverse IMP pyrophosphorolysis reaction. These results lend support to the fact that in both forward and reverse reactions, the process of chemical conversion (formation of IMP/hypoxanthine) is slow and the events of ligand association and dissociation are faster. PMID:26902413

  7. Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area

    PubMed Central

    Gama, Bianca Ervatti; de Oliveira, Natália K Almeida; Zalis, Mariano G; de Souza, José Maria; Santos, Fátima; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

    2009-01-01

    Background The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. Methods Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. Results Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. Conclusion This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed. PMID:19602248

  8. Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

    PubMed Central

    Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

    2014-01-01

    The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

  9. Host iron status and iron supplementation mediate susceptibility to erythrocytic stage Plasmodium falciparum.

    PubMed

    Clark, Martha A; Goheen, Morgan M; Fulford, Anthony; Prentice, Andrew M; Elnagheeb, Marwa A; Patel, Jaymin; Fisher, Nancy; Taylor, Steve M; Kasthuri, Raj S; Cerami, Carla

    2014-01-01

    Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria. PMID:25059846

  10. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion **

    PubMed Central

    Egan, Elizabeth S.; Jiang, Rays H.Y.; Moechtar, Mischka A.; Barteneva, Natasha S.; Weekes, Michael P.; Nobre, Luis V.; Gygi, Steven P.; Paulo, Joao A.; Frantzreb, Charles; Tani, Yoshihiko; Takahashi, Junko; Watanabe, Seishi; Goldberg, Jonathan; Paul, Aditya S.; Brugnara, Carlo; Root, David E.; Wiegand, Roger C.; Doench, John G.; Duraisingh, Manoj T.

    2015-01-01

    Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, precluding genetic manipulation in the cell where the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis. PMID:25954012

  11. Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

    NASA Astrophysics Data System (ADS)

    Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

    The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

  12. Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets

    PubMed Central

    2014-01-01

    Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets. PMID:24799897

  13. Antibodies to Pf155, a major antigen of Plasmodium falciparum: seroepidemiological studies in Haiti*

    PubMed Central

    Deloron, P.; Duverseau, Y. T.; Zevallos-Ipenza, A.; Magloire, R.; Stanfill, P. S.; Nguyen-Dinh, Phuc

    1987-01-01

    The presence of malaria parasites and the serological antibody responses against whole Plasmodium falciparum and the Pf155 antigen were studied in the population of a small rural locality in Haiti in December 1985. Only 7 (1.5%) of the individuals were found to be infected with P. falciparum, the only species observed. Antibodies to P. falciparum were detected in an ELISA in 38.2% of the sera, the positivity rates being age-related. Anti-Pf155 antibodies were detected in 12.5% and 13.6% of individuals by two different techniques used. The anti-Pf155 positivity rates increased only after 25 years of age. No trends were detected for a clear-cut protective value of Pf155 antibodies against clinical malaria and further longitudinally conducted field surveys are needed to satisfactorily assess the potential protective effect of Pf155 antibodies. ImagesFig. 2 PMID:3311436

  14. A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities

    NASA Technical Reports Server (NTRS)

    Li, Tao; Glushakova, Svetlana; Zimmerberg, Joshua

    2003-01-01

    Plasmodium falciparum replicates poorly in erythrocyte densities greater than a hematocrit of 20%. A new method to culture the major malaria parasite was developed by using a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100%. P. falciparum replicated equally well at all densities studied. This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof), because high yields ( approximately 10(10) in 4 days) could be prepared with less cost and labor. Concomitantly, secreted proteins were concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8%-12% with the 3D7 strain. The finding that P. falciparum can replicate at packed erythrocyte densities suggests that this system may be useful for study of the pathogenesis of fatal cerebral malaria, of which one feature is densely packed blood cells in brain microvasculature.

  15. Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide repeat.

    PubMed

    Koenen, M; Scherf, A; Mercereau, O; Langsley, G; Sibilli, L; Dubois, P; Pereira da Silva, L; Müller-Hill, B

    Plasmodium falciparum causes malaria infections in its human host. Its wide distribution in tropical countries is a major world health problem. Before a vaccine can be produced, the identification and characterization of parasite antigens is necessary. This can be achieved by the cloning and subsequent analysis of genes coding for parasite antigens. Recently established cDNA banks allow the expression of cDNA derived from the simian parasite Plasmodium knowlesi and P. falciparum in Escherichia coli. Recombinants encoding parasite antigens have been identified by immunodetection in both banks. Two of them contain repetitive units of 11 (ref. 7) or 12 (ref. 5) amino acids. We describe here the construction of an expression bank made directly from randomly generated fragments of P. falciparum genomic DNA. We detect several clones which react strongly with human African immune sera. One clone expresses an antigenic determinant composed of occasionally degenerated repeats of a peptide nonamer. PMID:6090935

  16. Synthesis of alpha-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors.

    PubMed

    Haemers, Timothy; Wiesner, Jochen; Van Poecke, Sara; Goeman, Jan; Henschker, Dajana; Beck, Edwald; Jomaa, Hassan; Van Calenbergh, Serge

    2006-04-01

    In view of the promising antimalarial activity of fosmidomycin or its N-acetyl homologue FR900098, the objective of this work was to investigate the influence of aromatic substituents in the alpha-position of the phosphonate moiety. The envisaged analogues were prepared using a linear route involving a 3-aryl-3-phosphoryl propanal intermediate. The activities of all compounds were evaluated on Eschericia coli 1-deoxy-d-xylulose 5-phosphate reductoisomerase and against two Plasmodium falciparum strains. Compared with fosmidomycin, several analogues displayed enhanced activity towards the P. falciparum strains. Compound 1e with a 3,4-dichlorophenyl substitution in the alpha-position of fosmidomycin emerged as the most potent analogue of this series. It is approximately three times more potent in inhibiting the growth of P. falciparum than FR900098, the most potent representative of this class reported so far. PMID:16439126

  17. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    SciTech Connect

    Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.; Clinch, Keith; Crump, Douglas R.; Rosario Jr., Irving; Merino, Emilio F.; Almo, Steve C.; Tyler, Peter C.; Schramm, Vern L.

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  18. Identification and initial characterisation of a Plasmodium falciparum Cox17 copper metallochaperone.

    PubMed

    Choveaux, David L; Krause, Robert G E; Przyborski, Jude M; Goldring, J P Dean

    2015-01-01

    Copper is an essential micronutrient for all living organisms as an important catalytic co-factor for key enzymes. In higher eukaryotes intracellular copper is distributed by copper metallochaperones. Copper chelators such as neocuproine and tetrathiomolybdate inhibit Plasmodium falciparum erythrocytic development, indicating a requirement for copper by the parasite. A screen of the P. falciparum genome database identified eight potential copper-requiring protein orthologs, including four candidate copper metallochaperones implicated in the delivery of copper to cytochrome-c oxidase. A P. falciparum Cox17 ortholog (PfCox17) was recombinantly expressed and the purified protein bound reduced copper in vitro. PfCox17 was localised to the parasite cytoplasm. Characterisation of plasmodial proteins involved in copper metabolism will help us understand the role of this essential microelement in plasmodial homeostasis. PMID:25447123

  19. Identification of a novel aminopeptidase P-like gene (OnAPP) possibly involved in Bt toxicity and resistance in a major corn pest (Ostrinia nubilalis).

    PubMed

    Khajuria, Chitvan; Buschman, Lawrent L; Chen, Ming-Shun; Siegfried, Blair D; Zhu, Kun Yan

    2011-01-01

    Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu(305) to Lys(305) and Arg(307) to Leu(307) in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains. PMID:21887358

  20. Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

    PubMed Central

    Wang, Shan-Qing; Wang, Guang-Ze; Li, Yu-Chun; Meng, Feng; Lin, Shi-Gan; Zhu, Zhen-Hu; Sun, Ding-Wei; He, Chang-Hua; Hu, Xi-Min; Du, Jian-Wei

    2015-01-01

    Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77×10-6 mol/L, 2.09×10-6 mol/L, 0.09×10-6 mol/L, and 0.05×10-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10-6 mol/L, 9.26×10-6 mol/L, 0.55×10-6 mol/L, and 0.07×10-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity. PMID:25748707

  1. Maternal-foetal transfer of Plasmodium falciparum and Plasmodium vivax antibodies in a low transmission setting.

    PubMed

    Charnaud, Sarah C; McGready, Rose; Herten-Crabb, Asha; Powell, Rosanna; Guy, Andrew; Langer, Christine; Richards, Jack S; Gilson, Paul R; Chotivanich, Kesinee; Tsuboi, Takafumi; Narum, David L; Pimanpanarak, Mupawjay; Simpson, Julie A; Beeson, James G; Nosten, François; Fowkes, Freya J I

    2016-01-01

    During pregnancy immunolglobulin G (IgG) antibodies are transferred from mother to neonate across the placenta. Studies in high transmission areas have shown transfer of P. falciparum-specific IgG, but the extent and factors influencing maternal-foetal transfer in low transmission areas co-endemic for both P. falciparum and P. vivax are unknown. Pregnant women were screened weekly for Plasmodium infection. Mother-neonate paired serum samples at delivery were tested for IgG to antigens from P. falciparum, P. vivax and other infectious diseases. Antibodies to malarial and non-malarial antigens were highly correlated between maternal and neonatal samples (median [range] spearman ρ = 0.78 [0.57-0.93]), although Plasmodium spp. antibodies tended to be lower in neonates than mothers. Estimated gestational age at last P. falciparum infection, but not P. vivax infection, was positively associated with antibody levels in the neonate (P. falciparum merozoite, spearman ρ median [range] 0.42 [0.33-0.66], PfVAR2CSA 0.69; P. vivax ρ = 0.19 [0.09-0.3]). Maternal-foetal transfer of anti-malarial IgG to Plasmodium spp. antigens occurs in low transmission settings. P. vivax IgG acquisition is not associated with recent exposure unlike P. falciparum IgG, suggesting a difference in acquisition of antibodies. IgG transfer is greatest in the final weeks of pregnancy which has implications for the timing of future malaria vaccination strategies in pregnant women. PMID:26861682

  2. Maternal-foetal transfer of Plasmodium falciparum and Plasmodium vivax antibodies in a low transmission setting

    PubMed Central

    Charnaud, Sarah C.; McGready, Rose; Herten-Crabb, Asha; Powell, Rosanna; Guy, Andrew; Langer, Christine; Richards, Jack S.; Gilson, Paul R.; Chotivanich, Kesinee; Tsuboi, Takafumi; Narum, David L.; Pimanpanarak, Mupawjay; Simpson, Julie A.; Beeson, James G.; Nosten, François; Fowkes, Freya J. I.

    2016-01-01

    During pregnancy immunolglobulin G (IgG) antibodies are transferred from mother to neonate across the placenta. Studies in high transmission areas have shown transfer of P. falciparum-specific IgG, but the extent and factors influencing maternal-foetal transfer in low transmission areas co-endemic for both P. falciparum and P. vivax are unknown. Pregnant women were screened weekly for Plasmodium infection. Mother-neonate paired serum samples at delivery were tested for IgG to antigens from P. falciparum, P. vivax and other infectious diseases. Antibodies to malarial and non-malarial antigens were highly correlated between maternal and neonatal samples (median [range] spearman ρ = 0.78 [0.57–0.93]), although Plasmodium spp. antibodies tended to be lower in neonates than mothers. Estimated gestational age at last P. falciparum infection, but not P. vivax infection, was positively associated with antibody levels in the neonate (P. falciparum merozoite, spearman ρ median [range] 0.42 [0.33–0.66], PfVAR2CSA 0.69; P. vivax ρ = 0.19 [0.09–0.3]). Maternal-foetal transfer of anti-malarial IgG to Plasmodium spp. antigens occurs in low transmission settings. P. vivax IgG acquisition is not associated with recent exposure unlike P. falciparum IgG, suggesting a difference in acquisition of antibodies. IgG transfer is greatest in the final weeks of pregnancy which has implications for the timing of future malaria vaccination strategies in pregnant women. PMID:26861682

  3. Impact of enhanced malaria control on the competition between Plasmodium falciparum and Plasmodium vivax in India.

    PubMed

    Prosper, Olivia; Martcheva, Maia

    2013-03-01

    The primary focus of malaria research and control has been on Plasmodium falciparum, the most severe of the four Plasmodium species causing human disease. However, the presence of both P. falciparum and Plasmodium vivax occurs in several countries, including India. We developed a mathematical model describing the dynamics of P. vivax and P. falciparum in the human and mosquito populations and fit this model to Indian clinical case data to understand how enhanced control measures affect the competition between the two Plasmodium species. Around 1997, funding for malaria control in India increased dramatically. Our model predicts that if India had not improved its control strategy, the two species of Plasmodium would continue to coexist. To determine which control measures contributed the most to the decline in the number of cases after 1997, we compared the fit of seven models to the 1997-2010 clinical case data. From this, we determined that increased use of bednets contributed the most to case reduction. During the enhanced control period, the best model predicts that P. vivax is out-competing P. falciparum. However, the reproduction numbers are extremely close to the invasion boundaries. Consequently, we cannot be confident that this outcome is the true future of malaria in India. We address this uncertainty by performing a parametric bootstrapping procedure for each of the seven models. This procedure, applied to the enhanced control period, revealed that the best model predicts that P. vivax outcompeting P. falciparum is the most likely outcome, whereas the remaining candidate models predict the opposite. Moreover, the predictions of the top model are counter to what one expects based on the case data alone. Although the proportion of cases due to falciparum has been increasing, the best fitting model reveals that this observation is insufficient to draw conclusions about the longterm competitive outcome of the two species. PMID:23261665

  4. Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

    PubMed

    Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

    2013-06-01

    Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. PMID:23615908

  5. Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum*

    PubMed Central

    Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luis

    2013-01-01

    Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions. PMID:23615908

  6. A Lectin-Like Receptor is Involved in Invasion of Erythrocytes by Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Jungery, M.; Pasvol, G.; Newbold, C. I.; Weatherall, D. J.

    1983-02-01

    Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

  7. Simple Molecular Methods for Early Detection of Chloroquine Drug Resistance in Plasmodium vivax and Plasmodium falciparum

    PubMed Central

    Singh, Raksha; Urhehar, Anant Dattatraya

    2016-01-01

    Introduction Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. Aim To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. Materials and Methods This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman’s method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. Results Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. Conclusion Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with

  8. Extraction of Hydrophilic Metabolites from Plasmodium falciparum-Infected Erythrocytes for Metabolomic Analysis

    PubMed Central

    Olszewski, Kellen L.; Llinás, Manuel

    2012-01-01

    Metabolomics is an increasingly common analytical approach for investigating metabolic networks of pathogenic organisms. This may be of particular use in the study of parasitic infections due to the intrinsic metabolic connection between the parasite and its host. In vitro cultures of the malaria parasite Plasmodium falciparum present a valuable platform to elucidate the structure and dynamics of the parasite’s metabolic network and to determine the mechanisms of action of antimalarial drugs and drug resistance mutations. Accurately measuring metabolite levels requires a reproducible method for quantifying intracellular metabolites. Here we present a simple protocol for extracting hydrophilic metabolites from P. falciparum-infected erythrocyte cultures. PMID:22990783

  9. Synthesis and in vitro evaluation of hydrazinyl phthalazines against malaria parasite, Plasmodium falciparum.

    PubMed

    Subramanian, Gowtham; Babu Rajeev, C P; Mohan, Chakrabhavi Dhananjaya; Sinha, Ameya; Chu, Trang T T; Anusha, Sebastian; Ximei, Huang; Fuchs, Julian E; Bender, Andreas; Rangappa, Kanchugarakoppal S; Chandramohanadas, Rajesh; Basappa

    2016-07-15

    In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liquids and demonstrate their ability to inhibit asexual stage development of human malaria parasite, Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects. These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel antimalarials. PMID:27261180

  10. Identification of Potent and Selective Non-covalent Inhibitors of the Plasmodium falciparum Proteasome

    PubMed Central

    2015-01-01

    We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host. PMID:25226494

  11. Synergistic Mortality Caused by Plasmodium falciparum during the 1918 Influenza Pandemic

    PubMed Central

    Shanks, G. Dennis

    2015-01-01

    At the end of World War I, British medical officers noted that soldiers infected with malaria were more likely to die during the 1918 influenza pandemic than those without malaria. This synergistic mortality appeared to be specific to Plasmodium falciparum and has not been generally noted since 1920. A possible explanation is that a malaria-induced procoagulant state enhanced the activation of influenza virus to increase inflammation and subsequent severe clinical outcomes. Falciparum proteins bind and likely inhibit antithrombin 3 and other factors. Pathogens interact in ways that may inform pathophysiology studies of remote epidemics. PMID:25802427

  12. Adaptation of the Panama II strain of Plasmodium falciparum to Panamanian owl monkeys.

    PubMed

    Rossan, R N; Baerg, D C

    1987-09-01

    The Panama II strain of Plasmodium falciparum, acquired at the second passage level in splenectomized Colombian owl monkeys, was adapted to owl monkeys of Panamanian origin. Patent infections were induced in 22 of 27 unaltered and 20 of 21 splenectomized recipients during 19 serial passages. The infections were significantly more virulent in splenectomized than normal Panamanian owl monkeys, however recrudescences in seven normal monkeys achieved peak parasitemias 48 times greater than in the primary attack. These results describe the first reproducible infections of indigenous falciparum malaria in Panamanian owl monkeys. PMID:3310680

  13. Synthesis and biological evaluation of cyclopropyl analogues of fosmidomycin as potent Plasmodium falciparum growth inhibitors.

    PubMed

    Devreux, Vincent; Wiesner, Jochen; Goeman, Jan L; Van der Eycken, Johan; Jomaa, Hassan; Van Calenbergh, Serge

    2006-04-20

    A series of fosmidomycin analogues featuring restricted conformational mobility has been synthesized and evaluated as inhibitors of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase and as growth inhibitors of P. falciparum. The enantiomerically pure trans-cyclopropyl N-acetyl analogue 3b showed comparable inhibitory activity as fosmidomycin toward E. coli DOXP reductoisomerase and proved equally active when tested in vitro for P. falciparum growth inhibition. Conversely, the alpha-phenyl cis-cyclopropyl analogue 4 showed virtually no inhibition of the enzyme. PMID:16610809

  14. Asexual and sexual stages of Plasmodium falciparum in Nigerian pregnant women attending antenatal booking clinic

    PubMed Central

    Balogun, S T; Fehintola, F A; Adeyanju, O A; Adedeji, A A

    2010-01-01

    Susceptibility to infection by Plasmodium falciparum is increased in pregnant women. In sub-Saharan Africa, the consequences of maternal malaria include preterm birth, fetal growth restriction and increased infant mortality. Malaria transmission requires the circulation of viable gametocytes that can be ingested by the female mosquito taking a blood meal. This study was conducted to evaluate the presence of asexual and sexual stages of P. falciparum in pregnant women attending antenatal booking clinics in south-western Nigeria, an area hyper-endemic for malaria. Gametocyte carriage was about 13%, similar to that documented for children symptomatic for malaria in our area of study.

  15. Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of leucine aminopeptidase (LAP) from the pepA gene of Xanthomonas oryzae pv. oryzae

    PubMed Central

    Huynh, Kim-Hung; Natarajan, Sampath; Choi, Jeongyoon; Song, Na-Hyun; Kim, Jeong-Gu; Lee, Byoung-Moo; Ahn, Yeh-Jin; Kang, Lin-Woo

    2009-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 Å resolution and belonged to the cubic space group P213. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit. PMID:19724142

  16. Development of 2-thioxoquinazoline-4-one derivatives as dual and selective inhibitors of dynamin-related protein 1 (Drp1) and puromycin-sensitive aminopeptidase (PSA).

    PubMed

    Numadate, Akiyoshi; Mita, Yusuke; Matsumoto, Yotaro; Fujii, Shinya; Hashimoto, Yuichi

    2014-01-01

    An established inhibitor of dynamin-related protein 1 (Drp1), 3-(2,4-dichloro-5-methoxyphenyl)-2-thioxoquinazoline-4-one (mdivi-1), was recently reported also to show potent puromycin-sensitive aminopeptidase (PSA)-inhibitory activity. Herein, we report structural development of mdivi-1 derivatives and structure-activity relationship (SAR) analysis of the synthesized compounds, as well as the structurally related PSA-specific inhibitor 3-(2,6-diethylphenyl)quinazoline-2,4-dione (PAQ-22), with the aim of identifying key structural features for inhibitory activity in order to develop selective inhibitors of Drp1, which is a potential target for treatment of Huntington's disease. Among the synthesized compounds, 3-(4-chloro-3-methoxyphenyl)-2-thioxoquinazoline-4-one (10g) exhibited more potent Drp1-inhibitory activity than mdivi-1 with high selectivity for Drp1 over PSA. PMID:25273056

  17. Endoplasmic reticulum aminopeptidase 1 function and its pathogenic role in regulating innate and adaptive immunity in cancer and major histocompatibility complex class I-associated autoimmune diseases.

    PubMed

    Fruci, D; Romania, P; D'Alicandro, V; Locatelli, F

    2014-08-01

    Major histocompatibility complex (MHC) class I molecules present antigenic peptides on the cell surface to alert natural killer (NK) cells and CD8(+) T cells for the presence of abnormal intracellular events, such as virus infection or malignant transformation. The generation of antigenic peptides is a multistep process that ends with the trimming of N-terminal extensions in the endoplasmic reticulum (ER) by aminopeptidases ERAP1 and ERAP2. Recent studies have highlighted the potential role of ERAP1 in reprogramming the immunogenicity of tumor cells in order to elicit innate and adaptive antitumor immune responses, and in conferring susceptibility to autoimmune diseases in predisposed individuals. In this review, we will provide an overview of the current knowledge about the role of ERAP1 in MHC class I antigen processing and how its manipulation may constitute a promising tool for cancer immunotherapy and treatment of MHC class I-associated autoimmune diseases. PMID:25066018

  18. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc{gamma} receptors CD32 and CD64

    SciTech Connect

    Riemann, Dagmar; Wulfaenger, Jens

    2005-06-17

    Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc{gamma} receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc{gamma} receptor I/CD64, but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc{gamma} receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.

  19. Binding of phylogenetically distant Bacillus thuringiensis cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in receptor binding.

    PubMed

    Shinkawa, A; Yaoi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-07-01

    We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. PMID:10387111

  20. Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

    PubMed

    Nakanishi, K; Yaoi, K; Shimada, N; Kadotani, T; Sato, R

    1999-06-15

    Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin. PMID:10366728

  1. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    PubMed

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN. PMID:9931470

  2. Therapeutic efficacy test in malaria falciparum in Antioquia, Colombia

    PubMed Central

    Blair, Silvia; Carmona-Fonseca, Jaime; Piñeros, Juan G; Ríos, Alexandra; Álvarez, Tania; Álvarez, Gonzalo; Tobón, Alberto

    2006-01-01

    Objective Evaluate the frequency of failure of eight treatments for non-complicated malaria caused by Plasmodium falciparum in patients from Turbo (Urabá region), El Bagre and Zaragoza (Bajo Cauca region), applying the 1998 protocol of the World Health Organization (WHO). Monotherapies using chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ) and sulphadoxine-pyrimethamine (SP), and combinations using chloroquine-sulphadoxine-pyrimethamine (CQ-SP), amodiaquine-sulphadoxine-pyrimethamine (AQ-SP), mefloquine-sulphadoxine-pyrimethamine (MQ-SP) and artesunate-sulphadoxine-pyrimethamine (AS-SP), were examined. Methodology A balanced experimental design with eight groups. Samples were selected based on statistical and epidemiological criteria. Patients were followed for 21 to 28 days, including seven or eight parasitological and clinical evaluations, with an active search for defaulting patients. A non-blinded evaluation of the antimalarial treatment response (early failure, late failure, adequate response) was performed. Results Initially, the loss of patients to follow-up was higher than 40%, but the immediate active search for the cases and the monetary help for transportation expenses of patients, reduced the loss to 6%. The treatment failure was: CQ 82%, AQ 30%, MQ 4%, SP 24%, CQ-SP 17%, AQ-SP 2%, MQ-S-P 0%, AS-SP 3%. Conclusion The characteristics of an optimal epidemiological monitoring system of antimalarial treatment response in Colombia are discussed. It is proposed to focus this on early failure detection, by applying a screening test every two to three years, based on a seven to 14-day follow-up. Clinical and parasitological assessment would be carried out by a general physician and a field microscopist from the local hospital, with active measures to search for defaulter patients at follow-up. PMID:16504002

  3. Functional Analysis of Sirtuin Genes in Multiple Plasmodium falciparum Strains

    PubMed Central

    Merrick, Catherine J.; Jiang, Rays H. Y.; Skillman, Kristen M.; Samarakoon, Upeka; Moore, Rachel M.; Dzikowski, Ron; Ferdig, Michael T.; Duraisingh, Manoj T.

    2015-01-01

    Plasmodium falciparum, the causative agent of severe human malaria, employs antigenic variation to avoid host immunity. Antigenic variation is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. The histone-modifying ‘sirtuin’ enzymes PfSir2a and PfSir2b have been implicated in this process. Disparate patterns of var expression have been reported in patient isolates as well as in cultured strains. We examined var expression in three commonly used laboratory strains (3D7, NF54 and FCR-3) in parallel. NF54 parasites express significantly lower levels of var genes compared to 3D7, despite the fact that 3D7 was originally a clone of the NF54 strain. To investigate whether this was linked to the expression of sirtuins, genetic disruption of both sirtuins was attempted in all three strains. No dramatic changes in var gene expression occurred in NF54 or FCR-3 following PfSir2b disruption, contrasting with previous observations in 3D7. In 3D7, complementation of the PfSir2a genetic disruption resulted in a significant decrease in previously-elevated var gene expression levels, but with the continued expression of multiple var genes. Finally, rearranged chromosomes were observed in the 3D7 PfSir2a knockout line. Our results focus on the potential for parasite genetic background to contribute to sirtuin function in regulating virulence gene expression and suggest a potential role for sirtuins in maintaining genome integrity. PMID:25780929

  4. Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase.

    PubMed

    Karmakar, Tarak; Roy, Sourav; Balaram, Hemalatha; Prakash, Meher K; Balasubramanian, Sundaram

    2016-08-22

    Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of the Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP, which triggers the opening of flexible loop II, the gateway to the bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to be released via the same path in all of the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from the active site; however, an alternate but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP, whereas the motion of PPi.2Mg away from the reaction center is guided by the negatively charged Asp and Glu and a few positively charged residues (Lys and Arg) that line the product release channels. Mutations of a few key residues present in loop II of Trypanosoma cruzi (Tc) HGPRT have been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT resulted in partial opening of the flexible loop (loop II), thus exposing the active site to bulk water, which offers a rationale for the reduced catalytic activity of these two mutant enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover. PMID:27404508

  5. Crystal structure of truncated aspartate transcarbamoylase from Plasmodium falciparum.

    PubMed

    Lunev, Sergey; Bosch, Soraya S; Batista, Fernando de Assis; Wrenger, Carsten; Groves, Matthew R

    2016-07-01

    The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure of PfATC is reported. The space group of the PfATC crystals was determined to be monoclinic P21, with unit-cell parameters a = 87.0, b = 103.8, c = 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presented PfATC model shares a high degree of homology with the catalytic domain of Escherichia coli ATC. There is as yet no evidence of the existence of a regulatory domain in PfATC. Similarly to E. coli ATC, PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described. PMID:27380369

  6. Calmidazolium evokes high calcium fluctuations in Plasmodium falciparum.

    PubMed

    Budu, Alexandre; Gomes, Mayrim M; Melo, Pollyana M; El Chamy Maluf, Sarah; Bagnaresi, Piero; Azevedo, Mauro F; Carmona, Adriana K; Gazarini, Marcos L

    2016-03-01

    Calcium and calmodulin (CaM) are important players in eukaryote cell signaling. In the present study, by using a knockin approach, we demonstrated the expression and localization of CaM in all erythrocytic stages of Plasmodium falciparum. Under extracellular Ca(2+)-free conditions, calmidazolium (CZ), a potent CaM inhibitor, promoted a transient cytosolic calcium ([Ca(2+)]cyt) increase in isolated trophozoites, indicating that CZ mobilizes intracellular sources of calcium. In the same extracellular Ca(2+)-free conditions, the [Ca(2+)]cyt rise elicited by CZ treatment was ~3.5 fold higher when the endoplasmic reticulum (ER) calcium store was previously depleted ruling out the mobilization of calcium from the ER by CZ. The effects of the Ca(2+)/H(+) ionophore ionomycin (ION) and the Na(+)/H(+) ionophore monensin (MON) suggest that the [Ca(2+)]cyt-increasing effect of CZ is driven by the removal of Ca(2+) from at least one Ca(2+)-CaM-related (CaMR) protein as well as by the mobilization of Ca(2+) from intracellular acidic calcium stores. Moreover, we showed that the mitochondrion participates in the sequestration of the cytosolic Ca(2+) elicited by CZ. Finally, the modulation of membrane Ca(2+) channels by CZ and thapsigargin (THG) was demonstrated. The opened channels were blocked by the unspecific calcium channel blocker Co(2+) but not by 2-APB (capacitative calcium entry inhibitor) or nifedipine (L-type Ca(2+) channel inhibitor). Taken together, the results suggested that one CaMR protein is an important modulator of calcium signaling and homeostasis during the Plasmodium intraerythrocytic cell cycle, working as a relevant intracellular Ca(2+) reservoir in the parasite. PMID:26689736

  7. Structure-Guided, Single-Point Modifications in the Phosphinic Dipeptide Structure Yield Highly Potent and Selective Inhibitors of Neutral Aminopeptidases

    DOE PAGESBeta

    Vassiliou, Stamatia; Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Mulligan, Rory; Joachimiak, Andrzej; Mucha, Artur

    2014-10-09

    Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor–enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extendedmore » structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. In conclusion, another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π–π stacking interaction between a pyridine ring and Tyr372.« less

  8. Food-restricted and dehydrated-induced anorexic rats present differential TRH expression in anterior and caudal PVN. Role of type 2 deiodinase and pyroglutamyl aminopeptidase II.

    PubMed

    Alvarez-Salas, E; Aceves, C; Anguiano, B; Uribe, R M; García-Luna, C; Sánchez, E; de Gortari, P

    2012-08-01

    TRH synthesized in hypothalamic paraventricular nucleus (PVN) regulates thyroid axis function and is also implicated in anorexigenic effects. Under energy deficit, animals present decreased PVN TRH expression and release, low TSH levels, and increased appetite. Dehydration-induced anorexia (DIA) model allows insight into underlying mechanisms of feeding regulation. Animals drinking a 2.5% NaCl solution for 7 d present body weight reduction; despite their negative energy balance, they avoid food and have increased PVN TRH expression and TSH serum levels. These findings support an inhibiting role of PVN TRH in feeding control. We compared TRH expression by in situ hybridization in PVN subdivisions of 7-d dehydrated male rats to those of a pair-fed group (forced food-restricted) with similar metabolic changes than DIA, but motivated to eat, and to controls. We measured peripheral deiodinase activities, and expression and activity of medial basal hypothalamic type 2 deiodinase and pyroglutamyl-aminopeptidase II, to understand their regulating role in PVN TRH changes between food restriction and anorexia. TRH mRNA levels increased in anterior (aPVN) and medial-caudal subdivisions in DIA rats, whereas it decreased in medial PVN in both experimental groups. We confirmed the nonhypophysiotropic nature of aPVN TRHergic cells by injecting ip fluorogold tracer. Findings support a subspecialization of TRHergic hypophysiotrophic cells that responded differently between anorexic and food-restricted animals; also, that aPVN TRH participates in food intake regulation. Increased type 2 deiodinase activity seemed responsible for low medial PVN TRH synthesis, whereas increased medial basal hypothalamic pyroglutamyl-aminopeptidase II activity in DIA rats might counteract their high TRH release. PMID:22719053

  9. The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

    PubMed

    Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

    2016-07-19

    It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell. PMID:27225936

  10. Identification and characterization of CaApe2--a neutral arginine/alanine/leucine-specific metallo-aminopeptidase from Candida albicans.

    PubMed

    Klinke, Thomas; Rump, Andreas; Pönisch, Roman; Schellenberger, Wolfgang; Müller, Eva-Christina; Otto, Albrecht; Klimm, Wolfgang; Kriegel, Thomas M

    2008-09-01

    The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2, which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. Taking into account the results of DNA and protein sequence analysis including inspection of the genomic region upstream of ORF CaO19.5197, the gene CaAPE2 is likely to consist of two exons linked by a phase-2 intron with exons 1 and 2 encoding a signal peptide and the amino acids 88-954 of ORF CaO19.5197, respectively. The isolated CaApe2 protein shares an equally high similarity with the gene products ScAap1 and ScApe2, suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role that secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans. PMID:18637841

  11. The type II secretion system of Legionella pneumophila elaborates two aminopeptidases, as well as a metalloprotease that contributes to differential infection among protozoan hosts.

    PubMed

    Rossier, Ombeline; Dao, Jenny; Cianciotto, Nicholas P

    2008-02-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts. PMID:18083880

  12. Associations Between Helminth Infections, Plasmodium falciparum Parasite Carriage and Antibody Responses to Sexual and Asexual Stage Malarial Antigens.

    PubMed

    Ateba-Ngoa, Ulysse; Jones, Sophie; Zinsou, Jeannot Fréjus; Honkpehedji, Josiane; Adegnika, Ayola Akim; Agobe, Jean-Claude Dejon; Massinga-Loembe, Marguerite; Mordmüller, Benjamin; Bousema, Teun; Yazdanbakhsh, Maria

    2016-08-01

    Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity. PMID:27273645

  13. Leucine aminopeptidase blood test

    MedlinePlus

    ... RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 21. Pratt DS. Liver chemistry and function tests. In: Feldman M, Friedman LS, Brandt LJ, ... 10th ed. Philadelphia, PA: Elsevier Saunders; 2016:chap ...

  14. Evaluation of the vapor-protection capabilities of the M17 respirator/hood assembly on the USAF ground-crew chemical defense ensemble. Interim report, 21 March-2 June 1988

    SciTech Connect

    Scott, W.R.; Simpson, R.E.

    1989-10-01

    The hood used with the M17 respirator on the regulation United States Air Force (USAF) ground-crew chemical defense ensemble (CDE) may have deficiencies, both in construction and usage, in its ability to protect the neck from chemical agent vapors. The purpose of this study was to quantify vapor penetration under the hood skirt to the neck and measure the effect of this vapor penetration on vapor carry-through into the Toxic Free Area (TFA) of the Survivable Collective Protection Shelter Contamination Control Area (SCPS-2B CCA) facility at Brooks AFB, Texas. Test subjects, wearing the regulation ground-crew CDE, performed light exercises in a simulant vapor (methyl salicylate). Vapor concentrations were measured at the neck with Tenax tubes and in the SCPS-2B with sequential impingers. When the hood skirt was worn outside the CDE jacket, the standard configuration, the mean simulant vapor level at the neck was 29.6% of the outside vapor concentration. Placing the hood skirt underneath the CDE jacket resulted in an 87% decrease in the neck vapor concentrations and a 50% reduction in TFA vapor carry-through.

  15. No Clinical or Molecular Evidence of Plasmodium falciparum Resistance to Artesunate-Mefloquine in Northwestern Brazil.

    PubMed

    Ladeia-Andrade, Simone; de Melo, Gladson Naber P; de Souza-Lima, Rita de Cássia; Salla, Laís C; Bastos, Melissa S; Rodrigues, Priscila T; Luz, Francisco das Chagas O; Ferreira, Marcelo U

    2016-07-01

    We evaluated the clinical efficacy of artesunate-mefloquine (ASMQ) fixed-dose combination to treat uncomplicated malaria in Juruá Valley, the main Plasmodium falciparum transmission hotspot in Brazil. Between November 2010 and February 2013, we enrolled 162 patients aged 4-73 years, with fever or a history of fever, and a single-species P. falciparum infection confirmed by microscopy and polymerase chain reaction (PCR). All 154 patients who completed the 42-day follow-up presented an adequate clinical and parasitologic response. ASMQ was well tolerated and no adverse event caused treatment interruption. Gametocytes were detected in 46.3% patients; 35.2% had gametocytes at enrollment, whereas others developed patent gametocytemia 1-14 days after starting ASMQ. By day 3 of treatment, all subjects had cleared asexual parasitemia, but parasite DNA remained PCR detectable in 37.6% of them. Day-3 PCR positivity was associated with prolonged gametocyte carriage. We found no molecular evidence of resistance to either MQ (pfmdr1 gene amplification) or AS (mutations in selected kelch13 gene domains known to be associated with AS resistance) in the local P. falciparum population. These results strongly support the use of ASMQ as a first-line regimen to treat uncomplicated P. falciparum malaria in northwestern Brazil, but underscore the need for gametocytocidal drugs to reduce the transmission potential of ASMQ-treated patients (ClinicalTrials.gov number NCT01144702). PMID:27068396

  16. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    PubMed

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria. PMID:27262062

  17. Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

    PubMed

    Kilunga Kubata, B; Eguchi, N; Urade, Y; Yamashita, K; Mitamura, T; Tai, K; Hayaishi, O; Horii, T

    1998-09-21

    Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum. PMID:9743538

  18. Plasmodium falciparum epigenome: A distinct dynamic epigenetic regulation of gene expression

    PubMed Central

    Rawat, Mukul; Bhosale, Madhvi A.; Karmodiya, Krishanpal

    2015-01-01

    Histone modification profiles are predictive of gene expression and most of the knowledge gained is acquired through studies done in higher eukaryotes. However, genome-wide studies involving Plasmodium falciparum, the causative agent of malaria, have been rather few, at lower resolution (mostly using ChIP-on-chip), and covering limited number of histone modifications. In our recent study [1], we have performed extensive genome-wide analyses of multiple histone modifications including the active (H3K4me2, H3K4me3, H3K9ac, H3K14ac, H3K27ac and H4ac), inactive (H3K9me3 and H3K27me3), elongation (H3K79me3) and regulatory element (H3K4me1) in a stage-specific manner. Furthermore, we used a ligation-based method suitable for sequencing homopolymeric stretches as seen in P. falciparum for next-generation sequencing library amplification [2], enabling highly quantitative analysis of the extremely AT-rich P. falciparum genome. Our recently published study suggests that transcription regulation by virtue of poised chromatin and differential histone modifications is unique to P. falciparum [1]. Here we describe the experiments, quality controls and chromatin immunoprecipitation-sequencing data analysis of our associated study published in Epigenetics and Chromatin [1]. Stage-specific ChIP-sequencing data for histone modifications is submitted to Gene Expression Omnibus (GEO) database under the accession number GSE63369. PMID:26981368

  19. Sequestration and Red Cell Deformability as Determinants of Hyperlactatemia in Falciparum Malaria

    PubMed Central

    Ishioka, Haruhiko; Ghose, Aniruddha; Charunwatthana, Prakaykaew; Maude, Richard; Plewes, Katherine; Kingston, Hugh; Intharabut, Benjamas; Woodrow, Charlie; Chotivanich, Kesinee; Sayeed, Abdullah Abu; Hasan, Mahtab Uddin; Day, Nicholas P.; Faiz, Abul; White, Nicholas J.; Hossain, Amir; Dondorp, Arjen M.

    2016-01-01

    Background. Hyperlactatemia is a strong predictor of mortality in severe falciparum malaria. Sequestered parasitized erythrocytes and reduced uninfected red blood cell deformability (RCD) compromise microcirculatory flow, leading to anaerobic glycolysis. Methods. In a cohort of patients with falciparum malaria hospitalized in Chittagong, Bangladesh, bulk RCD was measured using a laser diffraction technique, and parasite biomass was estimated from plasma concentrations of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). A multiple linear regression model was constructed to examine their associations with plasma lactate concentrations. Results. A total of 286 patients with falciparum malaria were studied, of whom 224 had severe malaria, and 70 died. Hyperlactatemia (lactate level, ≥4 mmol/L) was present in 111 cases. RCD at shear stresses of 1.7 Pa and 30 Pa was reduced significantly in patients who died, compared with survivors, individuals with uncomplicated malaria, or healthy individuals (P < .05, for all comparisons). Multiple linear regression analysis showed that the plasma PfHRP2 level, parasitemia level, total bilirubin level, and RCD at a shear stress of 1.7 Pa were each independently correlated with plasma lactate concentrations (n = 278; R2 = 0.35). Conclusions. Sequestration of parasitized red blood cells and reduced RCD both contribute to decreased microcirculatory flow in severe disease. PMID:26494775

  20. Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids.

    PubMed

    López, Mary Luz; Vommaro, Rossiane; Zalis, Mariano; de Souza, Wanderley; Blair, Silvia; Segura, Cesar

    2010-06-01

    Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC(50) between 20-87microM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment. PMID:20153445

  1. Haemodynamic and oxygen transport response during exchange transfusion for severe falciparum malaria.

    PubMed

    Beards, S C; Joynt, G M; Lipman, J

    1994-11-01

    We describe the haemodynamic and oxygen transport response in a patient undergoing exchange transfusion for severe falciparum malaria. We found that exchange transfusion produced a significant increase in left ventricular stroke work index, systemic oxygen delivery and oxygen consumption. This potentially beneficial effect of exchange transfusion has not been reported previously. PMID:7824413

  2. Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border

    PubMed Central

    2013-01-01

    Background The Caribbean coast of Central America remains an area of malaria transmission caused by Plasmodium falciparum despite the fact that morbidity has been reduced in recent years. Parasite populations in that region show interesting characteristics such as chloroquine susceptibility and low mortality rates. Genetic structure and diversity of P. falciparum populations in the Honduras-Nicaragua border were analysed in this study. Methods Seven neutral microsatellite loci were analysed in 110 P. falciparum isolates from endemic areas of Honduras (n = 77) and Nicaragua (n = 33), mostly from the border region called the Moskitia. Several analyses concerning the genetic diversity, linkage disequilibrium, population structure, molecular variance, and haplotype clustering were conducted. Results There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (He) results were similarly low for both populations. A moderate differentiation was revealed by the FST index between both populations, and two putative clusters were defined through a structure analysis. The main cluster grouped most of samples from Honduras and Nicaragua, while the second cluster was smaller and included all the samples from the Siuna community in Nicaragua. This result could partially explain the stronger linkage disequilibrium (LD) in the parasite population from that country. These findings are congruent with the decreasing rates of malaria endemicity in Central America. PMID:24093629

  3. Plasmodium falciparum Serine/Threonine Phosphoprotein Phosphatases (PPP): From Housekeeper to 'Holy Grail'

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Availability of complete genome sequence for Plasmodium falciparum has been useful in drawing a comprehensive metabolic map of the parasite. Distinct and unique metabolic characteristics of the parasite may be exploited as potential targets for new antimalarial drug discovery research. Reversible ph...

  4. Serotypes of Plasmodium falciparum defined by immune serum inhibition of in vitro growth*

    PubMed Central

    Chulay, J. D.; Haynes, J. D.; Diggs, C. L.

    1985-01-01

    In vitro growth inhibition assays were used to detect antigenic differences among geographically distinct strains of Plasmodium falciparum. Owl monkeys were immunized against the Camp and FCR-3/FMG strains of P. falciparum by infection, drug treatment, and rechallenge with homologous parasites. Camp-immune monkey serum was used to inhibit the in vitro growth of eight strains of P. falciparum. Inhibition was maximum for the homologous Camp strain (an average of 62% inhibition by 100 ml/litre Camp-immune serum). Four other strains were inhibited to a lesser degree, and three strains (FCR-3/FMG, FVO, and Smith) were not significantly inhibited by Camp-immune serum at concentrations as high as 400 ml/litre. FCR-3/FMG-immune serum at a concentration of 50 ml/litre caused significant inhibition of the FCR-3/FMG strain, but not the Camp strain. Thus Camp and FCR-3/FMG strains appear to bear distinct antigenic determinants recognized by the homologous, but not the heterologous, antiserum. Inhibition of in vitro growth by immune serum may be useful for serotyping P. falciparum and may have application in the selection of strains for inclusion in a malaria vaccine. PMID:3893775

  5. Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach

    PubMed Central

    2011-01-01

    Background Over its life cycle, the Plasmodium falciparum parasite is exposed to different environmental conditions, particularly to variations in O2 pressure. For example, the parasite circulates in human venous blood at 5% O2 pressure and in arterial blood, particularly in the lungs, at 13% O2 pressure. Moreover, the parasite is exposed to 21% O2 levels in the salivary glands of mosquitoes. Methods To study the metabolic adaptation of P. falciparum to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken. Results Even though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response. Conclusions These results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of P. falciparum. This study provides a better understanding of the adaptive capabilities of P. falciparum to environmental changes and may lead to the development of novel therapeutic targets. PMID:21223545

  6. Plasmodium falciparum Genetic Diversity in Bangladesh Does Not Suggest a Hypoendemic Population Structure.

    PubMed

    Alam, Mohammad Shafiul; Elahi, Rubayet; Mohon, Abu Naser; Al-Amin, Hasan Mohammad; Kibria, Mohammad Golam; Khan, Wasif A; Khanum, Hamida; Haque, Rashidul

    2016-06-01

    Despite the recommendation for the use of merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2), and glutamate-rich protein (glurp) genes as markers in drug efficacy studies by World Health Organization and their limited use in Bangladesh, the circulating Plasmodium falciparum population genetic structure has not yet been assessed in Bangladesh. This study presents a comprehensive report on the circulating P. falciparum population structure based on msp1, msp2, and glurp polymorphic gene markers in Bangladesh. Among the 130 pretreatment (day 0) P. falciparum samples from seven malaria-endemic districts, 14 distinct genotypes were observed for msp1, 20 for msp2, and 13 for glurp Polyclonal infection was reported in 94.6% (N = 123) of the samples. Multiplicity of infection (MOI) for msp1 was the highest (1.5) among the MOIs of the markers. The heterozygosity for msp1, msp2, and glurp was 0.89, 0.93, and 0.83, respectively. Data according to different malaria-endemic areas are also presented and discussed. Bangladesh is considered as a malaria-hypoendemic country. However, the prevalence of polyclonal infection and the genetic diversity of P. falciparum do not represent hypoendemicity. PMID:27139455

  7. Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

    PubMed Central

    Del Giudice, G; Verdini, A S; Pinori, M; Pessi, A; Verhave, J P; Tougne, C; Ivanoff, B; Lambert, P H; Engers, H D

    1987-01-01

    A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial. PMID:2432083

  8. Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor

    PubMed Central

    Pandey, Kishor; Ferreira, Pedro E.; Ishikawa, Takeshi; Nagai, Takeharu; Kaneko, Osamu; Yahata, Kazuhide

    2016-01-01

    Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (<10 μm) measurement of intracellular Ca2+ in Plasmodium is technically challenging, and thus Ca2+ regulation in this human pathogen is not well understood. Here we analyze Ca2+ homeostasis via a new approach using transgenic P. falciparum expressing the Ca2+ sensor yellow cameleon (YC)-Nano. We found that cytosolic Ca2+ concentration is maintained at low levels only during the intraerythrocytic trophozoite stage (30 nM), and is increased in the other blood stages (>300 nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening. PMID:27006284

  9. Ca(2+) monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor.

    PubMed

    Pandey, Kishor; Ferreira, Pedro E; Ishikawa, Takeshi; Nagai, Takeharu; Kaneko, Osamu; Yahata, Kazuhide

    2016-01-01

    Calcium (Ca(2+))-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (<10 μm) measurement of intracellular Ca(2+) in Plasmodium is technically challenging, and thus Ca(2+) regulation in this human pathogen is not well understood. Here we analyze Ca(2+) homeostasis via a new approach using transgenic P. falciparum expressing the Ca(2+) sensor yellow cameleon (YC)-Nano. We found that cytosolic Ca(2+) concentration is maintained at low levels only during the intraerythrocytic trophozoite stage (30 nM), and is increased in the other blood stages (>300 nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca(2+) level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca(2+) signaling in P. falciparum and is applicable for drug screening. PMID:27006284

  10. A non-pharmaceutical form of Artemisia annua is not effective in preventing Plasmodium falciparum malaria.

    PubMed

    Lagarce, Laurence; Lerolle, Nicolas; Asfar, Pierre; Le Govic, Yohann; Lainé-Cessac, Pascale; de Gentile, Ludovic

    2016-05-01

    Non-pharmaceutical forms of Artemisia annua (a Chinese plant containing artemisinin) are used by some travellers who believe these products are safer than anti-malarial drugs. We report two cases of severe Plasmodium falciparum malaria requiring hospitalization in an Intensive Care Unit following prophylaxis with non-pharmaceutical A. annua in French travellers. PMID:27432906

  11. Plasmodium falciparum malaria occurring 8 years after leaving an endemic area.

    PubMed

    Szmitko, Paul E; Kohn, Magdie L; Simor, Andrew E

    2009-01-01

    A 29-year-old patient who was born in Angola developed Plasmodium falciparum malaria 8 years after leaving Africa. She had not returned to a malaria-endemic area, and there were no apparent risks of local or nosocomial acquisition of malaria in Canada. She recovered after treatment with oral quinine sulfate and doxycycline. PMID:18945569

  12. Novel Mutations in K13 Propeller Gene of Artemisinin-Resistant Plasmodium falciparum

    PubMed Central

    Uemura, Haruki; Kimata, Isao; Ichinose, Yoshio; Logedi, John; Omar, Ahmeddin H.; Kaneko, Akira

    2015-01-01

    We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012–2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites. PMID:25695257

  13. Novel mutations in K13 propeller gene of artemisinin-resistant Plasmodium falciparum.

    PubMed

    Isozumi, Rie; Uemura, Haruki; Kimata, Isao; Ichinose, Yoshio; Logedi, John; Omar, Ahmeddin H; Kaneko, Akira

    2015-03-01

    We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012-2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites. PMID:25695257

  14. Homology-Based Prediction of Potential Protein–Protein Interactions between Human Erythrocytes and Plasmodium falciparum

    PubMed Central

    Ramakrishnan, Gayatri; Srinivasan, Narayanaswamy; Padmapriya, Ponnan; Natarajan, Vasant

    2015-01-01

    Plasmodium falciparum, a causative agent of malaria, is a well-characterized obligate intracellular parasite known for its ability to remodel host cells, particularly erythrocytes, to successfully persist in the host environment. However, the current levels of understanding from the laboratory experiments on the host–parasite interactions and the strategies pursued by the parasite to remodel host erythrocytes are modest. Several computational means developed in the recent past to predict host–parasite/pathogen interactions have generated testable hypotheses on feasible protein–protein interactions. We demonstrate the utility of protein structure-based protocol in the recognition of potential interacting proteins across P. falciparum and host erythrocytes. In concert with the information on the expression and subcellular localization of host and parasite proteins, we have identified 208 biologically feasible interactions potentially brought about by 59 P. falciparum and 30 host erythrocyte proteins. For selected cases, we have evaluated the physicochemical viability of the predicted interactions in terms of surface complementarity, electrostatic complementarity, and interaction energies at protein interface regions. Such careful inspection of molecular and mechanistic details generates high confidence on the predicted host–parasite protein–protein interactions. The predicted host–parasite interactions generate many experimentally testable hypotheses that can contribute to the understanding of possible mechanisms undertaken by the parasite in host erythrocyte remodeling. Thus, the key protein players recognized in P. falciparum can be explored for their usefulness as targets for chemotherapeutic intervention. PMID:26740742

  15. Immunogenicity and in vitro Protective Efficacy of a Recombinant Multistage Plasmodium falciparum Candidate Vaccine

    NASA Astrophysics Data System (ADS)

    Shi, Ya Ping; Hasnain, Seyed E.; Sacci, John B.; Holloway, Brian P.; Fujioka, Hisashi; Kumar, Nirbhay; Wohlhueter, Robert; Hoffman, Stephen L.; Collins, William E.; Lal, Altaf A.

    1999-02-01

    Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

  16. Hemoglobin consumption by P. falciparum in individual erythrocytes imaged via quantitative phase spectroscopy

    NASA Astrophysics Data System (ADS)

    Rinehart, Matthew T.; Park, Han Sang; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

    2016-04-01

    Plasmodium falciparum infection causes structural and biochemical changes in red blood cells (RBCs). To quantify these changes, we apply a novel optical technique, quantitative phase spectroscopy (QPS) to characterize individual red blood cells (RBCs) during the intraerythrocytic life cycle of P. falciparum. QPS captures hyperspectral holograms of individual RBCs to measure spectroscopic changes across the visible wavelength range (475–700 nm), providing complex information, i.e. amplitude and phase, about the light field which has interacted with the cell. The complex field provides complimentary information on hemoglobin content and cell mass, which are both found to dramatically change upon infection by P. falciparum. Hb content progressively decreases with parasite life cycle, with an average 72.2% reduction observed for RBCs infected by schizont-stage P. falciparum compared to uninfected cells. Infection also resulted in a 33.1% reduction in RBC’s optical volume, a measure of the cells’ non-aqueous components. Notably, optical volume is only partially correlated with hemoglobin content, suggesting that changes in other dry mass components such as parasite mass may also be assessed using this technique. The unique ability of QPS to discriminate individual healthy and infected cells using spectroscopic changes indicates that the approach can be used to detect disease.

  17. Hemoglobin consumption by P. falciparum in individual erythrocytes imaged via quantitative phase spectroscopy.

    PubMed

    Rinehart, Matthew T; Park, Han Sang; Walzer, Katelyn A; Chi, Jen-Tsan Ashley; Wax, Adam

    2016-01-01

    Plasmodium falciparum infection causes structural and biochemical changes in red blood cells (RBCs). To quantify these changes, we apply a novel optical technique, quantitative phase spectroscopy (QPS) to characterize individual red blood cells (RBCs) during the intraerythrocytic life cycle of P. falciparum. QPS captures hyperspectral holograms of individual RBCs to measure spectroscopic changes across the visible wavelength range (475-700 nm), providing complex information, i.e. amplitude and phase, about the light field which has interacted with the cell. The complex field provides complimentary information on hemoglobin content and cell mass, which are both found to dramatically change upon infection by P. falciparum. Hb content progressively decreases with parasite life cycle, with an average 72.2% reduction observed for RBCs infected by schizont-stage P. falciparum compared to uninfected cells. Infection also resulted in a 33.1% reduction in RBC's optical volume, a measure of the cells' non-aqueous components. Notably, optical volume is only partially correlated with hemoglobin content, suggesting that changes in other dry mass components such as parasite mass may also be assessed using this technique. The unique ability of QPS to discriminate individual healthy and infected cells using spectroscopic changes indicates that the approach can be used to detect disease. PMID:27087557

  18. A crucial piece in the puzzle of the artemisinin resistance mechanism in Plasmodium falciparum.

    PubMed

    Bozdech, Zbynek; Ferreira, Pedro E; Mok, Sachel

    2015-08-01

    The spread of resistance of malaria infections to artemisinin is a major concern for the future. The Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K) may be a potential target of artemisinin and effector of resistance. This could be mediated by the Kelch13 protein, the molecular marker of resistance that modulates PfPI3K ubiquitination. PMID:26169358

  19. Slow Clearance of Plasmodium falciparum in Severe Pediatric Malaria, Uganda, 2011-2013.

    PubMed

    Hawkes, Michael; Conroy, Andrea L; Opoka, Robert O; Namasopo, Sophie; Zhong, Kathleen; Liles, W Conrad; John, Chandy C; Kain, Kevin C

    2015-07-01

    Plasmodium falciparum resistance to artemisinin derivatives is emerging in Asia. We examined molecular markers of resistance in 78 children in Uganda who had severe malaria and were treated with intravenous artesunate. We observed in the K13-propeller domain, A578S, a low-frequency (3/78), nonsynonymous, single-nucleotide polymorphism associated with prolonged parasite clearance. PMID:26079933

  20. Slow Clearance of Plasmodium falciparum in Severe Pediatric Malaria, Uganda, 2011–2013

    PubMed Central

    Hawkes, Michael; Conroy, Andrea L.; Opoka, Robert O.; Namasopo, Sophie; Zhong, Kathleen; Liles, W. Conrad; John, Chandy C.

    2015-01-01

    Plasmodium falciparum resistance to artemisinin derivatives is emerging in Asia. We examined molecular markers of resistance in 78 children in Uganda who had severe malaria and were treated with intravenous artesunate. We observed in the K13-propeller domain, A578S, a low-frequency (3/78), nonsynonymous, single-nucleotide polymorphism associated with prolonged parasite clearance. PMID:26079933